Avian Influenza

Edited by David E. Swayne David E. Swayne, DVM, PhD, Diplomate, American practitioners for any particular patient. The publisher College of Veterinary Pathologists; Diplomate, and the editor make no representations or warranties American College of Poultry Veterinarians, is with respect to the accuracy or completeness of the Laboratory Director at the Southeast Poultry Research contents of this work and specifi cally disclaim all Laboratory, Agricultural Research Service, U.S. warranties, including without limitation any implied Department of Agriculture, in Athens, Georgia. warranties of fi tness for a particular purpose. In view of ongoing research, equipment modifi cations, ©2008 Blackwell Publishing changes in governmental regulations, and the constant All rights reserved fl ow of information relating to the use of medicines, equipment, and devices, the reader is urged to review Blackwell Publishing Professional and evaluate the information provided in the package 2121 State Avenue, Ames, Iowa 50014, USA insert or instructions for each medicine, equipment, or device for, among other things, any changes in the Orders: 1-800-862-6657 instructions or indication of usage and for added Offi ce: 1-515-292-0140 warnings and precautions. Readers should consult Fax: 1-515-292-3348 with a specialist where appropriate. The fact that an Web site: www.blackwellprofessional.com organization or Website is referred to in this work as Blackwell Publishing Ltd a citation and/or a potential source of further 9600 Garsington Road, Oxford OX4 2DQ, UK information does not mean that the editor or the Tel.: +44 (0)1865 776868 publisher endorses the information the organization or Website may provide or recommendations it may Blackwell Publishing Asia make. Further, readers should be aware that Internet 550 Swanston Street, Carlton, Victoria 3053, Australia Websites listed in this work may have changed or Tel.: +61 (0)3 8359 1011 disappeared between when this work was written and when it is read. No warranty may be created or Authorization to photocopy items for internal or extended by any promotional statements for this personal use, or the internal or personal use of work. Neither the publisher nor the editor shall be specifi c clients, is granted by Blackwell Publishing, liable for any damages arising herefrom. provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Library of Congress Cataloging-in-Publication Data Danvers, MA 01923. For those organizations that Avian infl uenza / edited by David E. Swayne.—1st ed. have been granted a photocopy license by CCC, a p. ; cm. separate system of payments has been arranged. The Includes bibliographical references and index. fee codes for users of the Transactional Reporting ISBN 978-0-8138-2047-7 (alk. paper) Service are ISBN-13: 978-0-8138-2047-7/2008. 1. Avian infl uenza. I. Swayne, David E. [DNLM: 1. Infl uenza in Birds–prevention & First edition, 2008 control. 2. Disease Outbreaks–prevention & control. 3. Infl uenza A Virus, H1N1 Subtype– Disclaimer pathogenicity. 4. Infl uenza, Human–prevention & The contents of this work are intended to further control. SF 995.6.I6 A9565 2008] general scientifi c research, understanding, and discussion only and are not intended and should not SF995.6.I6A92 2008 be relied upon as recommending or promoting a 636.5’0896203–dc22 specifi c method, diagnosis, or treatment by 2007039629 The last digit is the print number: 9 8 7 6 5 4 3 2 1 Dedication

This book is dedicated to the veterinarians and vet- • Richard Slemons, who in 1972 isolated the fi rst erinary scientists who have devoted their careers to infl uenza A virus from a free-living duck, thus the study of infl uenza for the benefi t of avian health. launching a new area of scientifi c investigation Their individual and collective contributions have (i.e., AI ecology in free-living waterfowl) profoundly affected our scientifi c understanding of • Bernard (Barney) C. Easterday, who extensively avian infl uenza (AI) ecological, epidemiological, surveyed wild birds for AI viruses and conducted clinical, virological, pathobiological, and genetic experimental infections in some wild bird features of the disease or virus. The discoveries of species these leaders of the veterinary profession are often • James E. Pearson and Dennis A. Senne, who stan- overlooked, but they have made critical and signifi - dardized diagnostic test protocols for AI viruses cant contributions to the One World, One Health and developed quality reagents for distribution concept of medical science in the area of infl uenza • Benjamin S. Pomeroy and Ray A. Bankowski, biology: who organized the fi rst International Symposium on Avian Infl uenza in 1981 • Edoardo Perroncito, whose astute clinical obser- • David A. Halvorson, who made the fi rst direct vations and microbiological investigations in 1878 epidemiological linkage between free-living lead to the identifi cation of the fi rst cases of highly waterfowl AI viruses and AI virus infections in pathogenic (HP) AI (i.e., fowl plague) and its dif- range-reared domestic turkeys in Minnesota ferentiation from other serious poultry diseases during 1980–1981 such as fowl cholera and fowl typhoid • Robert J. Eckroade, who in 1983 recognized and • Zvonimir Dinter, who diagnosed the fi rst case of described the fi rst clinical occurrence of the muta- low pathogenicity (LP) AI in poultry (chickens) tion of an LPAI virus to an HPAI virus in Germany during 1949 • Yoshihiro Kawaoka, who led the team that discov- • R.V.L. Walker and G.L. Bannister, who diagnosed ered the loss of a carbohydrate group on the hem- the fi rst LPAI case in domestic ducks in Canada agglutinin was responsible for the shift in virulence during 1953 of H5N2 LPAI to HPAI viruses in United States • Werner Shäfer, who discovered fowl plague was during 1983 caused by infl uenza A virus in 1955 • Dennis J. Alexander, who pioneered the holistic • Gerhard Lang, who diagnosed the fi rst LPAI case approach to AI diagnosis and control that resulted in turkeys in Canada during 1963 in rapid adoption of molecular basis for pathoge- • Charles W. Beard, who developed the agar gel nicity determination and the common use of immunodiffusion test for AI that has become sequencing of AI viruses for epidemiological the primary serological surveillance test for purposes gallinaceous poultry since its development in • Max Brugh, who developed the fi rst laboratory 1970 system to produce and study HP derivatives from • Rudolph Rott, who led the research team that H5 and H7 LPAI viruses determined in the 1970s that the cleavability of the • Michael L. Perdue, who discovered the mecha- hemagglutinin protein was the major determinate nism for insertions of extra codons in the hemag- of infl uenza A virus virulence in chickens glutinin proteolytic cleavage site

v Table of Contents

Contributors List ix Foreword xiii Preface xv

1. Infl uenza A Virus 3 David L. Suarez 2. Molecular Determinants of Pathogenicity for Avian Infl uenza Viruses 23 Michael L. Perdue 3. Ecology of Avian Infl uenza in Wild Birds 43 David E. Stallknecht and Justin D. Brown 4. Epidemiology of Avian Infl uenza in Agricultural and Other Man-Made Systems 59 David E. Swayne 5. Pathobiology of Avian Infl uenza Virus Infections in Birds and Mammals 87 David E. Swayne and Mary Pantin-Jackwood Color Plate Section 6. The Global Nature of Avian Infl uenza 123 David E. Swayne 7. The Beginning and Spread of Fowl Plague (H7 High Pathogenicity Avian Infl uenza) Across Europe and Asia (1878–1955) 145 Erhard F. Kaleta and Catherine P. A. Rülke 8. High Pathogenicity Avian Infl uenza in the Americas 191 David E. Swayne 9. Highly Pathogenic Avian Infl uenza Outbreaks in Europe, Asia, and Africa Since 1959, Excluding the Asian H5N1 Virus Outbreaks 217 Dennis J. Alexander, Ilaria Capua, and Guus Koch 10. Avian Infl uenza in Australia 239 Leslie D. Sims and Andrew J. Turner 11. Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 251 Leslie D. Sims and Ian H. Brown 12. Avian Infl uenza Control Strategies 287 David E. Swayne 13. Avian Infl uenza Diagnostics and Surveillance Methods 299 Erica Spackman, David L. Suarez, and Dennis A. Senne 14. Mass Depopulation as an Effective Measure for Disease Control Purposes 309 Elizabeth A. Krushinskie, Martin Smeltzer, Patrice Klein, and Harm Kiezenbrink

vii viii Contents

15. Methods for Disposal of Poultry Carcasses 333 Boris Brglez and John Hahn 16. Farm and Regional Biosecurity Practices 353 Carol J. Cardona 17. Farm Biosecurity Risk Assessment and Audits 369 David Shapiro and Bruce Stewart-Brown 18. Methods for Inactivation of Avian Infl uenza Virus in the Environment 391 Nathan G. Birnbaum and Bethany O’Brien 19. Vaccines, Vaccination, and Immunology for Avian Infl uenza Viruses in Poultry 407 David E. Swayne and Darrell R. Kapczynski 20. Public Health Implications of Avian Infl uenza Viruses 453 Nancy J. Cox and Timothy M. Uyeki 21. The Role of Educational Programs in the Control of Avian Infl uenza 485 Nathaniel L. Tablante 22. Trade and Food Safety Aspects for Avian Infl uenza Viruses 499 David E. Swayne and Colleen Thomas 23. Control of Low Pathogenicity Avian Infl uenza 513 David A. Halvorson 24. The Economics of Avian Infl uenza 537 Anni McLeod 25. Global Strategy for Highly Pathogenic Avian Infl uenza: Progressive Control and Eradication, and Postoutbreak Recovery 561 Juan Lubroth, Subhash Morzaria, and Alejandro B. Thiermann

Index 587 Contributors List

Dennis J. Alexander (retired) Carol J. Cardona Virology Department Veterinary Medicine Extension VLA Weybridge School of Veterinary Medicine Woodham Lane, Addlestone University of California, Davis Surrey KT15 3NB, United Kingdom Davis, California 95616, USA Nathan G. Birnbaum Nancy J. Cox National Center for Animal Health Director, Infl uenza Division Emergency Management Veterinary Director, WHO Collaborating Center for Services Surveillance, Epidemiology and Control of Animal and Plant Health Inspection Infl uenza Service Centers for Disease Control and Prevention U.S. Department of Agriculture 1600 Clifton Road NE, Mail Stop A-20 4700 River Road Atlanta, Georgia 30333, USA Riverdale, Maryland 20737, USA John W. Hahn (Retired) Boris Brglez Veterinary Services, Arkansas Area Offi ce 109 Bourke Place Animal and Plant Health Inspection Services Cary, North Carolina 27511, USA U.S. Department of Agriculture 9801 Holt Drive Ian H. Brown Rogers, Arkansas 72756, USA Veterinary Laboratories Agency-Weybridge Addlestone David A. Halvorson Surrey KT15 3NB, United Kingdom College of Veterinary Medicine University of Minnesota Justin D. Brown 1971 Commonwealth Avenue Southeastern Cooperative Wildlife Disease Saint Paul, Minnesota 55108, USA Study Department of Population Health Erhard F. Kaleta College of Veterinary Medicine Clinic for Birds, Reptilia, Amphibia and Fish The University of Georgia Justus Liebig University Giessen Athens, Georgia 30602, USA Veterinary Faculty Frankfurter Strasse 91–93 Ilaria Capua D-35392 Giessen, Germany OIE/FAO Reference Laboratory for Newcastle Disease and Avian Infl uenza Darrell R. Kapczynski Virology Department Southeast Poultry Research Laboratory Istituto Zooprofi lattico Sperimentale delle Venezie Agricultural Research Service (IZSVe) U.S. Department of Agriculture Viale dell’Università 10 934 College Station Road 35020 Legnaro, Padova, Italy Athens, Georgia 30605, USA

ix x Contributors List

Harm Kiezebrink Bethany O’Brien BFC Bird Flu Control GmbH National Center Animal Health Emergency Schäftlarnstrasse 132 Management 81371 Munich, Germany Veterinary Services Animal and Plant Health Inspection Patrice N. Klein Service Veterinary Services—National Center for Animal U.S. Department of Agriculture Health Programs 4700 River Road Animal Plant Health Inspection Service Riverdale, Maryland 20737, USA U.S. Department of Agriculture 4700 River Road Mary J. Pantin-Jackwood Riverdale, Maryland 20737, USA Southeast Poultry Research Laboratory Agricultural Research Service Guus Koch U.S. Department of Agriculture Central Institute for Animal Disease Control 934 College Station Road (CIDC-Lelystad) Athens, Georgia 30605, USA Wageningen University and Research Postbox 2004 Michael L. Perdue 8203 AA Lelystad, the Netherlands Department of Epidemic and Pandemic Alert and Response Elizabeth A. Krushinskie WHO Global Inﬂ uenza Programme, WHO/CDS/ Director of Quality Assurance and Food EPR/GIP Safety 1211 Geneva 27, Switzerland Mountaire Farms, Inc. Millsboro, Delaware 19966, USA Catherine P. A. Ruelke Clinic for Birds, Reptilia, Amphibia and Fish Juan Lubroth Justus Liebig University Giessen Veterinary Head, Infectious Diseases Group Faculty Animal Health Service Frankfurter Strasse 91–93 Animal Production and Health Division D-35392 Giessen, Germany Food and Agriculture Organization of the United Nations Dennis A. Senne Viale delle Terme di Caracalla National Veterinary Services Laboratories 00153 Rome, Italy Veterinary Services Animal and Plant Health Inspection Service Anni McLeod U.S. Department of Agriculture Senior Ofﬁ cer (Livestock Policy) 1800 Dayton Avenue Livestock Information, Sector Analysis and Policy Ames, Iowa 50010, USA Branch Animal Production and Health Division David Shapiro Food and Agriculture Organization of the United Director of Veterinary Services Nations Perdue Farms, Inc. Viale delle Terme di Caracalla PO Box 1537 00100 Rome, Italy Salisbury, Maryland 21802, USA

Subhash P. Morzaria Leslie D. Sims Regional Offi ce for Asia and the Pacifi c Asia Pacifi c Veterinary Information Services Pty Food and Agriculture Organisation (FAO) of the Ltd United Nations PO Box 344 39 Phra Athit Road Palm Cove Bangkok 10200, Thailand Queensland 4879, Australia Contributors List xi

Martin A. Smeltzer David E. Swayne Veterinary Services Southeast Poultry Research Laboratory Animal Plant Health Inspection Service Agricultural Research Service U.S. Department of Agriculture U.S. Department of Agriculture 953 College Station Road 934 College Station Road Athens, Georgia 30602, USA Athens, Georgia 30605, USA

Erica Spackman Nathaniel L. Tablante Southeast Poultry Research Laboratory Virginia-Maryland Regional College of Veterinary Agricultural Research Service Medicine U.S. Department of Agriculture University of Maryland College Park 934 College Station Road 8075 Greenmead Drive Athens, Georgia 30605, USA College Park, Maryland 20742, USA Colleen Thomas David E. Stallknecht Southeast Poultry Research Laboratory Southeastern Cooperative Wildlife Disease Agricultural Research Service Study U.S. Department of Agriculture Department of Population Health 934 College Station Road College of Veterinary Medicine Athens, Georgia 30605, USA The University of Georgia Athens, Georgia 30602, USA Andrew Turner Bruce Stewart-Brown Andrew Turner Consulting Pty Ltd Glen Lee 25 Garton Street Bruce Stewart-Brown Princes Hill, Victoria 3054, Australia Perdue Farms, Inc 31149 Old Ocean City Road Timothy M. Uyeki Salisbury, Maryland 21804, USA Epidemiology and Prevention Branch Inﬂ uenza Division David L. Suarez National Center for Immunization and Respiratory Southeast Poultry Research Laboratory Diseases Agricultural Research Service MS A-32 U.S. Department of Agriculture Centers for Disease Control and Prevention 934 College Station Road 1600 Clifton Road NE Athens, Georgia 30605, USA Atlanta, Georgia 30333, USA Foreword

The long history of avian infl uenza (AI), unlike continues the tradition of excellent, science-based many other diseases, is refl ected in a series of dis- educational publications by this organization. tinct outbreaks or epidemics, each of which is not The book was conceived and edited by Dr. David only unique but also a rich source of information. E. Swayne, who has devoted 21 years to the study Each provides lessons by which knowledge is of AI and leads the U.S. Department of Agriculture, expanded and strategies for control can be improved, Agricultural Research Service, Southeast Poultry justifying a systematic and detailed analysis. This Research Laboratory in Athens, Georgia, USA, disease has attracted the attention of poultry veteri- which has been a strong contributor to knowledge narians, virologists, and public health specialists, not in this fi eld. Under his leadership, an impressive to mention the poultry industry and the general group of highly distinguished and internationally public. This text brings together in a comprehensive representative authors have contributed chapters. manner the knowledge and experience gained over Many of these authors are specialists on AI and all the century and a quarter that this disease has been speak with authority on their respective subjects. recognized in poultry. In so doing, it goes well Dr. Swayne, with his considerable experience as beyond previous discussions of the disease found in a pathologist, researcher, international consultant, chapters and symposia proceedings and provides a research leader, and editor, has personally contrib- unique resource that should be valuable for years to uted to more than a quarter of the 25 chapters. come. This book will be valued by poultry veterinarians, This book fl ows from historical accounts with researchers, and regulatory offi cials who deal with emphasis on epidemiology and biology to control AI and other exotic poultry diseases, but it also will strategies and their components, to public health, be helpful to public health offi cials in understanding educational, trade, and economic concerns—all in a the animal health side of this disease. distinctly global context. It is the fi rst text to cover all relevant aspects of AI from the perspective of Richard L. Witter, DVM, PhD, Dipl. ACPV poultry health. Member, National Academy of Sciences (USA) Publication of this book is sponsored by the Amer- U.S. Department of Agriculture, Agricultural ican Association of Avian Pathologists, an organiza- Research Service, Avian Disease and Oncology tion that has long supported the publication of Laboratory information relevant to poultry medicine. This book East Lansing, Michigan, USA

xiii Preface

Avian infl uenza (AI) was fi rst identifi ed as a distinct and poster presentations from international AI meet- disease entity of poultry in 1878, and endemic infec- ings have been assembled such as the Proceedings of tions or sporadic outbreaks have been reported in First through Sixth International Symposia on Avian poultry populations globally through the 1990s. Infl uenza (U.S. Animal Health Association, Rich- During such times, AI remained a disease of birds, mond, Virginia, USA), Proceedings of the Frontis primarily known to a limited number of poultry vet- Workshop on Avian Infl uenza: Prevention and erinarians and scientists until 1997 when the fi rst Control (Springer, Dordrecht, the Netherlands), and H5N1 high pathogenicity (HP) AI cases in humans proceedings from multiple OIE and Food and were reported in Hong Kong. This incident pro- Agricultural Organization (FAO) meetings on AI pelled AI, or “bird fl u,” into a globally recognized around the world such as those recorded in Develop- disease among the general veterinary medical comments in Biologicals (Volumes 114, 119, and 124). munity and public health offi cials, and even among There has been an excellent, but brief, atlas pub- the lay public. Over the next 10 years, H5N1 HPAI lished on AI: A Colour Atlas and Text on Avian in poultry spread across Asia, Europe, and Africa, Infl uenza (by Capua and Mutinelli, 2001, Papi thrusting AI to international prominence with the Editore, Bologna, Italy). distinction of being the largest transboundary animal However, no single text has been published that health crisis of the past 100 years with unprece- systematically covered all areas of biology, virol- dented socioeconomic and animal welfare conse- ogy, diagnostics, ecology, epidemiology, clinical quences. In addition, the continued occurrence of medicine, and control of AI and has been focused zoonotic infections has created a public health crisis on the educational and training needs of those who with concerns that this virus potentially could reas- deal with poultry health needs—veterinarians, sort with a human infl uenza A virus to produce the poultry scientists, and government animal health next human infl uenza pandemic. offi cials. Such a comprehensive text on AI would However, written knowledge on AI ecology, epi- have been too large a task for one person, and help demiology, pathology, pathogenesis, diagnostics, was solicited from experts around the world to con- and control has been limited to individual chapters in tribute their knowledge in individual chapters on books, scientifi c conference proceedings, and peer- specifi c aspects of AI. This text was envisioned to reviewed research and review articles in journals. not only cover the latest discoveries in the basic Most of the individual book chapters have been brief sciences but also translate such information into and have been general overviews such as those applications for the clinical setting and for use of included in the texts Diseases of Poultry (editions 1 such information by fi eld veterinarians and govern- through 12; Iowa State University Press and Wiley- ment health professionals in the control of AI. In Blackwell Publishing, Ames, Iowa, USA), Poultry addition, we are confi dent, our colleagues in public Diseases (editions 1 through 5; Bailliere-Tindall, health and poultry science will benefi t greatly from London, United Kingdom), and Merck Veterinary the knowledge within this book. Manual (Merck, Whitehouse Station, New Jersey, This text was commissioned by the American USA), or in special issues on poultry diseases as in Association of Avian Pathologist (AAAP), a non- the World Organization for Animal Health (OIE) profi t educational foundation, whose mission is to Scientifi c and Technical Review (Volume 19, Issue promote research and apply such new knowledge to 2). In some instances, written documentation of oral solving poultry health problems, which includes

xv xvi Preface providing educational resources to poultry veterinar- the past 2 years is much appreciated. Finally, I per- ians and poultry health professionals around the sonally thank Drs. Richard D. Slemons, Charles W. world. The authors and editor of this book have Beard, and Max Brugh for introducing me to the received no fi nancial compensation from the sale of exciting world of AI research and for their continual this book, but we do acknowledge the valuable pro- career guidance and mentoring, which has made the fessional satisfaction of helping colleagues around past 21 years of researching the AI virus and the the world and advancing the discipline of poultry disease a daily, fun adventure. medicine. All profi ts have been used to further the Mention of trade names or commercial products educational programs of AAAP, including dona- in this book is solely for the purpose of providing tions of poultry health educational materials to specifi c information and does not imply recommen- developing countries. dation or endorsement by the authors. The content As editor, my special thanks go to Sarah Benner, of individual chapters is based upon the scientifi c who provided assistance in verifying and formatting literature and the knowledge and experience of the references, in corresponding with authors, and in individual authors and is not the offi cial position of database management. The Board of Directors for the U.S. Department of Agriculture or other employ- AAAP is thanked for commissioning this text, and ers of the individual authors. several colleagues are thanked for providing anonymous critique and review of some chapters to ensure David E. Swayne, DVM, PhD, accuracy. The highly skilled and professional assis- Dipl. ACVP, Dipl. ACPV tance of Wiley-Blackwell, especially of Erin Editor Gardner, Dede Andersen, and Carrie Sutton, over Athens, Georgia, USA Plate 1

David L. Suarez

INTRODUCTION type A infl uenza viruses (referred to only as infl u- Infl uenza A viruses are important veterinary and enza virus for the rest of the chapter) will be human health pathogens around the world. Avian addressed for the remainder of the paper. infl uenza (AI) virus in poultry is unusual in that it can cause a range of disease symptoms from a sub- Composition clinical infection to being highly virulent with 100% All infl uenza viruses have eight different gene seg- mortality. The difference between low pathogenicity ments that encode at least 10 different viral proteins. (LP) viruses and high pathogenicity (HP) viruses The structural proteins in the mature virion can be can be as small as a single amino acid change in the divided into the surface proteins that include the hemagglutinin (HA) fusion cleavage site. Therefore, HA, neuraminidase (NA), and membrane ion channel it is important not only to assess an AI virus’s ability (M2) proteins and the internal proteins, including to cause disease in poultry but also to assess the the nucleoprotein (NP), the matrix protein (M1), and potential for AI viruses to cause disease in poultry. the polymerase complex composed of the poly- AI viruses also have a wide host range, including merase basic protein 1 (PB1), polymerase basic mammals, and can represent a zoonotic risk. Addi- protein 2 (PB2), and polymerase acidic protein (PA) tionally, the main reservoir for the AI viruses is in (68). Two additional proteins produced by infl uenza wild aquatic birds; therefore, complete eradication viruses are the nonstructural proteins, nonstructural is not possible. All these factors make AI viruses an protein 1 (NS1) and nonstructural protein 2 (NS2), important but diffi cult pathogen to control in which is also known as the nuclear export protein poultry. (NEP) (64). The NS1 protein is considered to be a true nonstructural protein that is not found in the ETIOLOGY virus particle but is produced in large amounts in the host cell (8, 120). The NS2 protein is primarily Classifi cation found in host cells, but some protein can be found The AI virus belongs to the Orthomyxoviridae in the virion (68). One protein that is not found in family of segmented negative-sense RNA viruses all type A infl uenza viruses is the PB1-F2 protein, that are divided into fi ve different genera, including which is an 87–amino acid protein that is transcribed infl uenza types A, B, and C, Isavirus, and Thogoto- from a different reading frame from the PB1 protein. virus. The type A infl uenza viruses are the most This protein is thought to be involved in apoptosis widespread and important members of the group in host cells, and its role in pathogenesis is still being infecting many different avian and mammalian determined (14). species. Types B and C infl uenza viruses are human pathogens that rarely infect other species (68). The Morphology Isavirus group includes the important fi sh pathogen, The virus morphologically can be extremely vari- infectious salmon anemia virus (39), and the thogo- able, ranging from spherical particles with a diam- toviruses are tick-borne arboviruses that have been eter of 80 to 120 nm to fi lamentous forms that can isolated from both humans and livestock (45). Only be several microns in length. The fi lamentous forms

Avian Influenza Edited by David E. Swayne 3 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 4 Avian Influenza seem to predominate in clinical isolates, but after produces a trypsin-like protease as seen with mam- passage in cell culture or chicken eggs, the virus malian kidney cell cultures (40). Recently, however, often changes morphology to the spherical forms, at the use of chicken eggs has been found to be inad- least for human viruses (9, 68). The morphology equate for the isolation of some infl uenza viruses appears to be primarily controlled by the matrix 1 from turkeys, specifi cally swine infl uenza-like protein with two specifi c amino acids being identi- viruses, swine, and humans. As early as 1996, H3N2 fi ed as being important (9). The overall structure of variants were isolated in cell culture that no longer the virus includes a lipid membrane derived from grew well in chicken eggs without adaptation (139). the host cell that has three viral integral membrane For these viruses, isolation in mammalian cell proteins, the HA, NA, and matrix 2 proteins. The culture was more reliable for primary isolation HA protein exists as a trimer that appears as spikes (117), although in one case the use of egg yolk sac on the lipid membrane and is the most abundant route of inoculation instead of allantoic sac inocula- surface protein (15). The NA protein exists as tetra- tion resulted in a virus isolation (107). The same mers and forms more of a globular structure extend- viruses that no longer replicate well in chicken eggs ing from the lipid membrane. The M2 protein is a also no longer effi ciently hemagglutinate chicken small protein that functions as an ion channel that is red blood cells (RBCs), which has necessitated important for triggering viral uncoating. The M1 using alternative RBCs like turkey or guinea pig protein appears to be the primary bridge between the RBCs (59, 107). lipid membrane and the viral core of NP, viral RNA, and the polymerase complex. Nomenclature The nomenclature for describing infl uenza viruses Propagation has been standardized to provide a consistent and Infl uenza viruses are easily propagated in the labora- informative nomenclature for all infl uenza viruses. tory, which has allowed them to be widely studied. The features used to name all new infl uenza viruses Avian, human, swine, and equine viruses were all include (1) antigenic type (A, B, or C); (2) host originally propagated in embryonating chicken eggs, animal from which the virus was isolated, but for and this method is still commonly used for both human isolates this may be omitted and is simply diagnostic purposes and for vaccine production. implied; (3) geographic origin of the isolate, which Recently, there has been more emphasis, particu- can be a city, state, province, or country designation; larly for the mammalian viruses, to grow infl uenza (4) unique laboratory or other reference identifi ca- viruses in cell culture, in both primary and continu- tion number for each isolate; (5) the year of isola- ous cell lines, for both routine diagnostics and tion; and (6) the HA and NA subtypes (often included vaccine production (66, 139). Common cell lines for in parentheses at the end). For example, an infl uenza virus isolation and propagation are chicken embryo virus isolated from turkeys in Missouri would be fi broblast cells, chicken embryo kidney cells, Madin- A/turkey/Missouri/24093/1999 (H1N2). Darby canine kidney cells, Vero cells, and others. For AI viruses, the isolation and characterization of Pathotype viruses are most commonly performed in 9- to 11- The World Organization for Animal Health (formerly day-old embryonating chicken eggs by inoculation Offi ce Internationale des Epizooties, OIE) defi nes of the allantoic cavity. Embryonation of chicken the pathogenicity of AI viruses as follows (133): eggs provides the added advantage of allowing replication for both LPAI and HPAI (low pathogenicity 1. High Pathogenicity notifi able avian infl uenza AI and high pathogenicity AI, respectively) viruses (HPNAI) viruses have an intravenous pathoge- (25). Primary chicken embryo cell cultures are also nicity index (IVPI) in 6-week-old chickens used, but for LPAI virus, trypsin must be added to greater than 1.2 or, as an alternative, cause at the media for effi cient replication and plaque forma- least 75% mortality in 4- to 8-week-old chickens tion of the virus. Alternatively, the use of some cell infected intravenously. H5 and H7 viruses which culture systems, like primary chicken kidney cells, do not have an IVPI of greater than 1.2 or cause allows replication and plaquing of LPAI virus less than 75% mortality in an intravenous lethal- without additional trypsin, presumably because it ity test should be sequenced to determine whether 1 / Influenza A Virus 5

multiple basic amino acids are present at the infection. The pathology correlates to the expression cleavage site of the hemagglutinin molecule of α-2,3 sialic acid in alveolar type II cells in the (HA0); if the amino acid motif is similar to that lung (88). An additional factor is that the specifi city observed for other HPNAI isolates, the isolate of the HA for either type of sialic acid is not abso- being tested should be considered as HPNAI; lute, and some viruses can bind both α-2,3 and α-2,6 2. Low pathogenicity notifi able avian infl uenza sialic acid (138). In experimental studies in humans (LPNAI) viruses are all infl uenza A viruses of H5 and animals, replication can often occur with many and H7 subtype that are not HPNAI viruses. viruses if the subjects are given a large enough challenge dose (7, 29). In general terms, HPNAI viruses are synonymous The HA receptor specifi city for sialic acid is not with HPAI viruses by defi nition while LPAI viruses absolute and can change with as little as two amino are all AI viruses that are not HPAI (HPNAI) viruses acid substitutions at positions 226 and 228 (H3 using the above criteria. The LPAI pathotype can amino acid numbering) (16, 125). Amino acid include AI viruses from any of the 16 HA (H1-16) changes at these two positions can result in major and 9 NA (N1-9) subtypes while LPNAI viruses are changes of the phenotype of the virus. a subset of LPAI viruses: i.e. only the H5 and H7 Pigs have previously been suggested to be a major LPAI viruses. mixing vessel for human infl uenza and AI viruses because they express high levels of both α-2,3 and VIRUS LIFE CYCLE α-2,6 sialic acid in their respiratory epithelium. The The initial step in viral infection is the attachment theory was that pigs could be simultaneously infected of the viral HA protein to the host cell receptor sialic with human infl uenza and AI viruses and reassort- acid. Sialic acid is a general term for the terminal ment could occur between the two viruses, resulting sugars found in N- and O-linked glycoproteins that in a new virus that could result in a pandemic strain can be made of many derivatives of neuraminic acid. (84, 128). The pig as a mixing vessel has some Sialic acid molecules are often classifi ed as to how support from fi eld data and complex reassortant they are linked to the underlying sugars by the α-2 viruses have been isolated from pigs (34, 124). carbon. The most common linkages are the α-2,3 However, the recent outbreaks in humans with AI- and α-2,6 linkage (110). These different sialic acid like viruses (H5N1, H9N2, H7N7, and H7N3), linkages result in different conformations of the host although not resulting in a pandemic virus, show receptor that affects virus binding. The viral HA, that exposure to infected poultry and not exposure based on amino acid sequence, has strong specifi city to pigs was the main risk factor for infection (42, 70, for either the α-2,3 or α-2,6 linkage, and this can be 105, 123). one factor in host specifi city. Different animals will Once viral attachment has occurred, the virus is have different tissue patterns and levels of expres- endocytosed, and when the endosome becomes sion of α-2,3 and α-2,6 sialic acid, and because acidifi ed, that triggers the fusion domain of the HA infl uenza viruses have a strong preference for either protein to become active and the viral RNA is α-2,3 and α-2,6 sialic acid, most viruses can only released into the cytoplasm (99). The M2 protein effi ciently infect animals that express their prefer- plays a key role in the triggering process because it + ence of sialic acid. The α-2,3 sialic acid is typically is an integral membrane protein that allows H ions expressed in avian species and the α-2,6 sialic acid to enter into the virion, which causes a conforma- is expressed in humans. Sialic acid conformation tional change of the HA at the lower pH to allow the likely contributes to host specifi city, but it likely is fusion domain to become active (76). The adaman- only one of the factors that contributes to the species tane class of antiviral drugs act by blocking the barrier. For example, both human and at least some function of the M2 protein, which prevents the avian hosts, including quail, express both types of fusion of the HA within the endosome (27, 109). sialic acid, although with different tissue distribu- The fusion of the viral membrane and the endosomal tions (119, 126). This receptor distribution can membrane allows the release of the viral RNA-poly- directly affect pathogenesis as has been proposed for merase complex into the cytoplasm, which is then H5N1 infection in humans where pneumonia is actively transported to the nucleus because of nuclear commonly seen and not an upper respiratory tract localization signals in the NP (63). 6 Avian Influenza

The negative-sense viral RNA is copied into that have reduced NA activity will aggregate on the positive-sense mRNA by the polymerase complex, cell surface because of particles attaching to each which includes the three polymerase proteins and other, which can greatly reduce the effective titer of the NP, in the nucleus. The virus also uses host the virus (4). The loss of NA activity is not just a proteins to initiate mRNA synthesis, including RNA theoretical exercise, because one of the markers of polymerase II. The mRNA requires a 5′ capped AI virus adaptation to poultry is the presence of stalk primer that is stolen from host mRNA by the PB2 deletions of the NA protein (57). These stalk dele- protein in a process known as cap snatching (43). tions result in marked decrease in NA activity. The The positive-sense viral mRNA then migrates from infl uenza virus can at least partially compensate for the nucleus to begin viral protein translation in the this reduced NA activity by making changes in the cytoplasm using the host cellular machinery. The HA protein that reduce the affi nity of binding to positive-sense RNA also serves as a template to sialic acid, typically by the addition of additional produce the negative-sense viral RNA that will be glycosylation sites near the receptor binding sites packaged into the virion. (60). We currently do not understand the selective Two viral proteins, the M1 and NEP, are crucial advantage of NA stalk deletions in poultry. for traffi cking of viral proteins to and from the For LPAI viruses, the released viral particles are nucleus. The M1 protein also plays a critical role in not infectious until the HA protein is cleaved into the assembly and structure of the virion (9). The HA1 and HA2 subunits by trypsin or trypsin-like viral assembly process includes the three integral proteases. The role of HA cleavage will be discussed membrane proteins, HA, NA, and small amounts of in more detail in the pathogenesis section. the M2 protein, entering the endoplasmic reticulum, where they are folded and glycosylated before even- VIRUS GENETICS tually moving to the apical plasma membrane (5). The M1 protein is believed to be critical in bridging Ecology in Wild Birds the surface integral membrane proteins and the ribo- The natural host and reservoir for all type A infl u- nucleoprotein complex and each of the eight viral enza viruses are in wild birds, primarily in water- gene segments before the virion is complete. All fowl, gulls, and shorebirds (36, 90). In the natural eight viral gene segments have highly conserved host, the virus appears to be evolving slowly with regions, 13 and 12 nucleotides long, on the 5′ and most internal genes being highly conserved at the 3′ end of each segment, respectively, that are impor- amino acid level (101). The surface glycoproteins, tant packaging signals. The process of RNA packag- HA and NA, are much more variable in amino acid ing appears to be an ineffi cient process, and many sequence, demonstrating the greater diversity of viral particles do not package all eight gene seg- these genes. For both proteins, multiple antigenic ments, creating a high proportion of defective viral subtypes have been characterized, where antibody to particles. It has been estimated that greater than 90% one subtype will neutralize with high specifi city of viral particles are noninfectious (18, 20). The only viruses of that subtype. For the HA protein, 16 packaging process also may allow multiple gene subtypes of AI have been characterized (Fig. 1.1), segments, particularly of the smaller genes, to be and 9 subtypes have been characterized for the NA included in the virion. This multiple packaging may protein. At the amino acid level, the difference even affect the phenotype of the virus because it has between subtypes is as little 20%, but the most been hypothesized that when multiple copies of the divergent subtypes are up to 63% different. About NS gene are packaged per virion, an increased resis- 25% of the amino acids are conserved among all 16 tance to interferon will occur (86). HA subtypes (62). Similar comparisons are found The effi cient budding of the viral particle from the for the NA subtypes with amino acid differences of cellular membrane requires, among other things, the between 31% and 61%. enzymatic activity of the NA protein to remove In comparing the nucleotide sequence of most of sialic acid from the surface glycoproteins, specifi - the gene segments from wild bird isolates, including cally the HA protein. This prevents self-binding of within an HA and NA subtype, a clear separation the protein and the aggregation of the virus at the occurs among viruses isolated from Europe, Asia, cell surface (58, 87). In experimental studies, viruses Africa, and Australia (Eurasian lineage) and those 1 / Influenza A Virus 7

DK/WI/1938/80 H1 DK/Miyagi/66/77 H1 Gull/MD/19/77 H2 DK/Hong Kong/273/78 H2 TK/WI/68 CL H5 DK/Ireland/113/83 H5

Shearwater/Australia/1/72 H6 DK/Hong Kong/73/76 H6

DK/England/56 H11 CK/NJ/15906/96 H11 Gull/MD/704/77 H13

BHGull/Astrakhan/227/84 H13 Black headed Gull/Sweden/3/99 H16 Black-headed/Kittywake/295/75 H16 TK/Ontario/6118/68 H8 DK/Yangzhou/02/2005 H8 DK/Alberta/60/76 H12

Rednecked Stint/Australia/5745/1981 H12 TK/WI/66 H9 CK/Korea/96006/96 H9 DK/Memphis/928/74 H3 Aquatic bird/Hong Kong/399/99 H3 CK/Alabama/1/75 H4 Dk/New Zealand/31/76 H4 Mallard/Gurjev/244/82 H14 CK/NY/8030-2/96 H7

DK/Heinersdorf/S495/6/86 H7 Shearwater/WAustralia/2576/79 H15

CK/Germany/N/49 H10 Quail/NJ/25254-22/95 H10 50 changes

Figure 1.1. Phylogenetic tree of 16 hemagglutinin subtypes. The complete amino acid sequence of representative isolates for all 16 HA subtypes are included, including a representative North American and Eurasian isolate where available. Abbreviations include CK (chickens), DK (ducks), and TK (turkeys). Standard two-letter abbreviations are used for states from isolates from the United States.

isolated from the Americas (American lineage) lineages and between the Australian viruses and (101). At the amino acid level for the more conserved European and Asian viruses (106). The differentia- internal proteins, the distinctions between American tion of the wild bird isolates into distinct Old World and Eurasian lineages are lost. The HA and NA and New World lineages suggests that little transfer genes having greater nucleotide sequence diversity of infl uenza genes is occurring between these two still separate at the amino acid level into clear Eur- geographic regions. asian and American lineages. For the H7 subtype, As more sequence information becomes available where there is a large amount of sequence informa- from wild bird and poultry isolates, the general rule tion, a further division of lineages can be observed of American versus Eurasian lineage appears to have between the North American and South American some exceptions. For example, the H2 subtype infl u- 8 Avian Influenza enza viruses appear to follow the rule of American birds. The virus, if allowed to circulate long enough and Eurasian lineages for poultry and duck isolates, in the new host, becomes a human, chicken, or swine but the North American origin shorebird and gull adapted virus and results in unique phylogenetic lin- viruses are more closely related to Eurasian isolates eages being created (10, 24). Infl uenza viruses in a than to other North American H2 isolates (54, 83). new host change at a high and predictable level that Although the H2 shorebird and gull viruses are more is the result of the high error rate of the virus and similar to Eurasian viruses, they do cluster as a host selection pressures (11, 24, 85, 102). For species unique sublineage. A similar Eurasian-like gull and under immune pressure from natural infection and/ shorebird sublineage also exists for H6 infl uenza or vaccination, the changes in the HA and NA genes viruses from North America, but the internal genes, can occur at an even faster rate (22, 48). The changes including the matrix and nonstructural genes, have in both genes are concentrated in specifi c antigenic the anticipated American origin sequence (97). sites. For example, the human H3 protein has fi ve Therefore, these data likely represent a unique sub- antigenic sites that are binding sites for neutralizing population of the HA gene circulating in North antibody (127, 129). Even with our current level of America, and not evidence of recent movement of understanding, we cannot predict the changes that Eurasian lineage viral genes into the Americas. allow species adaptation or allow the virus to evade The complete host range of AI virus in wild birds the host immune response. is not known, but based on sampling studies, two Infl uenza viruses have become endemic in a orders of birds are most consistently infected, the number of species, including humans, swine, horses, Anseriformes and Charadriiformes. The Anseri- and poultry, and once a strain of infl uenza circulates formes include ducks, geese, and swans, but the in a particular species for an extended period of time incidence of infection appears to be highest in dab- (months to years), the virus becomes increasingly bling ducks, including mallards, pintails, and teal. species specifi c. So human infl uenza viruses do not The incidence of infection appears to be seasonal, usually infect swine, equine infl uenza viruses do not with the highest isolation rate being in juvenile birds infect turkeys, and poultry viruses do not infect in the fall of the year (98). A lower incidence of humans. However, this general rule of host-adapted infection occurs in Charadriiformes birds, which infl uenza viruses staying within a single species or include shorebirds and gulls. Wild bird AI viruses related species does have exceptions. For example, seem to pass easily between different bird species, classic swine H1N1 infl uenza viruses from North and it is not currently possible to predict the species America routinely cross the species barrier from from which the virus was isolated based on the swine to turkeys, causing costly disease outbreaks nucleotide sequence. The one possible exception to (28). The sporadic infection of humans with some this rule is that most H13 viruses are from gulls, and AI viruses (H5N1, H7N7, H7N3, and H9N2) from gulls also seem to have a predominant gull lineage poultry has been observed, and therefore AI viruses for at least some of the internal genes (24, 104) do present a public health threat as a zoonotic patho- (Fig. 1.2). The ecology of AI viruses in wild birds gen, although the risk is considered to be low (42, is discussed in detail in Chapter 3 (Ecology of Avian 70, 108, 123). Few experimental challenge studies Infl uenza in Wild Birds). of humans have been performed with AI viruses, but in general the viruses replicated poorly and caused Epidemiology in Man-made Systems little to no clinical disease (7). It is not understood AI viruses are unusual in that they can infect and if all HA and NA subtypes of AI viruses have the replicate in a wide variety of host species, including same ability to infect humans or other species. Cur- chickens, turkeys, swine, horses, humans, and a rently, only a limited number of subtypes have wide variety of other avian and mammalian species. become endemic in humans (H1, H2, H3, N1, and However, the amount of virus required to infect the N2) (135). host can vary greatly depending on the level of host The movement of AI viruses from wild birds to adaptation, which provides at least some level of domestic bird species is not uncommon but rarely species barrier (95, 121). The virus as it becomes results in viruses becoming endemic in poultry. adapted to the new host typically becomes less able Several routes of exposure of wild bird viruses to to replicate in the original host species like wild poultry have been documented or suspected of being 1 / Influenza A Virus 9

NY/923/2006 South Australia/47/2000 Guangdong/39/89 Bangkok/1/79 Udorn/72 Port Chalmers/1/73 Korea/426/68 Human lineage Leningrad/134/171/57 FW/1/50 USSR/90/77 Ft Monmouth/1/47 PR/8/34 Swine/1976/31 Swine/29/37 Swine/Hong Kong/273/94 Swine/Iowa/17672/88 Swine/Ontario/2/81 Classic Swine Swine/Tennessee/24/77 Swine/Wisconsin/1/61 Lineage Swine/May/54 Swine/Iowa/15/30 Anas acuta/Primorje/695/79 Budge/Hokkaido/1/77 Ck/Victoria/1/85 CK/Hong Kong/14/76 Duck/Hong Kong/193/77 Eurasian Avian Duck/Bavaria/2/77 Oystercatcher/Germany/87 Lineage TK/England/91 Duck/Nanchang/1749/92 Gull/MD/19/77 Shearwater/Australia/1/72 Duck/Czechoslovakia/56 CK/Brescia/1902 FPV/Weybridge Mallard/NY/6750/78 CK/NY/13833-7/95 CK/PA/11767-1/97 CK/PA/13552/98 North American TK/NY/4450/94 Seal/MA/3911/92 Lineage DK/MI/80 Tk/MN/166/81 TK/Ontario/66 TK/OR/71 Equine/KY/2/86 Equine/WI/1/2003 Equine Type 2 Equine/Hong Kong/1/92 Equine/Miami/63 Lineage Tk/MN/833/80 Mallard/WS/34/75 CK/Chiapas/15405/97 CK/Mexico/31382-7/94 TK/CO/13356/91 North American Rhea/NC/39482/93 Guinea Hen/NJ/04236/93 Lineage CK/PA/13609/93 DK/NY/13152/94 CK/PA/1370/83 HerringGull/DE/677/88 DK/OH/421/87 Gull/Massachusetts/26/80 Gull/MD/1815/79 Gull/MD/1824/78 Gull Lineage Gull/MN/945/80 Equine/Prague/1/56 Equine Type 1 10 changes Lineage Figure 1.2. Phylogenetic tree of the matrix gene. The tree is based on the complete nucleotide sequence of representative isolates for major groups of type A infl uenza viruses. The tree is rooted to equine/Prague/1/56, which is the most divergent type A infl uenza virus. Abbreviations include CK (chickens), DK (ducks), and TK (turkeys). Standard two-letter abbreviations are used for states from isolates from the United States. the origins of outbreaks. Direct exposure to wild and NA subtypes were isolated from turkeys in dif- birds is the most likely method, with some of the ferent outbreaks, and usually at times when wild best documented cases of exposure being in com- ducks were migrating to or from their summer mercial turkeys in Minnesota where multiple out- breeding grounds. During the migratory wild duck breaks of AI were observed yearly in the 1980s and season, turkeys were raised outside and the wild early 1990s (26). AI viruses of many different HA birds could fl y over or actually land in the turkey 10 Avian Influenza pens. During the 1990s, the management system was or river, are used for drinking water or other changed so that the turkeys were reared in confi ne- purposes. If the drinking water is not properly puri- ment for their entire lives, and the incidence of AI fi ed, AI viruses from wild birds can be introduced virus was greatly decreased (115). Limiting expo- to the poultry fl ock. The use of raw drinking water sure of poultry to wild birds through confi nement was suggested to be the source of AI outbreaks in rearing and other biosecurity measures provides an the United States, Australia, and Chile (30, 89, opportunity to reduce the risk of AI virus introduc- 106). tion from wild birds. At least one other common source of transmission Another source of introduction of AI virus to of infl uenza virus for turkeys is the exposure to pigs poultry is the live bird market (LBM) system, which infected with the swine infl uenza virus. Turkeys are is found in many countries around the world, includ- susceptible to swine infl uenza viruses, and having a ing the United States. LBMs typically offer a variety turkey farm and swine farm in close proximity is a of birds that can be slaughtered and used for human risk factor for the introduction of swine infl uenza food consumption. For many developing countries viruses. Infections with both classic H1N1 swine where refrigeration is not available, LBMs provide infl uenza and the more recent reassortant H1N2 and a way to maintain freshness until the product is sold. H3N2 swine infl uenza viruses in turkeys have been For other countries like the United States or Hong reported (28, 107, 136). The impact of man-made Kong, the LBM system caters to consumer prefer- systems on the epidemiology of AI is discussed in ences at a premium price for specifi c selection of a more detail in Chapter 4 (Epidemiology of Avian food bird compared to the purchase of a chilled or Infl uenza in Agricultural and Other Man-Made frozen bird from a supermarket. However, this mar- Systems). keting system provides an ideal environment to introduce and maintain AI viruses in the poultry CLINICAL DISEASE IN POULTRY population (44, 102). A common scenario is when domestic waterfowl, primarily ducks, are raised on Field Presentation ponds where exposure to wild ducks and other Infl uenza infections in poultry, primarily chickens aquatic birds is common (6). This creates a high risk and turkeys, can be asymptomatic, but it often causes of infection for domestic ducks, which can be trans- production losses and a range of clinical disease ported to the LBM system where there is close from mild to severe in affected fl ocks. The virus can contact with other poultry, including chickens, quail, be generally divided into viruses that cause mucosal and other gallinaceous birds. A constant supply of infection, in the respiratory and/or enteric tract, and AI virus–naive birds continues to enter the LBM those viruses that also cause systemic infections. system and provides the opportunity for viruses to The viruses that cause mucosal infections are usually become adapted to chickens and other avian species. referred to as low pathogenicity or mildly patho- Once AI virus becomes entrenched in the LBM genic AI (LPAI) viruses, and typically these viruses system, it provides an ongoing source of infection do not cause high mortality in affected fl ocks. The back to commercial poultry. One example is the viruses that cause systemic infections usually cause H7N2 AI virus that has been circulating in the north- high mortality and are referred to as highly patho- east United States since 1994 and has been associ- genic AI, high pathogenicity AI (HPAI), or histori- ated with at least fi ve different outbreaks in cally as fowl plague viruses (134). industrialized poultry in seven states (96). The The LPAI viruses can cause asymptomatic infec- concern for LBMs in the introduction of AI virus tions, but typically the most common symptoms are has resulted in Hong Kong banning the sale of live mild to severe respiratory disease. A decrease in ducks and geese in the markets, a comprehensive feed or water consumption is another common indi- surveillance program, and stricter sanitary require- cation of fl ock infection when careful records of ments (44). These changes have appeared effective consumption are kept. For layer fl ocks or breeder in reducing the incidence of infected birds in the fl ocks, drops in egg production can also be observed. markets. The drops in egg production can be severe with the An additional risk of introduction to farms is fl ocks never returning to full production, as is com- through the birds’ drinking water. Typically this monly seen in turkey breeders infected with swine- occurs when surface water sources, such as a lake like infl uenza viruses (28, 61). In large fl ocks, small 1 / Influenza A Virus 11 increases in daily mortality can be observed as the found within most cells in the host. Infl uenza viruses virus spreads through the fl ock. The LPAI infection must have the HA protein, which is produced as a at least contributes to this increased mortality single polypeptide, cleaved into the HA1 and HA2 because diagnostic testing of the daily mortality is subunits before it can become infectious. This cleav- considered to be a sensitive way to identify AI infec- age is necessary for the fusion domain to be acti- tion in fl ocks infected with LPAI virus (1, 103). In vated during the uncoating step of virus replication. some situations, infection with LPAI virus may Normally, trypsin or trypsin-like proteases (plasmin, result in high mortality, generally in association with blood clotting factor–like proteases, tryptase Clara, concurrent or secondary pathogens and/or poor bacterial proteases) cleave the HA protein by recog- environmental conditions (3). On rare occasions, nizing a single arginine in the extracellular environ- LPAI viruses may cause specifi c lesions in internal ment (25, 40, 41, 46). The distribution of LPAI organs, through either direct infection or other indi- viruses in the host is believed to be highly infl uenced rect causes (141). The disease and lesions caused by by the local availability of these trypsin-like prote- AI virus infections are discussed in more detail in ases in the respiratory and enteric tracts (41). Other Chapter 5 (Pathobiology of Avian Infl uenza Virus proteases can also cleave infl uenza, and in chick Infections in Birds and Mammals). embryos it is believed to be a prothrombin-like enzyme similar to blood clotting factor X (25). Molecular and Biological Features of Low and However, when multiple basic amino acids (lysine High Pathogenicity Avian Infl uenza Viruses and arginine) are present at the HA cleavage site, The LPAI viruses can be of many different HA and particularly by the insertion of multiple basic amino NA subtypes. The HPAI viruses, for unknown acids, the cleavage site becomes accessible to furin reasons, have been restricted to the H5 and H7 sub- or other ubiquitous proteases that are found in most types, but most H5 and H7 infl uenza viruses are of cells of the body (100). The HPAI viruses’ HA low pathogenicity. It is a rare occurrence that these protein is cleaved during the assembly stage of virus LPAI viruses mutate into the HPAI viruses. It is replication and therefore is infectious when it is generally believed that HPAI viruses arise from H5 released from the cell (99, 100). This allows the and H7 LPAI viruses that have been allowed to HPAI virus to greatly expand its ability to replicate circulate in poultry for extended periods of time. For in a number of different cell types, including a example, LPAI viruses circulated for several months variety of cell types in the brain, heart, skeletal to years in poultry fl ocks in the H5 outbreaks in muscle, and pancreas. The damage to critical organs Pennsylvania in 1983 and Mexico in 1994, and in or to endothelial cells lining blood vessels can cause the H7 outbreak in Italy in 1999, before the viruses a variety of disease symptoms that often lead to the mutated to become HPAI (32, 37, 140). The selec- death of the bird (72, 111). Other viral genes are also tion pressures for viruses to change from LPAI to thought to be important in the virulence of the virus, HPAI are not currently known, but the replication but the HA cleavage site is by far the most important of virus in gallinaceous birds, including chickens, virulence trait (52, 82). turkeys, and quail, are considered a critical part of the process. HPAI viruses are not believed to be Host and Virus Strain Impact on normally present in the wild bird host reservoir (80). Pathogenicity The recent outbreaks with mortality in wild birds The HPAI phenotype by defi nition causes high mor- with Asian H5N1 HPAI viruses are believed to be tality in 4- to 6-week-old specifi c pathogen–free related to spillover of HPAI virus from domestic chickens (133), but just because it is HPAI in chick- birds to wild birds, and it is currently unclear if this ens does not necessarily provide a predictor for lineage of HPAI has become endemic in some wild disease in other species. Few studies have character- bird species (53). ized pathogenicity of a single isolate in a number of different species after experimental challenge. One Cellular Pathobiology and Hemagglutinin of the broadest series of studies examined an H5N1 Cleavage HPAI 1997 chicken isolate from Hong Kong that The primary virulence characteristic that separates was used as an experimental inoculum for a variety the LPAI and the HPAI viruses is the ability of of avian species. The Hong Kong 97 strain caused HPAI viruses to be cleaved by ubiquitous proteases high mortality in all of the gallinaceous species 12 Avian Influenza tested, including chickens, turkeys, quail, and pheas- sequences of LPAI wild bird isolates of H5 and H7s ants, although differences in mean death time were viruses are different (80). H5s viruses typically have observed among species (72). Most other species a QRETR/G sequence with arginine at the -1 and -4 tested had less severe or, in some cases, no clinical position. H7s typically have NPKTR/G sequence disease signs, although most were infected based on with a lysine and arginine at the -1 and -3 positions. the ability to reisolate virus from challenged birds The change to virulence for H5s can occur by both (73–75). Predictions of virulence, outside of the gal- substitution of nonbasic to basic amino acids or by linaceous species, could not be made for different an insertion of basic and nonbasic amino acids at the orders of birds. For example, some geese when chal- cleavage site. The chicken/Scotland/59 H5N1 virus lenged had neurological signs and lesions that cor- has four basic amino acids at the cleavage site related with virus replication sites in the brain (73). RKKR/G (17), presumably through site substitution However, ducks tested from the same order of birds, that results in an HPAI phenotype. More commonly, Anseriformes, had limited infection in respiratory additional basic amino acids are inserted at the tract but did not show any evidence of disease (73). cleavage site, with both two, three, and four addi- It seems clear that the virulence associated with HA tional amino acids being observed. For example, the cleavability is not the only factor that decides viru- chicken/Hong Kong/97 H5N1 virus had a sequence lence in other species. This has been clearly shown of QRERRRKKR/G (105). The mechanism of inser- in ducks with the recent Asian H5N1 HPAI viruses. tion of amino acids is not clear, but a duplication In a 2-week-old Pekin duck model, the early H5N1 event appears likely for several of the H5 HPAI HPAI viruses from 1997 to 2001 could infect but viruses (71). Other parts of the HA protein can also did not cause morbidity or mortality. However, start- play a role in the phenotype of the virus. The best ing with some isolates in 2002, increased mortality example is the presence or absence of a glycosyl- was observed, with 100% mortality being seen with ation site at position 10–12 of the HA1 protein. In more recent viruses (112). The Asian H5N1 HPAI 1983, a LPAI H5N2 virus, chicken/Pennsylva- viruses all have an H5 gene from the same lineage nia/1/1983, was isolated that had four basic amino and an identical or nearly identical hemagglutin acids, QRKKR/G, at the cleavage site. Six months cleavage site sequence with an insert of multiple later, an HPAI virus emerged in Pennsylvania, basic amino acids, and all remain highly pathogenic chicken/Pennsylvania/1370/83, that had the same for chickens. The internal genes for these viruses, HA cleavage site, but this virus had lost a glycosyl- however, are variable, and it is believed that these ation site at position 10–12 in the HA1 protein. The internal gene differences account for the difference glycosylation site is structurally extremely close to in virulence (50). the HA cleavage site, and it is believed that the loss of the sugars allowed greater access to the cleavage Hemagglutinin Changes Associated with site, making it accessible to the ubiquitous proteases High Pathogenicity that changed the phenotype of the virus (37). This The HA cleavage site remains the best but not a and other glycosylation sites experimentally have perfect predictor of viral virulence in chickens and also been shown to be important in virulence (33). other gallinaceous birds. As previously mentioned, The change from LPAI to HPAI for H7 viruses the presence of multiple basic amino acids upstream appears to have several important differences. First, of the HA1 and HA2 cleavage is correlated with all HPAI H7 viruses have insertions of 2 to 10 addi- virulence (81). Only the H5 and H7 subtypes of AI tional basic amino acids at the cleavage site. The are currently known to have an HPAI phenotype, for mechanism for such insertions also appears to be reasons that are not readily apparent. Sequence com- different in many cases. Although a duplication parisons show the H5 and H7 subtypes to be dis- event appears likely for some viruses, in several tinctly different from each other. Although both H5 recent cases nonhomologous recombination is the and H7 proteins maintain the general principle of the likely method of insertion. In both the Chile out- cleavage site being between arginine and glycine break in 2002 and the Canadian outbreak in 2004, and multiple basic amino acids at the cleavage site an insertion of 30 nucleotides homologous from the resulting in HPAI, there are distinct differences NP gene and 24 nucleotides from the matrix gene, between the subtypes. The typical cleavage site respectively, resulted in the increase in virulence 1 / Influenza A Virus 13

(69, 106). Other cases of nonhomologous recombi- intravenously with the LPAI virus turkey/ nation have been seen in experimental studies where Oregon/1971, death occurred in seven of eight NP and host ribosomal RNA sequence was inserted chicks. When the same virus was administered to at the cleavage site (38, 65). In all four examples, 4-week-old chickens at the same dose and route, the insertions had some basic amino acids, but they mortality was seen in only one of eight chicks. In were a minority of the insert. In these examples, the this example, the virus replicated to high titer in the increased spacing in the cleavage site loop appear to kidney that resulted in renal failure leading to death be the more important factor for increasing virulence in most of the 1-day-old chicks. The same virus as opposed to just the addition of basic amino acids. given by the intrachoanal cleft (intranasal) at the Almost all of the H7 HPAI outbreak viruses appear same dose caused mortality in only one of eight 1- to have become HP by unique events at the cleavage day-old chicks (13). This example shows that mor- site, which makes the prediction of minimum tality can be greatly affected by the age of the bird changes to defi ne HPAI by sequence alone diffi cult and route of inoculation. The intravenous inocula- for H7s. The mechanisms of change in AI virus tion route, which is not a natural route of exposure, virulence is covered in Chapter 2 (Molecular Deter- likely seeded high levels of virus to the kidney, minants of Pathogenicity for Avian Infl uenza which led to the high mortality. The intravenous Viruses). route of challenge, the standard for in vivo pathotyping in chickens, can result in sporadic deaths with Other Variables That Impact Pathogenicity low pathogenic strains of AI virus, typically because The HPAI virus is defi ned either by an in vivo of the replication in the kidney resulting in kidney pathotyping test in chickens, applicable to any infl u- failure (91, 92, 114). Primary chicken kidney cells enza virus or a sequence analysis of the HA cleavage allow replication of LPAI viruses, presumably site for H5 and H7 infl uenza, or by both. The best because they produce trypsin-like enzymes that predicator of HPAI virus is when a suspect virus has cleave the HA protein, and this property allows the same cleavage site as another known HPAI LPAI viruses to be plaqued without the addition of virus. In such situations the virus is reportable to the trypsin in primary kidney embryo cell lines (13). World Organization of Animal Health (Offi ce Inter- Ducks also have been shown to have a marked nationale des Epizooties [OIE]) as an HPAI virus. difference in disease based on age, with younger However, a recent outbreak in the United States ducks being more susceptible to severe infection. (Texas) in 2004 may be the fi rst clear case where For example, several Asian H5N1 HPAI viruses the phenotype and the genotype did not match. In cause high mortality in 2-week-old ducks, but the this case, the Texas/04 isolate had the same cleavage same viruses in 4-week-old ducks produced much site sequence as the chicken/Scotland/59 virus and lower to no mortality (112). Increased virulence in was reported to OIE as an HPAI virus, but the virus younger animals is commonly seen, although the was LP in the standard intravenous chicken pathotyp- reasons for the differences are not clearly defi ned. ing test (51). Even though the two tests did not The immaturity of the immune response, both innate correlate and high virulence was not seen in the and adaptive, likely contributes to these differences. fi eld, the virus was still considered a virulent virus For example, the interferon response greatly increases that resulted in major trade sanctions on poultry in the embryo as it ages and presumably the peak exports for a limited period of time. Other examples interferon response is also after hatching (56). of discordance between phenotype and genotype In some cases, virulence can be greater in older have previously been described (131). Currently no birds or in birds in egg production. A common completely accurate prediction scheme has been example is swine-like infl uenza in turkeys. For determined for AI viruses. turkey breeders in production, infection can cause It is also clear from experimental studies that the severe drops in egg production, but for fl ocks not in age and route of inoculation, as well as species, can production, the birds often seroconvert with no clin- affect the virulence of AI virus in experimental ical signs of disease (2, 21, 28, 107). Increases in infections. The age effect has been seen in both mortality have also been seen in layers with egg yolk chickens and ducks. For example, when 1-day-old peritonitis after AI virus infection that is not seen in specifi c pathogen–free chickens were challenged immature birds (141). 14 Avian Influenza

ANTIGENIC DRIFT AND SHIFT same time. The viruses, however, do change at a Infl uenza viruses have two primary mechanisms to rapid and predictable rate, sometimes called a provide diversity in the viral population: a high molecular clock (11). The observed changes in the mutation rate and the ability to reassort gene seg- genome are not random but are concentrated primar- ments (55, 122). Both methods provide an opportu- ily in the surface glycoproteins (77). Infl uenza nity for the virus to rapidly change and adapt, which viruses, like other RNA viruses, lack a proofreading contributes in the ability of the viruses to establish mechanism in the replication of viral RNA, which infections in new host species. The rapid ability to results in errors in transcription leading to a high mutate and adapt is not unique among the RNA mutation rate (68). The high mutation rate provides viruses, but some viruses can tolerate higher levels the opportunity for change, but many of the changes of sequence changes in at least some viral genes. introduced by this error-prone transcription are del- Infl uenza viruses, as has been previously described, eterious to the virus because it creates premature can differ greatly in amino acid sequence, particu- stop codons, changes in amino acids so the virus is larly in the surface glycoproteins, HA and NA (62). less fi t, or changes in a regulatory signal that affects These differences in amino acid sequence result in virus replication (78). Most of the deleterious muta- differences in antigenicity, such that antibodies to tions are lost during the selection process to achieve H1 infl uenza will neutralize only H1 viruses, and not the fi ttest virus in a population. The mutation rate any other infl uenza viruses. These antigenic differ- for all eight gene segments is likely the same, but ences have major implications for vaccination, because of positive selection, more changes in the because vaccine protection is mediated primarily HA and NA genes are seen (77). by specifi c antibodies being produced to the HA One of the primary selective factors on the HA protein and, to a lesser extent, the NA protein protein is thought to be antibody pressure from the (49). Therefore, current vaccines are limited to pro- host, either from previous exposure to the virus or viding only subtype protection, and to provide com- by vaccination (77). For the human H3 protein, fi ve plete protection from AI would require the addition antigenic regions have been characterized where of 16 different antigens representing each HA antibody to these regions can be neutralizing to the subtype. virus and therefore would be protective for the host Although neutralizing antibodies to one HA during infection. These antigenic regions are on the subtype of infl uenza should neutralize all viruses globular head of the HA protein, with many close to within the same subtype, differences in the the receptor binding site (127, 129, 130). Antibodies specifi city of the antibody greatly affect the level of to the antigenic sites can be neutralizing because protection observed. The impact of antigenic drift they directly block access to the receptor binding on vaccination with human infl uenza is a well-char- site and prevent the virus from attaching to and acterized problem that requires the vaccine seed initiating infection in the host. These antigenic strain to be evaluated every year to try to achieve regions, however, can tolerate a signifi cant amount the best match with the circulating strain as possible of amino acid diversity, and when changes to key (93). Two different subtypes of type A infl uenza amino acids occur, one of the neutralizing epitopes viruses are endemic around the world in the human may be changed so that antibodies can no longer population, the H1N1 and H3N2. For both subtypes bind (127). These changes in specifi city of the anti- of virus, a single lineage of virus is present that can body can result in a virus being better able to escape be traced back to the time the virus was introduced the host’s ability to control infection, resulting in to the human population (11, 12, 24). Unlike what greater virus replication and transmission of these we see with animal infl uenza viruses, which will be escape mutants. The accumulation of these amino described in more detail later, these two subtypes of acid changes at these antigenic sites is the antigenic virus have evolved with little difference in sequence drift that results in vaccines for infl uenza being less based on geographic origins of the virus. This world- protective over time. For humans, the infl uenza wide distribution is likely the result of widespread vaccine seed strains are evaluated yearly to deter- and rapid movement of humans between regions that mine if the currently circulating fi eld strains are still effi ciently transmit the virus that allows only rela- neutralized effectively by antibody produced to the tively minor variants of the virus to circulate at the vaccine strain. Comparison of virus sequence is used 1 / Influenza A Virus 15 to identify when new viral variants are occurring and An additional concern with AI viruses is the wide at what frequency (93). From the sequence informa- diversity of viruses that can infect poultry. Because tion, representative strains are used to produce anti- most outbreaks of LPAI and HPAI result from inde- bodies to do more in-depth cross–hemagglutination pendent introductions of viruses from the diverse inhibition (HI) studies. If the fi eld strains in the wild bird reservoir, most epidemiologically unre- cross-HI studies show a 4-fold or greater differences lated outbreaks are antigenically different from each in inhibition, this is evidence that the current vaccine other even within the same subtype (23, 47). This seed strain may be ineffective. Vaccination for antigenic diversity as described earlier is broken human infl uenza requires a close match of vaccine down generally into North American and Eurasian to fi eld strain or protection from vaccination is lineages, and the selection of a vaccine seed strain adversely affected (31). Antigenic differences of should at a minimum consider matching the HA greater than 4-fold appear to be the range where the amino acid sequence as closely as possible to try and decrease in antibody specifi city affects the protec- get the best protection and reduction in shedding tion seen from vaccines. The seed strains are typi- (113). However, many different factors are involved cally changed every 3 to 4 years to compensate for in vaccine seed strain selection. this antigenic drift (93). One additional complication with AI viruses and For poultry, antigenic drift also occurs, but the other animal infl uenza infections is that if an out- interpretation and importance of antigenic drift are break becomes widespread, geographic separation much more complicated. The principles of changes of viral populations can occur because of limits on at antigenic sites affecting the specifi city of neutral- movement of animals and animal products that izing antibody are the same for the immune response allows separate evolutionary paths to occur. The in poultry, but the trigger for when antigenic change geographic separation has been observed with necessitates a vaccine change is not defi ned. In part several outbreaks, including H5N2 LPAI in Mexico, this is a difference in the pathobiology between H9N2 LPAI in the Middle East and Asia, and H5N1 infl uenza in humans and HPAI in chickens. With HPAI in Asia, Europe, and Africa (48, 132, 137). human infl uenza, viral infection is a mucosal infec- The issue of different lineages again complicates tion of the respiratory tract, and with HPAI, the virus vaccine selection, because antigenic drift can occur has both systemic and mucosal replication. The use within a clade or lineage, but it can also occur of killed vaccines, commonly used in humans and between clades. The current H5N1 HPAI viruses poultry, provides high levels of serum IgG (or IgY, circulating have separated into two major clades that the avian counterpart to mammalian IgG) antibody by amino acid sequence analysis differ by around but little, if any, secretory IgA, which is the most 6% and antigenically are up to 8-fold divergent in effective antibody for the control of infl uenza in HI titers (94, 132). experimental mouse models (79). The transudation For long-lived animals, an additional concern of IgG (IgY) that crosses the mucosal surface can with infl uenza infection is antigenic shift. Antigenic provide effective control of clinical disease, but it shifts are typically considered for human infl uenza, does not provide ideal protection (116). In chickens but antigenic shifts have also been seen in animals. with LPAI and for replication of HPAI viruses on Antigenic shift is where a large proportion of the the mucosal surface, a similar immune response host population has either previous exposure, by likely occurs. However, the severe clinical disease infection or vaccination, with a particular HA sub- seen with HPAI is primarily from the systemic rep- type, and then they become exposed to a different lication of the virus, and subtype-specifi c antibody HA subtype (19). Because the host population has appears to effi ciently block viremia and therefore the little or no protective immunity to the new virus, it systemic replication of the virus (49). The serum can rapidly spread in the new population, causing a antibody protection appears to be impacted less by widespread and sometimes severe outbreak of infl u- antigenic drift in its ability to block viremia and enza called a pandemic. For humans, three major prevent severe clinical disease, but it has been shown pandemics occurred in the 20th century. The most previously that the level of virus shedding is corre- severe was when an H1N1 virus emerged, likely lated to the relatedness of the vaccine to challenge replacing an H2 human infl uenza, in 1918 and strain (48, 113). resulted in a major pandemic that likely killed over 16 Avian Influenza

40 million people (118). The second pandemic of to cause disease and production losses, it is found the century occurred in 1957, when the H1N1 virus more widely than HPAI, and for LPAI H5s and H7s was supplanted by an H2N2 virus. The last pan- the potential to mutate to HPAI remains ever present. demic started in 1968, when an H3N2 virus sup- AI viruses are diffi cult to control because of the planted the H2N2 virus (135). The origins of a new wildlife reservoir, the adaptability of the virus, and pandemic virus are not clearly known, although it the lack of good control tools. Efforts to increase our appears that it can be caused by a completely new understanding of the virus and research to develop virus being introduced into the human population or new methods for control should be a priority for the by a reassortment event between the circulating veterinary community. human strain and another infl uenza virus (135). The 1918 H1N1 virus appeared to be a completely new REFERENCES virus, but the H2N2 and H3N2 viruses appeared to 1. Akey, B.L. 2003. Low-pathogenicity H7N2 avian be reassortant viruses that changed multiple genes, infl uenza outbreak in Virginia during 2002. Avian including, most importantly, the HA gene (135). Diseases 47:1099–1103. For poultry, antigenic shift has not been a major 2. Andral, B., D. Toquin, F. Madec, M. Aymard, issue because of the short production lives of most J.M. Gourreau, C. Kaiser, M. Fontaine, and M.H. commercially produced poultry. Because infection Metz. 1985. Disease in turkeys associated with with AI viruses had been uncommon, commercial H1N1 infl uenza virus following an outbreak of the poultry were not naturally exposed and vaccination disease in pigs. Veterinary Record 116:617–618. is still not widely practiced. Therefore, most poultry 3. Bano, S., K. Naeem, and S.A. Malik. 2003. Eval- are completely susceptible to infection with any uation of pathogenic potential of avian infl uenza virus serotype H9N2 in chickens. Avian Diseases infl uenza subtype. However, with some other animal 47:817–822. species, including horses and swine, antigenic shifts 4. Barman, S., L. Adhikary, A.K. Chakrabarti, C. have occurred that have required changes in the Bernas, Y. Kawaoka, and D.P. Nayak. 2004. Role types of vaccines used. For horses, H7N7 was the of transmembrane domain and cytoplasmic tail only recognized infl uenza virus in horses until the amino acid sequences of infl uenza A virus neur- early 1960s, when an H3N8 virus started to supplant aminidase in raft association and virus budding. the H7N7. In this case, the virus looked to be a Journal of Virology 78:5258–5269. completely new introduction of virus in the horse 5. Barman, S., A. Ali, E.K. Hui, L. Adhikary, and population, and eventually replaced the H7N7 D.P. Nayak. 2001. Transport of viral proteins to viruses (67). For swine in the United States, H1N1 the apical membranes and interaction of matrix was primarily the only strain of infl uenza that circu- protein with glycoproteins in the assembly of infl uenza viruses. Virus Research 77:61–69. lated from 1918 to the late 1990s. However, starting 6. Bean, W.J., Y. Kawaoka, and R.G. Webster. in 1998, H3N2 viruses started to be isolated in the 1986. Genetic characterization of H5N2 infl uenza United States. This virus was an unusual reassortant viruses isolated from poultry in 1986. In: B.C. virus that had H1N1 swine infl uenza virus–like Easterday and C.W. Beard (eds.). Proceedings of genes, human infl uenza virus–like genes, and AI the Second International Symposium on Avian virus–like genes. The H1N1, H3N2, and even other Infl uenza, Georgia Center for Continuing Educa- reassortant viruses (H1N2 and H3N1) currently co- tion, the University of Georgia, Athens, Georgia, circulate in the United States (34, 35). Because of USA, September 3–5, 1986. Symposium on the antigenic shift, vaccines for horses and swine Avian Infl uenza, US Animal Health Association: had to be updated to include the new viruses to Richmond, VA, pp. 207–214. achieve protection from vaccination. 7. Beare, A.S., and R.G. Webster. 1991. Replication of avian infl uenza viruses in humans. Archives of CONCLUSIONS Virology 119:37–42. 8. Birch-Machin, I., A. Rowan, J. Pick, J. Mumford, AI viruses remain a major health issue for poultry and M. Binns. 1997. Expression of the nonstruc- around the world. The greatest concern typically has tural protein NS1 of equine infl uenza A virus: been for highly pathogenic AI because of its severe detection of anti-NS1 antibody in post infection clinical disease and its effects on trade. However, equine sera. Journal of Virological Methods LPAI also remains a concern because of its ability 65:255–263. 1 / Influenza A Virus 17

9. Bourmakina, S.V., and A. Garcia-Sastre. 2003. 21. Ficken, M.D., J.S. Guy, and E. Gonder. 1989. An Reverse genetics studies on the fi lamentous mor- outbreak of infl uenza (H1N1) in turkey breeder phology of infl uenza A virus. Journal of General hens. Avian Diseases 33:370–374. Virology 84:517–527. 22. Fitch, W.M., R.M. Bush, C.A. Bender, K. Sub- 10. Buckler-White, A.J., and B.R. Murphy. 1986. barao, and N.J. Cox. 2000. The Wilhelmine E. Nucleotide sequence analysis of the nucleopro- Key 1999 Invitational Lecture. Predicting the tein gene of an avian and a human infl uenza virus evolution of human infl uenza A. Journal Heredity strain identifi es two classes of nucleoproteins. 91:183–185. Virology 155:345–355. 23. Garcia, M., D.L. Suarez, J.M. Crawford, J.W. 11. Buonagurio, D.A., S. Nakada, J.D. Parvin, M. Latimer, R.D. Slemons, D.E. Swayne, and M.L. Krystal, P. Palese, and W.M. Fitch. 1986. Evolu- Perdue. 1997. Evolution of H5 subtype avian tion of human infl uenza A viruses over 50 years: infl uenza A viruses in North America. Virus rapid, uniform rate of change in NS gene. Science Research 51:115–124. 232:980–982. 24. Gorman, O.T., W.J. Bean, Y. Kawaoka, and R.G. 12. Bush, R.M., C.A. Bender, K. Subbarao, N.J. Cox, Webster. 1990. Evolution of the nucleoprotein and W.M. Fitch. 1999. Predicting the evolution gene of infl uenza A virus. Journal of Virology of human infl uenza A. Science 286:1921–1925. 64:1487–1497. 13. Cauthen, A.N., D.E. Swayne, M.J. Sekellick, P.I. 25. Gotoh, B., T. Ogasawara, T. Toyoda, N.M. Ino- Marcus, and D.L. Suarez. 2007. Amelioration of cencio, M. Hamaguchi, and Y. Nagai. 1990. An infl uenza virus pathogenesis in chickens attrib- endoprotease homologous to the blood clotting uted to the enhanced interferon-inducing capacity factor X as a determinant of viral tropism in chick of a virus with a truncated NS1 gene. Journal of embryo. The EMBO Journal 9:4189–4195. Virology 81:1838–1847. 26. Halvorson, D.A., C.J. Kelleher, and D.A. Senne. 14. Chen, W., P.A. Calvo, D. Malide, J. Gibbs, U. 1985. Epizootiology of avian infl uenza: effect of Schubert, I. Bacik, S. Basta, R. O’Neill, J. season on incidence in sentinel ducks and domes- Schickli, P. Palese, P. Henklein, J.R. Bennink, tic turkeys in Minnesota. Applied Environmental and J.W. Yewdell. 2001. A novel infl uenza A Microbiology 49:914–919. virus mitochondrial protein that induces cell 27. Hay, A.J., A.J. Wolstenholme, J.J. Skehel, and death. Nature Medicine 7:1306–1312. M.H. Smith. 1985. The molecular basis of the 15. Compans, R.W., H.D. Klenk, L.A. Caliguiri, and specifi c anti-infl uenza action of amantadine. The P.W. Choppin. 1970. Infl uenza virus proteins. I. EMBO Journal 4:3021–3024. Analysis of polypeptides of the virion and iden- 28. Hinshaw, V.S., R.G. Webster, W.J. Bean, J. tifi cation of spike glycoproteins. Virology Downie, and D.A. Senne. 1983. Swine infl uenza- 42:880–889. like viruses in turkeys: potential source of virus 16. Connor, R.J., Y. Kawaoka, R.G. Webster, and for humans? Science 220:206–208. J.C. Paulson. 1994. Receptor specifi city in human, 29. Hinshaw, V.S., R.G. Webster, B.C. Easterday, avian, and equine H2 and H3 infl uenza virus iso- and W.J. Bean, Jr. 1981. Replication of avian lates. Virology 205:17–23. infl uenza A viruses in mammals. Infection and 17. De, B.K., G.G. Brownlee, A.P. Kendal, and M.W. Immunity 34:354–361. Shaw. 1988. Complete sequence of a cDNA clone 30. Hinshaw, V.S., R.G. Webster, and B. Turner. of the hemagglutinin gene of infl uenza A/chicken/ 1979. Water-bone transmission of infl uenza A Scotland/59 (H5N1) virus: comparison with con- viruses? Intervirology 11:66–68. temporary North American and European strains. 31. Hirota, Y., M. Kaji, S. Ide, J. Kajiwara, K. Nucleic Acids Research 16:4181–4182. Kataoka, S. Goto, and T. Oka. 1997. Antibody 18. Donald, H.B., and A. Isaacs. 1954. Counts of effi cacy as a keen index to evaluate infl uenza infl uenza virus particles. Journal of General vaccine effectiveness. Vaccine 15:962–967. Microbiology 10:457–464. 32. Horimoto, T., E. Rivera, J. Pearson, D. Senne, S. 19. Dowdle, W.R., and G.C. Schild. 1976. Infl uenza: Krauss, Y. Kawaoka, and R.G. Webster. 1995. its antigenic variation and ecology. Bulletin of Origin and molecular changes associated with Pan American Health Organization 10:193– emergence of a highly pathogenic H5N2 infl u- 195. enza virus in Mexico. Virology 213:223–230. 20. Enami, M., G. Sharma, C. Benham, and P. Palese. 33. Hulse, D.J., R.G. Webster, R.J. Russell, and D.R. 1991. An infl uenza virus containing nine different Perez. 2004. Molecular determinants within the RNA segments. Virology 185:291–298. surface proteins involved in the pathogenicity of 18 Avian Influenza

H5N1 infl uenza viruses in chickens. Journal of J.S. Peiris. 2003. The impact of a monthly rest Virology 78:9954–9964. day on avian infl uenza virus isolation rates in 34. Karasin, A.I., M.M. Schutten, L.A. Cooper, C.B. retail live poultry markets in Hong Kong. Avian Smith, K. Subbarao, G.A. Anderson, S. Carman, Diseases 47:1037–1041. and C.W. Olsen. 2000. Genetic characterization 45. Kuno, G., G.J. Chang, K.R. Tsuchiya, and B.R. of H3N2 infl uenza viruses isolated from pigs in Miller. 2001. Phylogeny of Thogoto virus. Virus North America, 1977–1999: evidence for wholly Genes 23:211–4. human and reassortant virus genotypes. Virus 46. Lazarowitz, S.G., A.R. Goldberg, and P.W. Research 68:71–85. Choppin. 1973. Proteolytic cleavage by plasmin 35. Karasin, A.I., K. West, S. Carman, and C.W. of the HA polypeptide of infl uenza virus: host cell Olsen. 2004. Characterization of avian H3N3 activation of serum plasminogen. Virology and H1N1 infl uenza A viruses isolated from pigs 56:172–180. in Canada. Journal of Clinical Microbiology 47. Lee, C.W., D.A. Senne, J.A. Linares, P.R. Wool- 42:4349–4354. cock, D.E. Stallknecht, E. Spackman, D.E. 36. Kawaoka, Y., T.M. Chambers, W.L. Sladen, and Swayne, and D.L. Suarez. 2004. Characteriza- R.G. Webster. 1988. Is the gene pool of infl uenza tion of recent H5 subtype avian infl uenza viruses viruses in shorebirds and gulls different from that from US poultry. Avian Pathology 33:288– in wild ducks? Virology 163:247–250. 297. 37. Kawaoka, Y., C.W. Naeve, and R.G. Webster. 48. Lee, C.W., D.A. Senne, and D.L. Suarez. 2004. 1984. Is virulence of H5N2 infl uenza viruses in Effect of vaccine use in the evolution of Mexican chickens associated with loss of carbohydrate lineage H5N2 avian infl uenza virus. Journal of from the hemagglutinin? Virology 139:303–316. Virology 78:8372–8381. 38. Khatchikian, D., M. Orlich, and R. Rott. 1989. 49. Lee, C.W., and D.L. Suarez. 2005. Avian infl u- Increased viral pathogenicity after insertion of a enza virus: prospects for prevention and control 28S ribosomal RNA sequence into the haemag- by vaccination. Animal Health Research Reviews glutinin gene of an infl uenza virus. Nature 6:1–15. 340:156–157. 50. Lee, C.W., D.L. Suarez, T.M. Tumpey, H.W. 39. Kibenge, F.S., K. Munir, M.J. Kibenge, T. Joseph, Sung, Y.K. Kwon, Y.J. Lee, J.G. Choi, S.J. Joh, and E. Moneke. 2004. Infectious salmon anemia M.C. Kim, E.K. Lee, J.M. Park, X. Lu, J.M. Katz, virus: causative agent, pathogenesis and immu- E. Spackman, D.E. Swayne, and J.H. Kim. 2005. nity. Animal Health Research Reviews 5:65–78. Characterization of highly pathogenic H5N1 40. Kido, H., Y. Yokogoshi, K. Sakai, M. Tashiro, Y. avian infl uenza A viruses isolated from South Kishino, A. Fukutomi, and N. Katunuma. 1992. Korea. Journal of Virology 79:3692–3702. Isolation and characterization of a novel trypsin- 51. Lee, C.W., D.E. Swayne, J.A. Linares, D.A. like protease found in rat bronchiolar epithelial Senne, and D.L. Suarez. 2005. H5N2 avian infl u- Clara cells. A possible activator of the viral fusion enza outbreak in Texas in 2004: the fi rst highly glycoprotein. Journal of Biological Chemistry pathogenic strain in the United States in 20 years? 267:13573–13579. Journal of Virology 79:11412–11421. 41. Klenk, H.D., R. Rott, M. Orlich, and J. Blodorn. 52. Li, S., J. Schulman, S. Itamura, and P. Palese. 1975. Activation of infl uenza A viruses by trypsin 1993. Glycosylation of neuraminidase determines treatment. Virology 68:426–439. the neurovirulence of infl uenza A/WSN/33 virus. 42. Koopmans, M., B. Wilbrink, M. Conyn, G. Journal of Virology 67:6667–6673. Natrop, H. van der Nat, H. Vennema, A. Meijer, 53. Liu, J., H. Xiao, F. Lei, Q. Zhu, K. Qin, X.W. J. van Steenbergen, R. Fouchier, A. Osterhaus, Zhang, X.L. Zhang, D. Zhao, G. Wang, Y. Feng, and A. Bosman. 2004. Transmission of H7N7 J. Ma, W. Liu, J. Wang, and G.F. Gao. 2005. avian infl uenza A virus to human beings during a Highly pathogenic H5N1 infl uenza virus infec- large outbreak in commercial poultry farms in the tion in migratory birds. Science 309:1206. Netherlands. Lancet 363:587–593. 54. Makarova, N.V., N.V. Kaverin, S. Krauss, D. 43. Krug, R.M. 1981. Priming of infl uenza viral RNA Senne, and R.G. Webster. 1999. Transmission of transcription by capped heterologous RNAs. Eurasian avian H2 infl uenza virus to shorebirds Current Topics of Microbiology and Immunology in North America. Journal of General Virology 93:125–149. 80:3167–3171. 44. Kung, N.Y., Y. Guan, N.R. Perkins, L. Bissett, T. 55. Manrubia, S.C., C. Escarmis, E. Domingo, and E. Ellis, L. Sims, R.S. Morris, K.F. Shortridge, and Lazaro. 2005. High mutation rates, bottlenecks, 1 / Influenza A Virus 19

and robustness of RNA viral quasispecies. Gene Spala, H. Rebelo de Andrade, P.R. Perez Brena, 347:273–282. J. Beytout, L. Brydak, D. Caraffa de Stefano, O. 56. Marcus, P.I., J.M. Rojek, and M.J. Sekellick. Hungnes, J. Kyncl, E. Montomoli, A. Gil de 2005. Interferon induction and/or production and Miguel, R. Vranckx, and A. Osterhaus. 2005. A its suppression by infl uenza A viruses. Journal of new European perspective of infl uenza pandemic Virology 79:2880–2890. planning with a particular focus on the role of 57. Matrosovich, M., N. Zhou, Y. Kawaoka, and R. mammalian cell culture vaccines. Vaccine Webster. 1999. The surface glycoproteins of H5 23:5440–5449. infl uenza viruses isolated from humans, chickens, 67. Paillot, R., D. Hannant, J.H. Kydd, and J.M. Daly. and wild aquatic birds have distinguishable prop- 2006. Vaccination against equine infl uenza: quid erties. Journal of Virology 73:1146–1155. novi? Vaccine 24:4047–4061. 58. Matrosovich, M.N., T.Y. Matrosovich, T. Gray, 68. Palese, P., and M.L. Shaw. 2007. Orthomyxo- N.A. Roberts, and H.D. Klenk. 2004. Neuramin- viridae: The Viruses and Their Replication, idase is important for the initiation of infl uenza In: D.M. Knipe and P.M. Howley (eds), Fields virus infection in human airway epithelium. Virology 5th ed. Lippincott Williams & Wilkins, Journal of Virology 78:12665–12667. Philadelphia 1647–1689. 59. Medeiros, R., N. Escriou, N. Naffakh, J.C. Manu- 69. Pasick, J., K. Handel, J. Robinson, J. Copps, D. guerra, and S. van der Werf. 2001. Hemagglutinin Ridd, K. Hills, H. Kehler, C. Cottam-Birt, J. residues of recent human A(H3N2) infl uenza Neufeld, Y. Berhane, and S. Czub. 2005. Inter- viruses that contribute to the inability to aggluti- segmental recombination between the haemag- nate chicken erythrocytes. Virology 289:74–85. glutinin and matrix genes was responsible for the 60. Mitnaul, L.J., M.N. Matrosovich, M.R. Castrucci, emergence of a highly pathogenic H7N3 avian A.B. Tuzikov, N.V. Bovin, D. Kobasa, and Y. infl uenza virus in British Columbia. Journal of Kawaoka. 2000. Balanced hemagglutinin and General Virology 86:727–731. neuraminidase activities are critical for effi cient 70. Peiris, M., K.Y. Yuen, C.W. Leung, K.H. Chan, replication of infl uenza A virus. Journal of Virol- P.L. Ip, R.W. Lai, W.K. Orr, and K.F. Shortridge. ogy 74:6015–6020. 1999. Human infection with infl uenza H9N2. 61. Mohan, R., Y.M. Saif, G.A. Erickson, G.A. Gus- Lancet 354:916–917. tafson, and B.C. Easterday. 1981. Serologic and 71. Perdue, M.L., M. Garcia, D. Senne, and M. epidemiologic evidence of infection in turkeys Fraire. 1997. Virulence-associated sequence with an agent related to the swine infl uenza virus. duplication at the hemagglutinin cleavage site of Avian Diseases 25:11–16. avian infl uenza viruses. Virus Research 49:173– 62. Nobusawa, E., T. Aoyama, H. Kato, Y. Suzuki, 186. Y. Tateno, and K. Nakajima. 1991. Comparison 72. Perkins, L.E., and D.E. Swayne. 2001. Pathobiol- of complete amino acid sequences and receptor- ogy of A/chicken/Hong Kong/220/97 (H5N1) binding properties among 13 serotypes of hem- avian infl uenza virus in seven gallinaceous agglutinins of infl uenza A viruses. Virology species. Veterinary Pathology 38:149–164. 182:475–485. 73. Perkins, L.E., and D.E. Swayne. 2002. Pathoge- 63. O’Neill, R.E., R. Jaskunas, G. Blobel, P. Palese, nicity of a Hong Kong-origin H5N1 highly patho- and J. Moroianu. 1995. Nuclear import of infl u- genic avian infl uenza virus for emus, geese, enza virus RNA can be mediated by viral nucleo- ducks, and pigeons. Avian Diseases 46:53–63. protein and transport factors required for protein 74. Perkins, L.E., and D.E. Swayne. 2002. Suscepti- import. Journal of Biological Chemistry 270: bility of laughing gulls (Larus atricilla) to H5N1 22701–22704. and H5N3 highly pathogenic avian infl uenza 64. O’Neill, R.E., J. Talon, and P. Palese. 1998. The viruses. Avian Diseases 46:877–885. infl uenza virus NEP (NS2 protein) mediates 75. Perkins, L.E., and D.E. Swayne. 2003. Varied the nuclear export of viral ribonucleoproteins. pathogenicity of a Hong Kong-origin H5N1 avian The EMBO Journal 17:288–296. infl uenza virus in four passerine species and bud- 65. Orlich, M., H. Gottwald, and R. Rott. 1994. Non- gerigars. Veterinary Pathology 40:14–24. homologous recombination between the hemag- 76. Pinto, L.H., and R.A. Lamb. 2007. Controlling glutinin gene and the nucleoprotein gene of an infl uenza virus replication by inhibiting its proton infl uenza virus. Virology 204:462–465. channel. Molecular Biosystems 3:18–23. 66. Oxford, J.S., C. Manuguerra, O. Kistner, A. 77. Plotkin, J.B., and J. Dushoff. 2003. Codon bias Linde, M. Kunze, W. Lange, B. Schweiger, G. and frequency-dependent selection on the hemag- 20 Avian Influenza

glutinin epitopes of infl uenza A virus. Proceed- enza virus receptors in the human airway. Nature ings of the National Academy of Sciences U S A 440:435–436. 100:7152–7157. 89. Sivanandan, V., D.A. Halvorson, E. Laudert, 78. Pybus, O.G., A. Rambaut, R. Belshaw, R.P. D.A. Senne, and M.C. Kumar. 1991. Isolation of Freckleton, A.J. Drummond, and E.C. Holmes. H13N2 infl uenza A virus from turkeys and surface 2007. Phylogenetic evidence for deleterious water. Avian Diseases 35:974–977. mutation load in RNA viruses and its contribution 90. Slemons, R.D., D.C. Johnson, J.S. Osborn, and F. to viral evolution. Molecular Biology and Evolu- Hayes. 1974. Type-A infl uenza viruses isolated tion 24:845–852. from wild free-fl ying ducks in California. Avian 79. Renegar, K.B., P.A. Small, Jr., L.G. Boykins, and Diseases 18:119–124. P.F. Wright. 2004. Role of IgA versus IgG in the 91. Slemons, R.D., and D.E. Swayne. 1990. Replica- control of infl uenza viral infection in the murine tion of a waterfowl-origin infl uenza virus in the respiratory tract. Journal of Immunology 173: kidney and intestine of chickens. Avian Diseases 1978–1986. 34:277–284. 80. Rohm, C., T. Horimoto, Y. Kawaoka, J. Suss, and 92. Slemons, R.D., and D.E. Swayne. 1995. Tissue R.G. Webster. 1995. Do hemagglutinin genes of tropism and replicative properties of waterfowl- highly pathogenic avian infl uenza viruses consti- origin infl uenza viruses in chickens. Avian Dis- tute unique phylogenetic lineages? Virology eases 39:521–527. 209:664–670. 93. Smith, D.J. 2003. Applications of bioinformatics 81. Rott, R., H.D. Klenk, Y. Nagai, and M. Tashiro. and computational biology to infl uenza surveil- 1995. Infl uenza viruses, cell enzymes, and patho- lance and vaccine strain selection. Vaccine genicity. American Journal of Respiratory and 21:1758–1761. Critical Care Medicine 152:S16–S19. 94. Smith, G.J., X.H. Fan, J. Wang, K.S. Li, K. Qin, 82. Salomon, R., J. Franks, E.A. Govorkova, N.A. J.X. Zhang, D. Vijaykrishna, C.L. Cheung, K. Ilyushina, H.L. Yen, D.J. Hulse-Post, J. Humberd, Huang, J.M. Rayner, J.S. Peiris, H. Chen, R.G. M. Trichet, J.E. Rehg, R.J. Webby, R.G. Webster, Webster, and Y. Guan. 2006. Emergence and pre- and E. Hoffmann. 2006. The polymerase complex dominance of an H5N1 infl uenza variant in China. genes contribute to the high virulence of the Proceedings of the National Academy of Sci- human H5N1 infl uenza virus isolate A/ ences U S A 103:16936–16941. Vietnam/1203/04. Journal of Experimental Med- 95. Spackman, E., K.G. McCracken, K. Winker, and icine 203:689–697. D.E. Swayne. 2006. H7N3 avian infl uenza virus 83. Schafer, J.R., Y. Kawaoka, W.J. Bean, J. Suss, D. found in a South American wild duck is related Senne, and R.G. Webster. 1993. Origin of the to the Chilean 2002 poultry outbreak, contains pandemic 1957 H2 infl uenza A virus and the per- genes from equine and North American wild bird sistence of its possible progenitors in the avian lineages, and is adapted to domestic turkeys. reservoir. Virology 194:781–788. Journal of Virology 80:7760–7764. 84. Scholtissek, C., H. Burger, O. Kistner, and K.F. 96. Spackman, E., D.A. Senne, S. Davison, and D.L. Shortridge. 1985. The nucleoprotein as a possible Suarez. 2003. Sequence analysis of recent H7 major factor in determining host specifi city of avian infl uenza viruses associated with three dif- infl uenza H3N2 viruses. Virology 147:287–294. ferent outbreaks in commercial poultry in the United 85. Schultz, U., W.M. Fitch, S. Ludwig, J. Mandler, States. Journal of Virology 77:13399–13402. and C. Scholtissek. 1991. Evolution of pig infl u- 97. Spackman, E., D.E. Stallknecht, R.D. Slemons, enza viruses. Virology 183:61–73. K. Winker, D.L. Suarez, M. Scott, and D.E. 86. Sekellick, M.J., S.A. Carra, A. Bowman, D.A. Swayne. 2005. Phylogenetic analyses of type A Hopkins, and P.I. Marcus. 2000. Transient resis- infl uenza genes in natural reservoir species in tance of infl uenza virus to interferon action attrib- North America reveals genetic variation. Virus uted to random multiple packaging and activity Research 114:89–100. of NS genes. Journal of Interferon Cytokine 98. Stallknecht, D.E. 1998. Ecology and epidemi- Research 20:963–970. ology of avian infl uenza viruses in wild bird 87. Seto, J.T., and R. Rott. 1966. Functional signifi - populations: waterfowl, shorebirds, pelicans, cor- cance of sialidose during infl uenza virus multipli- morants, etc. In: D.E. Swayne and R.D. Slemons cation. Virology 30:731–737. (eds.). Proceedings of the Fourth International 88. Shinya, K., M. Ebina, S. Yamada, M. Ono, N. Symposium on Avian Infl uenza. USAHA: Rich- Kasai, and Y. Kawaoka. 2006. Avian fl u: infl u- mond, Virginia, pp. 61–69. 1 / Influenza A Virus 21

99. Steinhauer, D.A. 1999. Role of hemagglutinin infl uenza viruses. Biological Pharmacy Bulletin cleavage for the pathogenicity of infl uenza virus. 28:399–408. Virology 258:1–20. 111. Swayne, D.E. 1997. Pathobiology of H5N2 100. Stieneke-Grober, A., M. Vey, H. Angliker, E. Mexican avian infl uenza virus infections of Shaw, G. Thomas, C. Roberts, H.D. Klenk, and chickens. Veterinary Pathology 34:557–567. W. Garten. 1992. Infl uenza virus hemagglutinin 112. Swayne, D.E., and M. Pantin-Jackwood. 2006. with multibasic cleavage site is activated by furin, Pathogenicity of avian infl uenza viruses in a subtilisin-like endoprotease. The EMBO Journal poultry. Developments in Biologicals (Basel) 11:2407–2414. 124:61–67. 101. Suarez, D.L. 2000. Evolution of avian infl uenza 113. Swayne, D.E., M.L. Perdue, J.R. Beck, M. Garcia, viruses. Veterinary Microbiology 74:15–27. and D.L. Suarez. 2000. Vaccines protect chickens 102. Suarez, D.L., M. Garcia, J. Latimer, D. Senne, against H5 highly pathogenic avian infl uenza in and M. Perdue. 1999. Phylogenetic analysis of the face of genetic changes in fi eld viruses over H7 avian infl uenza viruses isolated from the live multiple years. Veterinary Microbiology 74:165– bird markets of the Northeast United States. 172. Journal of Virology 73:3567–3573. 114. Swayne, D.E., and R.D. Slemons. 1990. Renal 103. Suarez, D.L., C.W. Lee, and D.E. Swayne. 2006. pathology in specifi c-pathogen-free chickens Avian infl uenza vaccination in North America: inoculated with a waterfowl-origin type A infl u- strategies and diffi culties. Developments in Bio- enza virus. Avian Diseases 34:285–294. logicals (Basel) 124:117–124. 115. Swayne, D.E., and D.L. Suarez. 2005. U.S. Strat- 104. Suarez, D.L., and M.L. Perdue. 1998. Multiple egies for Controlling Avian Infl uenza in Agricul- alignment comparison of the non-structural genes tural Systems. In: S.L. Knobler, A. Mack, A. of infl uenza A viruses. Virus Research 54:59– Mahmoud, and S.M. Lemon (eds.). The Threat of 69. Pandemic Infl uenza. Are We Ready? National 105. Suarez, D.L., M.L. Perdue, N. Cox, T. Rowe, C. Academies Press: Washington, D.C., pp. 233– Bender, J. Huang, and D.E. Swayne. 1998. Com- 242. parisons of highly virulent H5N1 infl uenza A 116. Tamura, S., and T. Kurata. 2004. Defense mech- viruses isolated from humans and chickens from anisms against infl uenza virus infection in the Hong Kong. Journal of Virology 72:6678–6688. respiratory tract mucosa. Japanese Journal of 106. Suarez, D.L., D.A. Senne, J. Banks, I.H. Brown, Infectious Diseases 57:236–247. S.C. Essen, C.W. Lee, R.J. Manvell, C. Mathieu- 117. Tang, Y., C.W. Lee, Y. Zhang, D.A. Senne, R. Benson, V. Moreno, J.C. Pedersen, B. Panigrahy, Dearth, B. Byrum, D.R. Perez, D.L. Suarez, and H. Rojas, E. Spackman, and D.J. Alexander. Y.M. Saif. 2005. Isolation and characterization of 2004. Recombination resulting in virulence shift H3N2 infl uenza A virus from turkeys. Avian Dis- in avian infl uenza outbreak, Chile. Emerging eases 49:207–213. Infectious Diseases 10:693–699. 118. Taubenberger, J.K., and D.M. Morens. 2006. 107. Suarez, D.L., P.R. Woolcock, A.J. Bermudez, 1918 Infl uenza: the mother of all pandemics. and D.A. Senne. 2002. Isolation from turkey Emerging Infectious Diseases 12:15–22. breeder hens of a reassortant H1N2 infl uenza 119. Thompson, C.I., W.S. Barclay, M.C. Zambon, virus with swine, human, and avian lineage genes. and R.J. Pickles. 2006. Infection of human airway Avian Diseases 46:111–121. epithelium by human and avian strains of infl u- 108. Subbarao, K., A. Klimov, J. Katz, H. Regnery, enza A virus. Journal of Virology 80:8060–8068. W. Lim, H. Hall, M. Perdue, D. Swayne, C. 120. Tumpey, T.M., R. Alvarez, D.E. Swayne, and Bender, J. Huang, M. Hemphill, T. Rowe, M. D.L. Suarez. 2005. Diagnostic approach for dif- Shaw, X. Xu, K. Fukuda, and N. Cox. 1998. ferentiating infected from vaccinated poultry on Characterization of an avian infl uenza A (H5N1) the basis of antibodies to NS1, the nonstructural virus isolated from a child with a fatal respiratory protein of infl uenza A virus. Journal of Clinical illness. Science 279:393–396. Microbiology 43:676–683. 109. Sugrue, R.J., and A.J. Hay. 1991. Structural char- 121. Tumpey, T.M., D.R. Kapczynski, and D.E. acteristics of the M2 protein of infl uenza A Swayne. 2004. Comparative susceptibility of viruses: evidence that it forms a tetrameric chickens and turkeys to avian infl uenza A H7N2 channel. Virology 180:617–624. virus infection and protective effi cacy of a com- 110. Suzuki, Y. 2005. Sialobiology of infl uenza: mercial avian infl uenza H7N2 virus vaccine. molecular mechanism of host range variation of Avian Diseases 48:167–176. 22 Avian Influenza

122. Turner, P.E. 2003. Searching for the advantages of H5N1 avian infl uenza viruses in Asia. Emerg- of virus sex. Origins of Life and Evolution of the ing Infectious Diseases 11:1515–1521. Biosphere 33:95–108. 133. World Organization for Animal Health. 2006. 123. Tweed, S.A., D.M. Skowronski, S.T. David, A. Avian infl uenza 2.7.12, Terrestrial Animal Health Larder, M. Petric, W. Lees, Y. Li, J. Katz, M. Code—2006. World Organization for Animal Krajden, R. Tellier, C. Halpert, M. Hirst, C. Health: Paris, France. Astell, D. Lawrence, and A. Mak. 2004. Human 134. World Organization for Animal Health. 2005. illness from avian infl uenza H7N3, British Avian infl uenza. In: Manual of Diagnostics Tests Columbia. Emerging Infectious Diseases and Vaccines for Terrestrial Animals, Chapter 10:2196–2199. 2.7.12. World Organization of Animal Health: 124. Van Reeth, K. 2007. Avian and swine infl uenza Paris, France. viruses: our current understanding of the zoonotic 135. Wright, P.F., G. Neumann, and Y. Kawaoka. risk. Veterinary Research 38:243–260. 2007. Orthomyxoviruses. In: D.M. Knipe and 125. Vines, A., K. Wells, M. Matrosovich, M.R. Cas- P.M. Howley (eds.). Fields Virology, 5th ed., vol. trucci, T. Ito, and Y. Kawaoka. 1998. The role of 2. Lippincott Williams & Wilkins: Philadelphia, infl uenza A virus hemagglutinin residues 226 and pp. 1691–1740. 228 in receptor specifi city and host range restric- 136. Wright, S.M., Y. Kawaoka, G.B. Sharp, D.A. tion. Journal of Virology 72:7626–7631. Senne, and R.G. Webster. 1992. Interspecies 126. Wan, H., and D.R. Perez. 2006. Quail carry sialic transmission and reassortment of infl uenza A acid receptors compatible with binding of avian viruses in pigs and turkeys in the United States. and human infl uenza viruses. Virology 346:278– American Journal of Epidemiology 136:488– 286. 497. 127. Webster, R.G., and W.G. Laver. 1980. Determi- 137. Xu, K.M., K.S. Li, G.J. Smith, J.W. Li, H. Tai, nation of the number of nonoverlapping antigenic J.X. Zhang, R.G. Webster, J.S. Peiris, H. Chen, areas on Hong Kong (H3N2) infl uenza virus hem- and Y. Guan. 2007. Evolution and molecular epi- agglutinin with monoclonal antibodies and the demiology of H9N2 infl uenza A viruses from selection of variants with potential epidemiologi- quail in southern China, 2000 to 2005. Journal of cal signifi cance. Virology 104:139–148. Virology 81:2635–2645. 128. Webster, R.G., K.F. Shortridge, and Y. Kawaoka. 138. Yamada, S., Y. Suzuki, T. Suzuki, M.Q. Le, 1997. Infl uenza: interspecies transmission and C.A. Nidom, Y. Sakai-Tagawa, Y. Muramoto, emergence of new pandemics. FEMS Immunol- M. Ito, M. Kiso, T. Horimoto, K. Shinya, ogy and Medical Microbiology 18:275–279. T. Sawada, M. Kiso, T. Usui, T. Murata, Y. Lin, 129. Wiley, D.C., I.A. Wilson, and J.J. Skehel. 1981. A. Hay, L.F. Haire, D.J. Stevens, R.J. Russell, Structural identifi cation of the antibody-binding S.J. Gamblin, J.J. Skehel, and Y. Kawaoka. sites of Hong Kong infl uenza haemagglutinin and 2006. Haemagglutinin mutations responsible their involvement in antigenic variation. Nature for the binding of H5N1 infl uenza A viruses 289:373–378. to human-type receptors. Nature 444:378– 130. Wilson, I.A., J.J. Skehel, and D.C. Wiley. 1981. 382. Structure of the haemagglutinin membrane glyco- 139. Zambon, M. 1999. Cell culture for surveillance protein of infl uenza virus at 3 A resolution. Nature on infl uenza. Developments of Biological Stan- 289:366–373. dards 98:65–71; discussion 73–74. 131. Wood, G.W., J. Banks, J.W. McCauley, and D.J. 140. Zanella, A., P. Dall’Ara, and P.A. Martino. 2001. Alexander. 1994. Deduced amino acid sequences Avian infl uenza epidemic in Italy due to serovar of the haemagglutinin of H5N1 avian infl uenza H7N1. Avian Diseases 45:257–261. virus isolates from an outbreak in turkeys in 141. Ziegler, A.F., S. Davison, H. Acland, and R.J. Norfolk, England. Archives of Virology 134:185– Eckroade. 1999. Characteristics of H7N2 (non- 194. pathogenic) avian infl uenza virus infections in 132. World Health Organization Global Infl uenza commercial layers, in Pennsylvania, 1997–98. Program Surveillance Network. 2005. Evolution Avian Diseases 43:142–149. 2 Molecular Determinants of Pathogenicity for Avian Influenza Viruses

Michael L. Perdue

INTRODUCTION in waterfowl with a lower mutation rate, but clearly The avian infl uenza (AI) viruses present some inter- this is not the case with the currently circulating high esting challenges to current ideas regarding viral pathogenicity (HP) H5N1 viruses. Since their detec- evolution. One general consensus (49) is that when tion in 1996–1997, this group of viruses has evolved emerging pathogens jump species barriers, they extensively into geographic and antigenic clades that begin life in their new species as a virulent parasite are defi nitely not in evolutionary stasis (1, 14, 85). and then adapt over time to become less virulent and In this chapter, we discuss aspects of the molecu- more adapted to the new host. This process has been lar biology of AI viruses that have profound effects well documented for a few viruses and other patho- on the virulence and pathogenicity of the viruses and gens, and the logic behind the consensus is that the thus their ecological and pathobiological relation- pathogen would not “want” to destroy its new host ships with their hosts. species by remaining virulent. Unfortunately, many strains of AI viruses have not heard of this paradigm HOW AVIAN INFLUENZA VIRUSES and have jumped species barriers as mildly or non- EXPRESS VIRULENCE AND pathogenic strains, where they should remain happily PATHOGENICITY circulating in the new host. However, they then fi nd a way to become more virulent and (witnessing the Genetic Characteristics of Avian Infl uenza current H5N1 outbreak) can cause a devastating epi- Viruses zootic in their new host. This rapid “virulence shift” As described elsewhere in this book, the AI viruses has been reasonably well documented on at least six are segmented, negative-sensed, RNA-containing occasions now by molecular analysis of the viruses viruses whose genes are located on eight different that were circulating naturally during the shift pieces of RNA, or RNA gene segments. There are (69). 10 well-established and characterized genes, with The second challenge to current thinking posed the two smallest RNA segments each coding for two by AI viruses is the idea or theory of “evolutionary different genes (47). Additionally an 11th gene stasis” reached by AI viruses in migratory waterfowl product was recently discovered (19) but has not (98). Based on prior molecular epidemiology and been fully characterized. The negative-sense RNA assessments of viral mutation rates in some species, has to be copied into a coding mRNA after the virus it was proposed that AI viruses (infl uenza A viruses) enters the cell, and this process is part of a critical had reached a fully host adapted status in Anseri- aspect of the virus life cycle. When the virus is formes and Charadriiformes orders of birds. endocytosed into a susceptible cell, the RNA seg- Theoretically, many strains have indeed stabilized ments are released into the cell to initiate infection

Avian Influenza Edited by David E. Swayne 23 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 24 Avian Influenza by a process that involves lowering the pH within into humans from animals (e.g., measles and HIV- the endocytic vesicle and fusing the viral membrane AIDS). This mutability is almost certainly the reason with the vesicle membrane (12, 47, 83, 84, 96). This that infl uenza A viruses have been able to adapt and releases the viral RNA as a RNA-nucleoprotein circulate in several mammalian species (47, 73, (NP) complex into the cell along with the other 101). proteins that it needs to go to the nucleus and start This characteristic is attributed primarily to the making new virus particles. Most of the infl uenza polymerase complex of the virus, the RNA synthesis virus’s genetic information is geared toward estab- genes. The polymerase of RNA viruses has been lishing this early event and initiating RNA synthesis. termed faulty or error prone because they do not The three large polymerase proteins (about 50% of contain the equivalent activity found in DNA poly- total genetic information) begin operating as soon as merases that checks for mistakes made when copying the viral RNA reaches the nucleus, by making posi- genetic templates (27, 86). The mistakes occur as tive- or messenger-sensed RNA as well as the tem- ribonucleotides are added to the growing chain using plate from which to make new antisense RNA the template RNA molecules from the parent virus. molecules that will be packaged in the newly devel- If the polymerase complex sees a uracil (U), for oping virus particles. Getting the RNA-nucleopro- example, it is instructed to add an adenosine (A). If tein (NP)-polymerase complex to work effi ciently is a mistake in copying is made and a guanosine (G) key to getting the virus rapidly growing to high titers is added instead, the resulting protein coding in the host (58). sequence might be abruptly terminated and progeny Other important gene functions help regulate the virus particle(s) would be unable to replicate and production and assembly of virus particles after the would not survive in the population. If a less sig- process of messenger RNA production begins, but nifi cant mistake were made, the structure of the the second major component or activity, once an protein could change only slightly, making the infl uenza virus infects a cell, is the production of progeny virus now either less fi t or more fi t to adapt new proteins in the cytoplasm of the infected cell. to their environment. This process of making mis- Synthesis of the so-called structural proteins of the takes goes on continuously in RNA viruses resulting virus is specifi ed predominantly by four genes: (1) in what is know as a quasi-species population, that the major surface protein genes, hemagglutinin (HA) is, never being genetically homogeneous (9, 11). and neuraminidase (NA), and (2) the major internal protein genes, matrix (M1) and NP. Importance of Mutations and Their Effects So the three major categories of genes in infl uenza As mentioned previously, quasi-species of RNA A viruses may be broadly classifi ed as (1) RNA viruses have probably evolved as a mechanism to synthesis genes, (2) regulatory genes, and (3) struc- provide a broad range of subpopulations of viruses tural protein genes. These categories are important that may be capable of adapting to the new environ- to remember as we begin to discuss the various ments into which they are placed. For the infl uenza genetic determinants of pathogenicity for AI viruses. A viruses, this can have rapid and profound effects. Of particular importance are the types of genetic For example, populations of circulating H5 subtype variability that occur in these various genes that AI viruses have been demonstrated to have sub- infl uence the various phases of the virus life cycle. populations of viruses already resistant to antiviral drugs used for the treatment of infl uenza infections RNA Viruses and Genetic Variability (97). This resistance was present long before the The RNA viruses are the most mutable of all bio- antiviral drug was ever in use. Use of one of the logical entities (23, 33, 55). Their high mutability drugs approved for treatment of human infections, has been theorized to have evolved as a natural part Flumadine (rimantidine), is currently precluded of the life cycle of RNA viruses (55), allowing them when humans are infected with either the circulating to evade immune response of their current hosts or H3N2 seasonal strains or the current H5N1 strains, quickly adapt to new hosts. The majority of the because resistant populations are already, or would viruses that are currently “emerging” and zoonotic, quickly become, predominant in the environmental for example, are RNA viruses (63), and this high pressure of the presence of the drug. These rimanti- mutability may have formed the basis for their entry dine-resistance mutations can occur at several sites 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 25 along one of the proteins of the virus (M2 protein) specifying virulence or pathogenicity of AI viruses (35, 70), yielding a new structure no longer affected in birds. Figure 2.1 outlines the essential elements by the drug. We know that other small but distinct of the HA gene and protein to be discussed in this changes must be occurring in all of the viral proteins chapter. The HA protein occurs in the virus as a type synthesized by the same molecular processes, so I membrane protein, existing as a trimer, embedded there are always minority subpopulations of viruses in the lipid bilayer of the viral envelope. The protein waiting to be selected out of the majority population is synthesized as a 580- to 585-amino-acid polymer, by some selective environmental pressure. is cotranslationally modifi ed by N-link glycosylation at fi ve to seven positions as it transits the Evaluating Changes in Virulence and endoplasmic reticulum and cytoplasm to the mem- Pathogenicity in Avian Infl uenza Viruses brane and must be cleaved by a protease activity into The subject of this chapter centers on the observa- two subunits (HA-1 and HA-2), before it can realize tions over the past 50 years of profound changes in its full function(s) (47, 107). virulence seen among two subtypes of AI viruses, As one of the major surface glycoproteins of the H7 and the H5 subtypes. During this period, two infl uenza A viruses, the HA actually specifi es several major infl uenza A pandemics in humans occurred, major functions. The globular tip of the HA protein involving viruses that have picked up AI virus genes contains the receptor for binding to the host cell during their evolution (142, 154). Thus, we are surface (104). Antibodies made by the infected host acutely aware of the importance of following the and directed at the globular tip effectively attack the mutational changes in AI viruses. To this end, scores HA and prevent binding and entry. However, this of laboratories around the world are increasing sur- globular head also changes due to mutations rapidly veillance and closely following the genetic changes enough to produce novel structural components that that are occurring particularly in the H5N1 strains. are no longer recognized by host cell antibodies. Further, manipulation of the genome of AI viruses This “antigenic drift” in effect gives the virus an using “reverse genetics” to evaluate the meaning or “escape” mechanism from antibodies to once again effect of genetic changes has allowed better insight allow effi cient binding and entry to the cell (107). into the mutational effects. Much of the information This feature of the HA is the reason that twice yearly available regarding mutations and virulence of AI the World Health Organization convenes experts to viruses has come from descriptive biology of the evaluate antigenic and genomic changes and recom- naturally circulating strains. By performing sequence mend modifi cations to human vaccine strains accord- analysis of genes from viruses with different pheno- ing to the changes in the HA in seasonal human types, phylogenetic trees can be generated and infl uenza strains. viruses evaluated for relatedness. The sequence The HA protein also functions to allow fusion of information can identify genetic changes causing the virus membrane with the host cell membrane and virulence shifts in isolates that are closely related allow release of the genetic information to initiate geographically, temporally, and genetically. Fur- new virus synthesis. As mentioned, this process is thermore, complete sequence analysis should be mediated by a highly conserved region of the HA performed on all genome segments, as we know that called the “fusion peptide” (12, 83, 84, 96). The reassortment of segments can result in shuffl ing of fusion peptide is located on the HA-2 region of the genes between host species and these can have pro- mature protein and is exposed following cleavage found effects on virulence and host range. into HA-1 and HA-2 subunits. After the virus particle is endocytosed in an endocytic vesicle and the CENTRAL ROLE OF THE pH is lowered, the HA-2 undergoes a dramatic con- HEMAGGLUTININ IN PATHOGENICITY formational change that brings the fusion peptide OF AI VIRUSES adjacent to the vesicle membrane, allowing fusion and extrusion of the contents into the cellular cyto- Structure and Functions of the Avian Infl uenza plasm. Thus, cleavage of the HA polyprotein is Viral Hemagglutinin crucial for the exposure of the “fusion peptide” and Numerous studies over the past three decades have the released fusion peptide is absolutely crucial for clearly identifi ed the HA protein as the primary gene the initiation of infection. 26 Avian Influenza

Figure 2.1. Important elements of the AI hemagglutinin protein. The trimeric HA is embedded in the lipid bilayer of the virus particle (electron micrograph courtesy of U.S. Centers for Disease Control and Prevention, Atlanta, GA). This trimeric HA has two basic components, the HA1 (light) and the HA2 (dark). The HA1 is made up mostly of globular stretches of amino acids that undergo mutational changes that often affect the antigenicity of the virus and reaction with host antibodies. In order to become functional, the HA must be cleaved yielding the HA1 and HA2 (see monomer), thus releasing a “fusion peptide” that mediates fusion and release of the viral genome. The cleavage occurs at the PCS and in the case of HP strains is speciﬁ ed by the presence of multibasic amino acids. Carbohydrates added along the HA1 (balls) can also have an inﬂ uence on pathogenicity.

Hemagglutinin Proteolytic Cleavage Site sequence of the HA1 (66). Some of the amino acids This crucial cleavage step in the virus life cycle on either side of this sequence are likewise con- is specifi ed by the region between the HA1 and served, but a search through the available H5 HA2 commonly called the proteolytic cleavage site sequences in the public protein sequence database (PCS) (30, 43, 48). A typical HA cleavage site for reveals a remarkably conserved stretch of these 10 low pathogenicity (LP) H5 subtype infl uenza A amino acids in the LP H5 subtypes. There can be viruses has the amino acid sequence: . . . PQRETR/ minor differences, of course, among the various LP GLFG . . . , where the actual cleavage point is strains in this stretch, but the amino acids that differ between the R (arginine) and G (glycine), with the are almost always in a similar class as the amino R being lost in the cleavage. The N-terminal region acids replaced (i.e., neutral amino acid replaces of the fusion peptide thus includes the GLFG . . . , neutral amino acid). which ultimately allows fusion with the host mem- Low pathogenicity H7 subtype viruses have a brane. This 10-amino-acid sequence (PQRETR/ slightly different comparable conserved PCS region. GLFG) is very well conserved among LP H5 subtype In most, if not all, isolates, the comparable con- AI viruses at position 321–330 in the numbered served region is 11 amino acids with the general 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 27 motif of PEXPKXR/GLFG (153). Here the X can organisms, through evolution, try to expand their be a neutral or a basic amino acid, and there also environments or niches to promote increased fi tness appears to be a bit more variability within the site of their species. So the questions become, Why than seen with the H5 AI viruses (see later). and how do AI viruses alter their cleavage site by Other subtypes also appear to have their own con- mutation? served cleavage sites such as the human H3 strains: The question of “how” the increased number of PEKQTRG(L/I)GF. So it seems that cleavage sites basic amino acids fi nd their way into the cleavage likely evolve and adapt to the peculiar structure of site has not been answered empirically but rather by the HA in which they are found. The interesting descriptive analysis of the gene structure of many question is why have we only seen such dramatic HPAI isolates over the past 30 years. There are at changes thus far in only two avian subtypes and not least four mechanisms by which AI viruses acquire in mammalian strains? basic amino acids at the cleavage site. These are outlined in Table 2.1, which presents the best Amino Acid Changes at the Cleavage Site and evidence for each mechanism and also many rela- Virulence ted references that support the likelihood of the The primary virulence factor for AI viruses is the mechanism. structure of the PCS (37, 66, 78, 106). Alteration of the amino acid content of the PCS by either site Simple Site Mutations mutation or insertions of amino acids or both, result- This is the process of single nucleotide changes that ing in an increase in the number of basic amino acids occur naturally and often in RNA viruses, where (arginine and lysine) at the site provides a different mistakes are made that do not detrimentally affect template for cleavage by proteases. The cellular the fi tness of the virus and become fi xed in the enzymes that cleave the consensus sites of the LP population. Because this process occurs continually, H5 and H7 AI viruses have not been fully identifi ed. it also infl uences all the other proposed mechanisms Because trypsin-like enzymes have been clearly below and in fact data support this. This mechanism shown to cleave infl uenza A virus HAs, the trypsin- of virulence acquisition is probably best exemplifi ed like enzymes at the mucosal surface are assumed to by the H5N2 outbreak that began in the United play prominent roles in infection (44). Recent States in Pennsylvania in 1983 (22). This was a very studies, however, have shown that mammalian type signifi cant outbreak for the U.S. poultry industry II transmembrane serine proteases are capable of resulting in the culling of 17 million birds and supporting multicycle replication of infl uenza A costing $63 milllion. The extensive surveillance viruses (10). Thus, the array of enzymes involved in activities of the University of Pennsylvania and the cleavage of the LPAI viruses have not been fully U.S. Department of Agriculture and others followed identifi ed, but there is unique and limited tissue dis- by the excellent molecular detective work in the tribution of the enzymes which cleave the LP strains. laboratory of Robert Webster resulted in a clear Most tissues in the bird do not support replication picture of what happened (7, 21, 22, 40, 41, 102). A of these viruses, except some epithelial cell types in virus of LP, most likely originating from live poultry the respiratory and intestinal tracts. markets of the northeastern United States, infected In contrast, the widely distributed furin-like or commercial fl ocks and, following circulation in the subtilisin-like endoproteases are capable of cleaving fl ocks, mutated to suddenly become HP and caused HA proteins that have acquired, via mutation, addi- a devastating high-mortality disease in the region. tional basic amino acids at the cleavage site (30, 38, Although clearly other molecular attributes can 74). Thus, when viruses acquire these basic amino affect virulence (see later) and may have in this acids, they are then capable of replication in a much outbreak, the central role of the HA cleavage site wider range of tissues in the bird than previously and stood out dramatically after the analysis of the data. this is the fundamental factor for increased virulence Although the original infecting LPAI virus strain by the strains. Simply, the virus now obtains a much was not found, it is widely assumed that the standard wider range of host tissues to infect, giving it broader PQRETR amino acid sequence common to the H5 replication possibilities. This is a logical extension LP strains must have been the progenitor sequence. of the generally accepted biological paradigm that However, by site mutation basic amino acids must 28 Avian Influenza

Table 2.1. Proposed types of mutational changes in HA cleavage site (co-circulating virus strains). Likely AI Strain: HPAI LP Precursor Proposed Genetic Change at Molecular Related Outbreak Strain Proteolytic Cleavage Site Mechanism References

A/chicken/ A/chicken/ (PQRETR*G)→PQRKKR*G Site mutation(s) 20, 59, 66, Pennsylvania/ Pennsylvania/ (+ loss of CHO) 78, 102, 1370/83 H5N2 21525/83 H5N2 105, 106 A/turkey/Ontario/ ?(A/turkey/ PQRETR*G→PQRRKKR*G Accumulated 6, 20, 57, 7732/66 H5N9 Ontario/ single 66, 78, 6213/66 nucleotide 102, 106 H5N9)? insertions A/chicken/Jalisco/ A/chicken/ PQRETR*G→ Tandem 28, 29, 65, 14588–660/94 Queretaro/ PQRKRKRKTR*G duplication/ 66, 68 H5N2 7653–20/95 insertion H5N2 A/chicken/Chile/ A/chicken/Chile/ PEKPKTR*G→ RNA/RNA 36, 42, 50, 176822/02 4977/02 H7N3 PEKPKTCSPLSRCRETR*G recombination 60, 88 H7N3

have accumulated at the cleavage site so the fi rst based on data current in 1992, that these insertions analyzed LPAI isolate had multiple basic amino were a likely mechanism of generating HP strains acids at the cleavage site (PQRKKR). The HP strain from non-HP strains, but with the exception of iso- had an additional mutation that removed a glycosyl- lates from turkeys in England in 1979, there were ation site that, based on its position on the HA mol- no likely paired precursor-progeny strains of the ecule would have stearically interfered with the same subtype before the H7N1 outbreak in Italy in protease function at the PCS (102). 1999 (6). The outbreak in Italy in 1999 again showed that Accumulated Nucleotide Insertion(s) some LP viruses could rapidly change to become Resulting in Amino Acid Codon Addition HP. In this case, 12 nucleotides were added, result- The second proposed mechanism would be accumu- ing in a longer PCS with more basic amino acids, lated insertions of nucleotides which would ulti- but they likely would not have been added by the mately result in a functional codon, that is, tandem duplication model described below. It is not accumulation of three successive nucleotide inser- clear how such an insertion occurred, and none was tions. It is very likely that this mechanism occurs, postulated by the authors. However, it is possible although the evidence is less compelling. In order that this virulence transition could have been gener- for this single insertion mechanism to work, the ated by an RNA/RNA recombination event (see virus population would presumably have to carry later). along mutants in the quasi-species population that would be out of the coding frame until the third Tandem Duplications of Stretches nucleotide was added. The HPAI outbreak in turkeys of Purines in Ontario in 1966 yielded a virus with a single A likely mechanism for insertion of basic amino amino acid insertion at the cleavage site that could acids at the PCS arose from studies on the 1994– have been obtained via this mechanism (57, 66). 1995 H5N2 outbreak of AI in central Mexico (28, Also, several of the H7 virus isolates obtained before 66, 68). Here, LP and HPAI virus isolates were the 1963 turkey virus outbreak in England had single available from within weeks of each other from amino acid insertions. Wood et al. (106) postulated, central Mexico. Analysis of three isolates demon- 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 29 strated one site mutation and clear successive tandem dissimilar from any other HP viruses and did not duplications of the sequence AAAGAA at the cleav- contain a preponderance of basic amino acids. age site, resulting in the repeating motif of R-K-R- Several years later, investigators in the same lab K-R-K. The likelihood that single nucleotide selected and characterized a similar HP H7 variant insertions resulting in this large six-amino-acid of a seal isolate and showed that nonhomologous insertion would yield the duplicated nucleotide recombination at the cleavage site had occurred with sequence is virtually nil, and a model suggesting an insertion of 60 nucleotides most likely coming how the tandem nucleotide duplications could have from the nucleoprotein gene of the same strain occurred was proposed (28). When other cleavage (50, 60). site sequences were compared over the years, it was Infl uenza researchers were amazed but perplexed clear that tandem duplication of existing sequences as to what signifi cance these laboratory-derived must be occurring in several HP strains (66). Because mutants isolated under selective pressure might have purines predominate in the region, duplication of for infl uenza A ecology. Then an outbreak in poultry purines would more likely result in more basic in Chile occurred in May 2002, the fi rst documented amino acids (e.g., arginine and lysine whose codons outbreak of AI on the South American continent are composed predominantly of purines), whether or since pre-1959 (88). The fi rst virus recovered in not they were duplicated in-frame. Thus, the model May was an LP H7N3 subtype from a broiler- proposed a sort of purine enrichment site where, if breeder fl ock. During June, however, the disease the polymerase made slippage mistakes, basic amino pattern changed with a sudden increase in mortality, acids would most likely arise. Some evidence further and several HP variants were isolated from the same suggested that HPAI strains with such tandem dupli- fl ock. Just as in the outbreak in Mexico, when the cations also showed increased secondary structural LP precursor strain and the subsequent HP isolates constraints near the cleavage site that might affect were characterized, it was clear that a rapid dramatic the performance of the polymerase complex (68). change had occurred at the PCS. This time, and for In another recent HP H7 outbreak in the Nether- the fi rst time in a natural outbreak, the HA cleavage lands, no direct nonpathogenic precursor was avail- site had been lengthened, almost surely, by recom- able but evidence was quite strong that the bination with the nucleoprotein RNA in the same introduction of the virus came from migratory virus. The 30-nucleotide insert shared 100% identity waterfowl and that again mutation at the cleavage with nucleotides 1268–1297 of the NP RNA. site occurred to yield the HP variant. So clearly the Two years later, this scenario was repeated in an pattern of mutations resulting in more basic amino H7N3 AI outbreak in British Columbia with a dra- acids, occurring after viruses enter poultry, is estab- matic increase in mortality in chickens directly lished. In many, but certainly not all, cases, basic related to a 21-nucleotide insertion at the PCS, this amino acid insertions at the PCS in the HP strains time identical to a region of the viral M gene (36). of both the H5 and H7 subtypes can be explained by This time, two workers involved in the depopulation a tandem duplication event and generally follow the efforts (19 million birds were culled in the Fraser proposed model (28). Unfortunately, as is the general Valley) became ill following direct exposure to case for infl uenza viruses, when one tries to make a infected poultry on the affected farms (95). Symp- “rule” for them to follow, they fi nd a way to break it. toms included conjunctivitis, headache, and coryza 1 to 3 days later, and the viruses recovered were the RNA/RNA Recombination Events same as the poultry isolates with the notable excep- In 1989, in vitro passage of an H7 strain from tion that the new cleavage site had undergone further turkeys, in attempts to make it more pathogenic, site mutation, yielding a different amino acid in each resulted in the insertion of 54 nucleotides (18 amino patient. acids) at the HA PCS (42). The insertion appeared As mentioned earlier, it is also possible that the to have come from 28S ribosomal RNA as it shared 1999 H7N1 Italy outbreak could have occurred by 53 of 54 nucleotides with one region of mammalian recombination with a smaller piece of RNA because 28S RNA. This virus did indeed cleave in the the tandem duplication did not occur, and again, it absence of trypsin and showed increased pathoge- is diffi cult to understand how nine successive inser- nicity even though the inserted sequence was quite tions of nucleotides could occur. 30 Avian Influenza

Other Variation in the Hemagglutinin and This has also been an extremely important proce- the Effects dure for the development of human and animal vac- Although the dramatic shifts in virulence of some cines against HP strains (62). Using the reverse H5 and H7 AI strains in poultry populations can be genetics system, the polybasic cleavage site can be clearly linked to changes at the cleavage site, other removed from the HA of an HP candidate vaccine features of the HA certainly infl uence pathogenicity. strain and replaced with a nonvirulent version. This Glycosylation at various sites along the HA mole- allows production of vaccines under lowered bio- cule have been shown to have distinct effects on safety containment as well as possibly enhancing the virus phenotypes and may also affect or mediate the production of egg-grown virus preparations by adaptation of the virus to new species (5, 45, 56, 67, allowing increasing egg survival and higher growth 98). The effect of removing glycosylation near the titers (2). cleavage site was discussed earlier as a clear pathogenicity determinant. Experimental studies have ROLES OF OTHER GENES IN AVIAN shown that glycosylation near the receptor site INFLUENZA VIRULENCE AND increased lethality in birds (67). PATHOGENICITY The structure of receptor site could affect pathogenicity as the site has evolved to bind to receptors Interactions of Proteins in Infl uenza Viruses: specifi c for avian cells but can change to become The “Optimum Gene Constellation” Theory more adapted to humans. As the receptor site (25, 61, 75) mutates and changes, simply lowering the affi nity The differing effects of changes in the HA gene on for avian receptors would lower the fi tness for virus replication stability and pathogenicity also replication and thus the pathogenicity. Additionally, point out a central dogma explaining effi ciency of one study found that specifi c amino acids in infl uenza virus replication and pathogenicity, namely other regions of the globular head were clearly that the viral proteins do not function independently. associated with differences in pathogenicity of The most dramatic recent demonstrations of this are H5N1 isolates. Using reverse genetics and recombi- the reports on the rebuilding of the H1N1 infl uenza nant HAs (see later), amino acids 97, 108, 126, 138, virus that was responsible for the so-called 1918 212, and 217 were linked to expression of pathoge- Spanish Infl uenza Pandemic. In these elegant studies, nicity in chickens (39). Along the same lines, other investigators found that the full virulence potential mutations in functional areas such as the HA stalk, of the rebuilt or rescued virus was reached only after or changes affecting the internal association of all of the original eight gene segments were rescued the HA with M protein, or the stability of the glob- together. Replacement of any of the genes always ular head, likewise could affect replication and resulted in lowering the virulence of the virus in pathogenicity. mice (93). Thus, the complete set of interactions of all the Manipulation of the Hemagglutinin in viral proteins as they evolved naturally also contrib- the Laboratory utes to the virulence and this must be kept in mind Many recent studies have pointed out the importance when evaluating results from reverse genetics exper- of the various parts of the HA by performing “reverse iments. A forced mutation in one of the proteins that genetics” studies (26, 34, 71). These involve manip- changes the virulence phenotype of the mutant does ulating the structure of the HA in vitro by purpose- not necessarily mean the change resulted in an alter- fully mutating a plasmid insert containing the DNA ation of the proteins activity or virulence expression copy of the HA and then rescuing the gene as a within the cell. The induced mutation may have functional RNA segment in a new virus and measur- resulted in a change in the interaction of the mutant ing the resulting phenotypic changes. These studies protein with another viral protein, perhaps destabi- have shown that subtle changes can have profound lizing the virus or otherwise reducing replication effects and have verifi ed without doubt that the potential. While this induced mutation may certainly cleavage site of the HA is key to pathogenicity be called an experimental virulence factor, the virus expression both in avian and in mammalian cell may never have allowed such a mutation to occur systems (37). naturally and survive. 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 31

Neuraminidase Gene Asian strain in chickens (52). The elegant studies The continued in-depth analysis of naturally occur- deciphering the functions of the two NS proteins ring fi eld strains of AI viruses is necessary to build (NS1 and NS2) as regulatory proteins have shown the catalogue of pertinent mutations that are occur- that they can effect many intracellular functions for ring. This is clearly shown by the discovery in H5N1 the virus, including regulation of RNA splicing, viruses of “natural” deletions occurring in the neur- RNA binding, M1 protein binding and nuclear trans- aminidase gene. This deletion of 19 or 20 amino port (export) of proteins, inhibition of cellular mRNA acids occurs in the stalk portion of the N1 neur- capping and polyadenylation, and inhibition of the aminidase and may be associated with the adaptation cellular interferon response (47, 107). Variation in of viruses from wild waterfowl to domestic poultry. any of these functions could have dramatic effects on It is not entirely clear if the deletion is related to virus replication and as such on pathogenicity. The pathogenicity, but it is a central feature of the “Z” latter function or activity related to inhibiting the genotype of viruses that have established themselves interferon (IFN) α/β functional cascade has been in Asia and elsewhere and have been the predomi- suggested as a defi nite virulence or pathogenicity nant genotype infecting humans (see later). determinant, because overcoming the host cellular Several studies have implicated the NA as an antiviral response could clearly be considered a important gene in expressing increased pathogenic- pathogenicity determinant (47, 79, 80, 107), but data ity (4, 8, 18, 25, 39, 61, 75, 108). In one recent study, are equivocal with respect to its importance. Like all a glycosylation site near the globular head of the NA other proposed pathogenicity factors, its impact is of H5N1 strains was associated with increased interdependent on the overall functionality of the pathogenicity in chickens (39). The mechanism by viral genome and protein complex, that is, optimal which such glycosylation might enhance pathoge- constellation. Finally, a deletion of fi ve amino acids nicity is unknown, but in general it could be theo- in the NS gene has been characterized as a stable part rized that changes that might enhance activation of of the Z genotype of the H5N1 strains, although no proteases in the cell (77) by providing more optimal role in virulence has been identifi ed (85). sialidase activity of the NA itself could result in In addition to the large database of existing NS viruses with a better capacity to spread through host gene sequences, a recent large-scale sequencing tissues. project (59) looking at hundreds of infl uenza isolates So the two major structural surface genes clearly and thousands of viral genes from various species play important roles in specifying virulence in AI clearly showed the variability of the NS gene. The viruses. The other two major structural proteins, the study also pinpointed a PDZ ligand motif at the C nucleoprotein (NP) and the matrix (M) protein of the terminus of the NS1 protein that was associated with virus, have more conserved sequences than the HA the 1918 pandemic strain and HPAI strains that have and the NA, which are the most genetically variable. been causing deaths in humans but not in human Mutations in those proteins can also certainly affect strains of lower pathogenicity. PDZ domains are virulence of the virus (92), but again it is often dif- conserved 80- to 90-amino-acid stretches, often in fi cult to assess whether this is due to destabilizing multiple copies, in proteins associated with the important structural protein-protein interactions plasma membrane (24). The identifi ed NS ligand within the virus or otherwise altering functions of binds to PDZ domains (59) and thus may have many the proteins in the cell. intracellular effects, including having effects on the PDZ domain–containing proteins regulating activity Nonstructural Gene and traffi cking of membrane proteins. Numerous studies have implicated the NS gene in either enhancing or attenuating pathogenicity. For Polymerase Complex example, one study showed that increased virulence The polymerase complex, or those genes and pro- in chicken embryos was clearly linked to two amino teins responsible for making the viral RNA, might acid differences in the NS2 protein (64). Another seem an unlikely set of genes to express virulence recent publication showed that a single amino acid or pathogenicity factors because they are fairly well change at position 149 of the NS1 protein had dra- conserved and have a defi ned role, namely, making matic effects on the virulence expression by an H5N1 messenger RNA and viral RNA. These would appear 32 Avian Influenza to be more or less generic roles that would have to such mutations occur are dead end hosts and the be conserved to allow effi cient replication of any virus cannot “fi x” in the broader population. This is infl uenza A virus. In addition to the three poly- not easy to understand as it would seem logical that merase proteins (PB-1, PB-2, and PA), the PB1 gene changes such as wider recognition of the PCS in also codes for a fourth protein called the PB1-F2 other bird tissues would confer a selective advantage (19). The protein is 87 amino acids long and the for progeny viruses. Perhaps it could be explained region coding for this protein is under intense selec- by the observation that such changes have thus far tive pressure, so its purported role in apoptosis in been restricted to H5 and H7 strains. Perhaps these the cell may be critical, and although not found in strains have not been as capable as competing with all infl uenza A strains, it could certainly be consid- other subtypes and are “replaced” in these orders of ered a potential pathogenicity factor. birds by more fi t nonpathogenic subtypes. We know The polymerase complex has clearly been associ- that migratory birds can yield multiple subtypes ated with measured variations in pathogenicity in the from single samples and that routine surveillance in laboratory (76), and one well-reported molecular birds in contact with migratory waterfowl indicates determinant has been identifi ed both in the labora- that different subtypes predominate in species at dif- tory and in the fi eld as a likely determinant associ- ferent sampling times (32). ated with pathogenicity of AI viruses in mammalian In addition to the large database of existing avian hosts. A single change from glutamic acid (E) to and other animal infl uenza A virus gene sequences, lysine (K) at position 627 of the PB-2 has been the recent large-scale sequencing project (59) associated with both increased virulence and adapta- looking at hundreds of infl uenza isolates and thou- tion to mammals (89). Interestingly although viruses sands of viral genes from various species identifi ed containing the E at 627 can replicate in mice, this new allelic relationships among several genes, muta- ability was also correlated with another change in tions that compensated for other mutations and the duck viruses at position 701 that allowed replication persistence of some genotypes. The study also iden- and lethality (51). More studies are needed, in par- tifi ed, to a degree, the types and extent of variability ticular, crystallization or molecular modeling of each of the genes, supporting previously estab- studies, to reveal the role of the polymerase complex lished evolutionary rates but also demonstrating that in adaptation and pathogenicity. the HA and NA and NS1 RNAs contribute the most to variability of genomes of avian strains. The vari- Adaptation of Avian Infl uenza Viruses and ation in the surface proteins is understandable, but Migratory Waterbirds that identifi ed for the NS gene was not easily explain- The primordial reservoir for AI viruses has always able by any selective pressure. This latter fi nding is been considered to be migratory waterfowl, primar- signifi cant because it indicates with all other adapta- ily two orders of the class Aves, Anseriformes tions and virulence factors that emerge in AI viruses, (ducks and geese) and Charadriiformes (shorebirds the variation of the NS1 protein appears to have an and relatives). All of the 16 HA and 9 NA subtypes importance in the avian virus life cycle and may varieties have been isolated from birds over the past clearly have signifi cant effects on pathogenicity. 50 years (69). However, only limited numbers of subtypes have been isolated from other animals. Finding New Host Species: Adaptation and Until the recent widespread H5N1 outbreaks, the Pathogenesis large number of descriptive studies that have been The current concerns surrounding AI viruses and done have suggested that viruses isolated from these their pathogenesis, of course, are that they have orders exhibit two common characteristics: They are ways of entering new host species and becoming nonpathogenic or of LP in birds and other animals adapted. There are no better examples of this than and they appear to be fully adapted or evolutionarily the adaptation over the years of a limited number of stable, that is, no longer under selective pressure. subtypes of infl uenza A in humans and swine, where One conclusion from these studies could be that they currently circulate and cause disease and death. appearance of new pathogenicity determinants in the Interestingly, when the viruses do become fully populations, such as basic amino acids at the PCS, adapted, they then lose their capacity to replicate is not accepted or either birds or species in which effi ciently in the original host. However, this may 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 33 not be entirely dogma because it is not really known Studies on evolution and persistence of these what the original species was for the currently cir- viruses have taken place in many laboratories, and culating human and swine strains. It has been evaluation of the overall evolution and acquisition assumed that the original donor species was a bird of important mutations is far from fi nished. The best species and likely a waterfowl species. Unlike our evidence indicates that a sort of “index virus” exists understanding of what makes AI viruses pathogenic in the form of the goose/Guandong/96 virus that was for birds, understanding the changes that occurred isolated a year before the Hong Kong outbreak. This to allow adaptation of such a bird virus to mammals virus or a close relative appears to have donated the is not clear. HA gene (as well as the NA) that has been undergo- Several investigators have suggested a defi ned set ing considerable evolution ever since (15, 31). The of mutations that must occur in order to make a bird evolution of the so-called Z genotype has been virus become a human virus. Some have even tried covered elsewhere in this publication and others and to quantify the number needed for the current H5N1 yields the best course of genetic events relating to strains to become a human pandemic virus. Although the origin and expansion. there are some clear candidates for mutations that We know that considerable variation in the H5N1 would make the current H5N1 more closely fi t the strains has occurred yielding multiple subclades that current or past human circulating strains, it is simply are now poorly cross-reacting antigenically (2, 53, not known which of these proposed mutations would 85), that the viruses now are clearly capable of exist- even be acceptable to the virus in the context of the ing as the HP form in migratory waterfowl (15, 17, optimal constellation theory. Likewise, unless it is 103), and that a very limited number of species have accepted that the H5N1 virus has a direction, that is, been able to spread the virus over long distances for some reason it is under selective pressure to (16). These fi ndings have indicated clear uncon- “seek new hosts” (even though it has billions upon trolled evolution of these viruses as well as a change billions of more likely avian hosts to choose from), in tissue tropism of H5N1 in migrating birds and then mutations moving away from a human pan- raise many questions as to where the virus is headed demic strain would be just as likely to occur as those ecologically. moving the virus toward a pandemic strain. Hemaglutinin Cleavage Site Variation in the TRACKING THE CHANGES IN Current H5N1 Strains and Implications MOLECULAR DETERMINANTS: HP H5N1 In terms of what is happening with respect to patho- STRAINS AND THEIR GLOBAL IMPACT genicity determinants, the PCS of the H5N1 viruses has remained remarkably stable (Table 2.2). Table Origin(s) of the Currently Circulating Highly 2.2 illustrates the PCS of some 65 isolates from 26 Pathogenic H5N1 Viruses countries that have been recently sequenced and are The widespread distribution on three continents of compared with the index goose/Guandong/96 virus. the HP H5N1 strains that have infected and killed The list is not exhaustive but represents a subset of humans has mobilized animal health and human isolates from the various species and countries. public health agencies around the world. Since the Although an LP precursor to the HA of the goose/ original avian outbreak in Hong Kong in 1997 and Guangdong virus has not been identifi ed, the tandem the following human infections, considerable pro- repeated sequences found in the virus coding for the spective and retrospective analysis of the H5N1 arginines in the PCS suggest the polymerase slip- strains has occurred. There are over 1000 complete page mechanism could have generated the HP HA. or nearly complete H5 HA gene sequences now pub- Review of the variation occurring in the cleavage licly available from more than 15 laboratories around site in isolates after 1996 (Table 2.2) shows that not the world. The H5N1 viruses never disappeared more than one deletion relative to the original pro- after the 1997 Hong Kong outbreak, but close rela- totype virus has yet been characterized in over a tives reappeared in 1999, 2001, and 2003 in Hong thousand isolates, that the variation that is occurring Kong before the rapid spread of the virus in late is skewed toward the N terminus end of the PCS, 2003 (1, 13, 14, 46, 53, 72, 81, 82, 87, 90, 94, 100, and that the PCS sequence that fi rst appeared 103, 155). at Qinghai Lake in China in 2005 has remained Table 2.2. Variations in proteolytic cleavage site (PCS) of H5N1 HPAI viruses from 1996 to 2006. HP-PCS Sequence GenBank H5N1 HP Isolates (Type/Species/Place/ (Nonpathogenic: Accession Country Designation/Year of Isolation) PQRETRGLFG) Number

China A/goose/Guangdong/1/96 PQRERRRKKRGLFG NC007362 A/chicken/Hong Kong/915/97 PQRERRRKKRGLFG AF046100 A/environment/Hong Kong/437–10/99 PQRERRRKKRGLFG AF216737 A/duck/Zhejiang/52/00 PQREIRRKKRGLFG AY585377 A/chicken/Jiande/1218/01 PQRERRRKKRGLFG DQ003215 A/duck/Hong Kong/821/02 PQIERRRKKRGLFG AY676033 A/duck/Shanghai/xj/02 PQRERRRKKRGLFG DQ997531 A/chicken/Jilin/hn/03 PQREIRRKKRGLFG DQ997352 A/chicken/Hong Kong/NT93/03 PQRERRRKKRGLFG AY651354 A/goose/Fujian/bb/03 PQRERRRKKRGLFG DQ997405 A/blackbird/Hunan/1/04 PQRERRRKKRGLFG AY741213 A/swine/Henan/wy/04 PQRERRRKKRGLFG DQ997253 A/grey heron/Hong Kong/837/04 PQRERRRKKRGLFG DQ320924 A/bar-headed goose/Qinghai/0510/05 PQGERRRKKRGLFG DQ137873 A/Zhejiang/16/06 PLRERRR_KRGLFG DQ643809 Vietnam A/goose/Vietnam/113/01 PRIERRRKKRGLFG ISDN38260 A/chicken/Vietnam/8/03 PQRERRRKKRGLFG DQ497693 A/chicken/Vietnam/39/04 PQRERRRKKRGLFG AY651342 A/quail/Vietnam/177/04 PQRERRRKKRGLFG DQ497715 A/chicken/Vietnam/147/04 PQRERIRKKRGLFG DQ492818 A/duck/Vietnam/AG40–02/05 PQRE_RRKKRGLFG AM183676 A/duck/Vietnam/568/05 PQRERRRKKRGLFG DQ320939 A/Vietnam/CL105/05 PQKERRRKKRGLFG DQ497726 Thailand A/chicken/Lopburi/CU-38/04 PQRERKRKKRGLFG DQ083576 A/cat/Thailand/KU-02/04 PQRERRRKKRGLFG DQ236077 A/pigeon/Thailand/KU-03/04 PQRERRRKKRGLFG DQ236085 A/dog/Thailand/KU-08 PQRERRRKKRGLFG DQ530173 A/white peafowl/Bangkok/CU-29/04 PQRERKRKKRGLFG DQ083573 A/tiger/Thailand/CU-T5/04 PQRERRRKKRGLFG AY972540 A/chicken/Kamphaengphet-3-03/05 PQRERRRKKRGLFG DQ291756 A/Thailand/NK165/05 PQREKRRKKRGLFG DQ372591 Indonesia A/chicken/Indonesia/5/04 PQRERRRKKRGLFG AY651325 A/chicken/Kulon Progo/BBVet-XII-2/04 PQRERRR_KRGLFG DQ497650 A/chicken/Tarutung/BPPVI/05 PQRERRRKKRGLFG DQ497669 A/Indonesia/CDC184/05 PQRESRRKKRGLFG CY014197 A/feline/Indonesia/CDC1/06 PQRESRRKKRGLFG CY014208 Japan A/chicken/Yamaguchi/7/04 PQRERRRKKRGLFG AB166862 A/blow ﬂ y/Kyoto/93/04 PQRE_RRKKRGLFG AB212649 Mongolia A/whooper swan/Mongolia/6/05 PQGERRRKKRGLFG AB233322 Korea A/chicken/Korea/ES/03 PQRE_KRKKRGLFG AY676035 Laos A/duck/Laos/3295/06 PLRE_RRRKRGLFG DQ845348 Iran A/whooper swan/Iran/754/06 PQGERRRKKRGLFG CY016779 Afghanistan A/chicken/Afghanistan/1207/06 PQGERRRKKRGLFG CY016787

34 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 35

Table 2.2. Continued HP-PCS Sequence GenBank H5N1 HP Isolates (Type/Species/Place/ (Nonpathogenic: Accession Country Designation/Year of Isolation) PQRETRGLFG) Number

Egypt A/duck/Egypt/2253–3/06 PQGERRRKKRGLFG CY016899 Iraq A/domestic goose/Iraq/812/06 PQGERRRKKRGLFG DQ435201 Germany A/swan/Germany/R65/06 PQGERRRKKRGLFG DQ464354 Italy A/mallard/Italy/835/06 PQGERRRKKRGLFG CY016795 Croatia A/mute swan/Croatia/1/05 PQGERRRKKRGLFG CY016819 Romania A/chicken/Romania/4793/T1/05 PQGDRRRKKRGLFG DQ991231 Slovenia A/swan/Slovenia/760/06 PQGERRRKKRGLFG CY017043 France A/turkey/France/06222/06 PQGERRRKKRGLFG AM236074 Turkey A/turkey/Turkey/1/05 PQGERRRKKRGLFG DQ407519 Czech Republic A/mute swan/Czech Republic/5170/06 PQGERRRKKRGLFG DQ515984 Russia A/swan/Astrakhan/Nov-2/05 PQGERRRKKRGLFG DQ840533 A/goose/Krasnoozerka/627/05 PQGERRRKKRGLFG DQ676840 A/grebe/Tyva/Tyv06–1/06 PQGERRRKKRGLFG DQ914808 A/cat/Dagestan/87/06 PQGERRRKKRGLFG DQ864720 Ukraine A/chicken/Crimea/04/05 PQGERRRKKRGLFG DQ650659 A/goose/Crimea/615/05 PQGERRRKKRGLFG DQ864717 Nigeria A/chicken/Lagos.NIE/8.06/SO494/06 PQGERRRKKRGLFG AM262572 A/chicken/Nigeria/1047–34/06 PQGERRRKKRGLFG CY016947 Sudan A/chicken/Sudan/1784–10/06 PQGEGRRKKRGLFG CY016300 A/chick/Côte d’Ivoire/1787–34/06 PQGERRRKKRGLFG CY016811 Niger A/duck/Niger/914/06 PQGERRRKKRGLFG CY017027 Djibouti A/Djibouti/5691NAMRU3/06 PQGERRRKKRGLFG DQ666146

virtually constant throughout 2006 as the virus has cleavage site. The Asian H5N1 strains have become appeared in more than 18 countries and in more than more rather than less virulent as they have spread 10 new species (Table 2.2). This variation certainly around the globe (54, 91). Likewise as outlined, refl ects the other variation going on in the virus, but deletions in the NS gene and in the NA gene repre- it is interesting and unprecedented that these strains sent novel changes with phenotypic ramifi cations. have retained their HP phenotype for so long over The point is, we know we have had to deal with so large a geographical expanse and in so many infl uenza A viruses changing constantly on the species. surface of their HA protein as a means of evading the immune system (e.g. continually changing our SUMMARY AND CONSIDERATIONS FOR vaccine selection and formulation). In addition, we THE FUTURE must also deal with and rapidly understand the The experiences with AI viruses over the past three changes in the entire genome. Several efforts have decades have made it very clear to the world that arisen to expand the infl uenza sequence databases, these viruses are an ecological force that must be particularly for the avian viruses that infect humans, addressed. Just as an example, if one looks at the and make them accessible to a larger contingent of changes in the HA cleavage site, from isolates over investigators, particularly bio-informaticists. the last century, the changes have become increas- While one hesitates to embrace the dire predic- ingly dramatic, with insertions ranging up to 30 tions of human deaths due to a “bird fl u pandemic” nucleotides long occurring naturally and longer that have been made by some, the AI viruses do have stretches of basic amino acids appearing at the to now occupy a special place in our ecological 36 Avian Influenza study matrix. We ignore them at our peril. Much magglutinin and the neuraminidase genes prior to more funding has been made available at many the emergence of highly pathogenic H7N1 avian levels to respond to the outbreaks as well as trying infl uenza viruses in Italy. Archives of Virology to understand the viruses and how and why they 146:963–973. infect humans. Proposed global surveillance activi- 7. Bean, W.J., Y. Kawaoka, J.M. Wood, J.E. Pearson, and R.G. Webster. 1985. Characteriza- ties have expanded to unprecedented levels. tion of virulent and avirulent A/chicken/Pennsyl- However, we need to develop better coordination of vania/83 infl uenza A viruses: potential role of surveillance activities and analysis activities so that defective interfering RNAs in nature. Journal of countries dealing with outbreaks of AI can conduct Virology 54:151–160. risk assessments and develop informed decisions on 8. Bender, C., H. Hall, J. Huang, A. Klimov, N. how best to expend animal health and public health Cox, A. Hay, V. Gregory, K. Cameron, W. Lim, funds. and K. Subbarao. 1999. Characterization of the Better correlates of virulence for humans and surface proteins of infl uenza A (H5N1) viruses other mammals are needed so when we identify isolated from humans in 1997–1998. Virology crucial mutational changes in AI viruses and make 254:115–123. informed decisions. This requires good animal 9. Biebricher, C.K., and M. Eigen. 2006. What is a quasispecies? Current Topics in Microbiology models, more research into the mechanisms involved and Immunology 299:1–31. in generating and expressing the molecular determi- 10. Bottcher, E., T. Matrosovich, M. Beyerle, H.D. nants of pathogenesis and better bioinformatics Klenk, W. Garten, and M. Matrosovich. 2006. approaches for digesting high throughput whole Proteolytic activation of infl uenza viruses by genome sequencing. As the only zoonotic virus with serine proteases TMPRSS2 and HAT from human proven pandemic potential, notwithstanding SARS, airway epithelium. Journal of Virology 80:9896– the AI viruses hold a unique place for both the vet- 9898. erinary and medical worlds. 11. Bull, J.J., L.A. Meyers, and M. Lachmann. 2005. Quasispecies made simple. PLoS Computational Biology 1:e61. REFERENCES 12. Bullough, P.A., F.M. Hughson, J.J. Skehel, and 1. Anonymous. 2005. Evolution of H5N1 avian D.C. Wiley. 1994. Structure of infl uenza haemag- infl uenza viruses in Asia. Emerging Infectious glutinin at the pH of membrane fusion. Nature Diseases 11:1515–1521. 371:37–43. 2. Anonymous. 2006. Antigenic and genetic charac- 13. Cauthen, A.N., D.E. Swayne, S. Schultz-Cherry, teristics of H5N1 viruses and candidate H5N1 M.L. Perdue, and D.L. Suarez. 2000. Continued vaccine viruses developed for potential use as circulation in China of highly pathogenic avian pre-pandemic vaccines. Weekly Epidemiological infl uenza viruses encoding the hemagglutinin Record 81:325–340. gene associated with the 1997 H5N1 outbreak in 3. Anonymous. 2006. Epidemiology of WHO-con- poultry and humans. Journal of Virology 74: fi rmed human cases of avian infl uenza A (H5N1) 6592–6599. infection. Weekly Epidemiological Record 14. Chen, H., G. Deng, Z. Li, G. Tian, Y. Li, P. Jiao, 81:249–257. L. Zhang, Z. Liu, R.G. Webster, and K. Yu. 4. Baigent, S.J., and J.W. McCauley. 2001. Glyco- 2004. The evolution of H5N1 infl uenza viruses in sylation of haemagglutinin and stalk-length of ducks in southern China. Proceedings of the neuraminidase combine to regulate the growth of National Academy of Sciences U S A 101:10452– avian infl uenza viruses in tissue culture. Virus 10457. Research 79:177–185. 15. Chen, H., Y. Li, Z. Li, J. Shi, K. Shinya, G. Deng, 5. Banks, J., and L. Plowright. 2003. Additional gly- Q. Qi, G. Tian, S. Fan, H. Zhao, Y. Sun, and Y. cosylation at the receptor binding site of the hem- Kawaoka. 2006. Properties and dissemination of agglutinin (HA) for H5 and H7 viruses may be an H5N1 viruses isolated during an infl uenza out- adaptation to poultry hosts, but does it infl uence break in migratory waterfowl in western China. pathogenicity? Avian Diseases 47(Suppl):942– Journal of Virology 80:5976–5983. 950. 16. Chen, H., G.J. Smith, K.S. Li, J. Wang, X.H. Fan, 6. Banks, J., E.S. Speidel, E. Moore, L. Plowright, J.M. Rayner, D. Vijaykrishna, J.X. Zhang, L.J. A. Piccirillo, I. Capua, P. Cordioli, A. Fioretti, Zhang, C.T. Guo, C.L. Cheung, K.M. Xu, L. and D.J. Alexander. 2001. Changes in the hae- Duan, K. Huang, K. Qin, Y.H. Leung, W.L. Wu, 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 37

H.R. Lu, Y. Chen, N.S. Xia, T.S. Naipospos, K. 1999. Rescue of infl uenza A virus from recombi- Y. Yuen, S.S. Hassan, S. Bahri, T.D. Nguyen, nant DNA. Journal of Virology 73:9679–9682. R.G. Webster, J.S. Peiris, and Y. Guan. 2006. 27. Furio, V., A. Moya, and R. Sanjuan. 2005. The Establishment of multiple sublineages of H5N1 cost of replication fi delity in an RNA virus. Pro- infl uenza virus in Asia: implications for pandemic ceedings of the National Academy of Sciences control. Proceedings of the National Academy of U S A 102:10233–10237. Sciences U S A 103:2845–2850. 28. Garcia, M., J.M. Crawford, J.W. Latimer, E. 17. Chen, H., G.J. Smith, S.Y. Zhang, K. Qin, J. Rivera-Cruz, and M.L. Perdue. 1996. Heteroge- Wang, K.S. Li, R.G. Webster, J.S. Peiris, and Y. neity in the haemagglutinin gene and emergence Guan. 2005. Avian fl u: H5N1 virus outbreak in of the highly pathogenic phenotype among recent migratory waterfowl. Nature 436:191–192. H5N2 avian infl uenza viruses from Mexico. 18. Chen, J.M., H.C. Ma, J.W. Chen, Y.X. Sun, J.M. Journal of General Virology 77(Pt 7):1493– Li, and Z.L. Wang. 2007. A preliminary pan- 1504. orama of the diversity of N1 subtype infl uenza 29. Garcia, M., D.L. Suarez, J.M. Crawford, J.W. viruses. Virus Genes, 35(1):33–40. Latimer, R.D. Slemons, D.E. Swayne, and M.L. 19. Chen, W., P.A. Calvo, D. Malide, J. Gibbs, U. Perdue. 1997. Evolution of H5 subtype avian Schubert, I. Bacik, S. Basta, R. O’Neill, J. infl uenza A viruses in North America. Virus Schickli, P. Palese, P. Henklein, J.R. Bennink, Research 51:115–124. and J.W. Yewdell. 2001. A novel infl uenza A 30. Garten, W., F.X. Bosch, D. Linder, R. Rott, and virus mitochondrial protein that induces cell H.D. Klenk. 1981. Proteolytic activation of the death. Nature Medicine 7:1306–1312. infl uenza virus hemagglutinin: the structure of the 20. De, B.K., G.G. Brownlee, A.P. Kendal, and cleavage site and the enzymes involved in cleav- M.W. Shaw. 1988. Complete sequence of a age. Virology 115:361–374. cDNA clone of the hemagglutinin gene of in - 31. Guan, Y., J.S. Peiris, L.L. Poon, K.C. Dyrting, fl uenza A/chicken/Scotland/59 (H5N1) virus: T.M. Ellis, L. Sims, R.G. Webster, and K.F. comparison with contemporary North American Shortridge. 2003. Reassortants of H5N1 infl uenza and European strains. Nucleic Acids Research viruses recently isolated from aquatic poultry in 16:4181–4182. Hong Kong SAR. Avian Diseases 47(Suppl):911– 21. Deshpande, K.L., V.A. Fried, M. Ando, and R.G. 913. Webster. 1987. Glycosylation affects cleavage of 32. Halvorson, D.A., et al. 1992. Infl uenza in Turkeys an H5N2 infl uenza virus hemagglutinin and regu- in the USA (1987–1991). In: B.C. Easterday (ed.). lates virulence. Proceedings of the National Proceedings of the Third International Sympo- Academy of Sciences U S A 84:36–40. sium on Avian Infl uenza, May 27–29, 1992, 22. Eckroade, R.J., et al. 1986. In: B.C. Easterday and Madison, Wisconsin. Symposium on Avian Infl u- C.W. Beard (eds.). Proceedings of the Second enza, US Animal Health Association: Richmond, International Symposium on Avian Infl uenza, VA, pp. 33–42. Georgia Center for Continuing Education, the 33. Hanada, K., Y. Suzuki, and T. Gojobori. 2004. A University of Georgia, Athens, Georgia, USA, large variation in the rates of synonymous substi- September 3–5, 1986. Symposium on Avian tution for RNA viruses and its relationship to a Infl uenza, US Animal Health Association: Rich- diversity of viral infection and transmission mond, VA, pp. 22–51. modes. Molecular Biology and Evolution 23. Elena, S.F., P. Carrasco, J.A. Daros, and R. 21:1074–1080. Sanjuan. 2006. Mechanisms of genetic robustness 34. Hatta, M., G. Neumann, and Y. Kawaoka. 2001. in RNA viruses. EMBO Reports 7:168–173. Reverse genetics approach towards understand- 24. Fanning, A.S., and J.M. Anderson. 1999. PDZ ing pathogenesis of H5N1 Hong Kong infl uenza domains: fundamental building blocks in the A virus infection. Philosophical Transactions of organization of protein complexes at the plasma the Royal Society London B: Biological Sciences membrane. Journal of Clinical Investigation 356:1841–1843. 103:767–772. 35. Hay, A.J., M.C. Zambon, A.J. Wolstenholme, J.J. 25. Florent, G., M. Lobmann, A.S. Beare, and N. Skehel, and M.H. Smith. 1986. Molecular basis Zygraich. 1977. RNAs of infl uenza virus recom- of resistance of infl uenza A viruses to amanta- binants derived from parents of known virulence dine. Journal of Antimicrobial Chemotherapy for man. Archives of Virology 54:19–28. 18(Suppl B):19–29. 26. Fodor, E., L. Devenish, O.G. Engelhardt, P. 36. Hirst, M., C.R. Astell, M. Griffi th, S.M. Cough- Palese, G.G. Brownlee, and A. Garcia-Sastre. lin, M. Moksa, T. Zeng, D.E. Smailus, R.A. Holt, 38 Avian Influenza

S. Jones, M.A. Marra, M. Petric, M. Krajden, D. D.M. Knipe and P.M. Howley (eds.). Fields Lawrence, A. Mak, R. Chow, D.M. Skowronski, Virology, 4th ed. Lippincott Williams & Wilkins: S.A. Tweed, S. Goh, R.C. Brunham, J. Robinson, Philadelphia, pp. 1487–1532. V. Bowes, K. Sojonky, S.K. Byrne, Y. Li, D. 48. Lazarowitz, S.G., A.R. Goldberg, and P.W. Kobasa, T. Booth, and M. Paetzel. 2004. Novel Choppin. 1973. Proteolytic cleavage by plasmin avian infl uenza H7N3 strain outbreak, British of the HA polypeptide of infl uenza virus: host cell Columbia. Emerging Infectious Diseases activation of serum plasminogen. Virology 56: 10:2192–2195. 172–180. 37. Horimoto, T., and Y. Kawaoka. 1994. Reverse 49. Lederberg, J. 1998. Emerging infections: an evo- genetics provides direct evidence for a correlation lutionary perspective. Emerging Infectious Dis- of hemagglutinin cleavability and virulence of an eases 4:366–371. avian infl uenza A virus. Journal of Virology 50. Li, S.Q., M. Orlich, and R. Rott. 1990. Generation 68:3120–3128. of seal infl uenza virus variants pathogenic for 38. Horimoto, T., and Y. Kawaoka. 1995. The hem- chickens, because of hemagglutinin cleavage site agglutinin cleavability of a virulent avian infl u- changes. Journal of Virology 64:3297–3303. enza virus by subtilisin-like endoproteases is 51. Li, Z., H. Chen, P. Jiao, G. Deng, G. Tian, Y. Li, infl uenced by the amino acid immediately down- E. Hoffmann, R.G. Webster, Y. Matsuoka, and K. stream of the cleavage site. Virology 210:466– Yu. 2005. Molecular basis of replication of duck 470. H5N1 infl uenza viruses in a mammalian mouse 39. Hulse, D.J., R.G. Webster, R.J. Russell, and D.R. model. Journal of Virology 79:12058–12064. Perez. 2004. Molecular determinants within the 52. Li, Z., Y. Jiang, P. Jiao, A. Wang, F. Zhao, G. surface proteins involved in the pathogenicity of Tian, X. Wang, K. Yu, Z. Bu, and H. Chen. 2006. H5N1 infl uenza viruses in chickens. Journal of The NS1 gene contributes to the virulence of Virology 78:9954–9964. H5N1 avian infl uenza viruses. Journal of 40. Kawaoka, Y., C.W. Naeve, and R.G. Webster. Virology 80:1115–1123. 1984. Is virulence of H5N2 infl uenza viruses in 53. Ma, H.C., J.M. Chen, J.W. Chen, Y.X. Sun, J.M. chickens associated with loss of carbohydrate Li, and Z.L. Wang. 2006. The panorama of the from the hemagglutinin? Virology 139:303–316. diversity of H5 subtype infl uenza viruses. Virus 41. Kawaoka, Y., and R.G. Webster. 1985. Evolution Genes 34:283–287. of the A/chicken/Pennsylvania/83 (H5N2) infl u- 54. Maines, T.R., X.H. Lu, S.M. Erb, L. Edwards, J. enza virus. Virology 146:130–137. Guarner, P.W. Greer, D.C. Nguyen, K.J. Szretter, 42. Khatchikian, D., M. Orlich, and R. Rott. 1989. L.M. Chen, P. Thawatsupha, M. Chittaganpitch, Increased viral pathogenicity after insertion of a S. Waicharoen, D.T. Nguyen, T. Nguyen, H.H. 28S ribosomal RNA sequence into the haemag- Nguyen, J.H. Kim, L.T. Hoang, C. Kang, L.S. glutinin gene of an infl uenza virus. Nature Phuong, W. Lim, S. Zaki, R.O. Donis, N.J. Cox, 340:156–157. J.M. Katz, and T.M. Tumpey. 2005. Avian infl u- 43. Klenk, H.D. 1980. Processing of the infl uenza enza (H5N1) viruses isolated from humans in virus hemagglutinin. European Journal of Cell Asia in 2004 exhibit increased virulence in Biology 22:795–796. mammals. Journal of Virology 79:11788–11800. 44. Klenk, H.D., and R. Rott. 1988. The molecular 55. Manrubia, S.C., C. Escarmis, E. Domingo, and E. biology of infl uenza virus pathogenicity. Advances Lazaro. 2005. High mutation rates, bottlenecks, in Virus Research 34:247–281. and robustness of RNA viral quasispecies. Gene 45. Klenk, H.D., R. Wagner, D. Heuer, and T. Wolff. 347:273–282. 2002. Importance of hemagglutinin glycosylation 56. Matrosovich, M., N. Zhou, Y. Kawaoka, and for the biological functions of infl uenza virus. R. Webster. 1999. The surface glycoproteins of Virus Research 82:73–75. H5 infl uenza viruses isolated from humans, 46. Kwon, Y.K., H.W. Sung, S.J. Joh, Y.J. Lee, M.C. chickens, and wild aquatic birds have distinguish- Kim, J.G. Choi, E.K. Lee, S.H. Wee, and J.H. able properties. Journal of Virology 73:1146– Kim. 2005. An outbreak of highly pathogenic 1155. avian infl uenza subtype H5N1 in broiler breeders, 57. Narayan, O., G. Lang, and B.T. Rouse. 1969. A Korea. Journal of Veterinary Medical Science new infl uenza A virus infection in turkeys. V. 67:1193–1196. Pathology of the experimental disease by strain 47. Lamb, R.A., and R.M. Krug. 2001. Orthomyxo- turkey-Ontario 7732–66. Archiv Fur Die Gesamte viridae: The viruses and their replication. In: Virusforschung 26:166–182. 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 39

58. Noda, T., H. Sagara, A. Yen, A. Takada, H. Kida, plasmid-based reverse genetics system for infl u- R.H. Cheng, and Y. Kawaoka. 2006. Architecture enza A virus. Journal of Virology 70:4188– of ribonucleoprotein complexes in infl uenza A 4192. virus particles. Nature 439:490–492. 72. Puthavathana, P., P. Auewarakul, P.C. Charoeny- 59. Obenauer, J.C., J. Denson, P.K. Mehta, X. Su, S. ing, K. Sangsiriwut, P. Pooruk, K. Boonnak, R. Mukatira, D.B. Finkelstein, X. Xu, J. Wang, J. Khanyok, P. Thawachsupa, R. Kijphati, and P. Ma, Y. Fan, K.M. Rakestraw, R.G. Webster, E. Sawanpanyalert. 2005. Molecular characteriza- Hoffmann, S. Krauss, J. Zheng, Z. Zhang, and tion of the complete genome of human infl uenza C.W. Naeve. 2006. Large-scale sequence analysis H5N1 virus isolates from Thailand. Journal of of avian infl uenza isolates. Science 311:1576– General Virology 86(Pt 2):423–433. 1580. 73. Rott, R. 1997. [Infl uenza, a special form of zoo- 60. Orlich, M., H. Gottwald, and R. Rott. 1994. Non- nosis]. Berliner unc Munchener Tierarztliche homologous recombination between the hemag- Wochenschrift 110:241–246. glutinin gene and the nucleoprotein gene of an 74. Rott, R., H.D. Klenk, Y. Nagai, and M. Tashiro. infl uenza virus. Virology 204:462–465. 1995. Infl uenza viruses, cell enzymes, and patho- 61. Oxford, J.S., D.J. McGeoch, G.C. Schild, and genicity. American Journal of Respiratory and A.S. Beare. 1978. Analysis of virion RNA seg- Critical Care Medicine 152(Pt 2):S16–S19. ments and polypeptides of infl uenza A virus 75. Rott, R., M. Orlich, and C. Scholtissek. 1979. recombinants of defi ned virulence. Nature Correlation of pathogenicity and gene constella- 273:778–779. tion of infl uenza A viruses. III. Non-pathogenic 62. Palese, P. 2006. Making better infl uenza virus recombinants derived from highly pathogenic vaccines? Emerging Infectious Diseases 12:61–65. parent strains. Journal of General Virology 63. Perdue, M.L. 2007. H5N1 avian infl uenza: 44:471–477. responding to a zoonotic virus with pandemic 76. Salomon, R., J. Franks, E.A. Govorkova, N.A. potential. US Infectious Diseases, 1(March):23– Ilyushina, H.L. Yen, D.J. Hulse-Post, J. Humberd, 25. M. Trichet, J.E. Rehg, R.J. Webby, R.G. Webster, 64. Perdue, M.L. 1992. Naturally occurring NS gene and E. Hoffmann. 2006. The polymerase com- variants in an avian infl uenza virus isolate. Virus plex genes contribute to the high virulence of Research 23:223–240. the human H5N1 infl uenza virus isolate A/ 65. Perdue, M.L., M. Garcia, J. Beck, M. Brugh, and Vietnam/1203/04. Journal of Experimental Med- D.E. Swayne. 1996. An Arg-Lys insertion at the icine 203:689–697. hemagglutinin cleavage site of an H5N2 avian 77. Schulman, J.L., and P. Palese. 1977. Virulence infl uenza isolate. Virus Genes 12:77–84. factors of infl uenza A viruses: WSN virus neur- 66. Perdue, M.L., M. Garcia, D. Senne, and M. Fraire. aminidase required for plaque production in 1997. Virulence-associated sequence duplication MDBK cells. Journal of Virology 24:170–176. at the hemagglutinin cleavage site of avian infl u- 78. Senne, D.A., B. Panigrahy, Y. Kawaoka, J.E. enza viruses. Virus Research 49:173–186. Pearson, J. Suss, M. Lipkind, H. Kida, and R.G. 67. Perdue, M.L., J.W. Latimer, and J.M. Crawford. Webster. 1996. Survey of the hemagglutinin 1995. A novel carbohydrate addition site on the (HA) cleavage site sequence of H5 and H7 avian hemagglutinin protein of a highly pathogenic H7 infl uenza viruses: amino acid sequence at the HA subtype avian infl uenza virus. Virology 213(1): cleavage site as a marker of pathogenicity poten- 276–281. tial. Avian Diseases 40:425–437. 68. Perdue, M.L., and D.L. Suarez. 2000. Structural 79. Seo, S.H., E. Hoffmann, and R.G. Webster. 2002. features of the avian infl uenza virus hemaggluti- Lethal H5N1 infl uenza viruses escape host anti- nin that infl uence virulence. Veterinary Microbi- viral cytokine responses. Nature Medicine 8:950– ology 74:77–86. 954. 69. Perdue, M.L., and D.E. Swayne. 2005. Public 80. Seo, S.H., E. Hoffmann, and R.G. Webster. 2004. health risk from avian infl uenza viruses. Avian The NS1 gene of H5N1 infl uenza viruses circum- Diseases 49:317–327. vents the host anti-viral cytokine responses. Virus 70. Pinto, L.H., and R.A. Lamb. 1995. Understanding Research 103:107–113. the mechanism of action of the anti-infl uenza virus 81. Shinya, K., M. Hatta, S. Yamada, A. Takada, S. drug amantadine. Trends Microbiology 3:271. Watanabe, P. Halfmann, T. Horimoto, G. 71. Pleschka, S., R. Jaskunas, O.G. Engelhardt, T. Neumann, J.H. Kim, W. Lim, Y. Guan, M. Peiris, Zurcher, P. Palese, and A. Garcia-Sastre. 1996. A M. Kiso, T. Suzuki, Y. Suzuki, and Y. Kawaoka. 40 Avian Influenza

2005. Characterization of a human H5N1 infl u- 91. Swayne, D.E., and M. Pantin-Jackwood. 2006. enza A virus isolated in 2003. Journal of Virology Pathogenicity of avian infl uenza viruses in poultry. 79:9926–9932. Developmental Biology (Basel) 124:61–67. 82. Shortridge, K.F., N.N. Zhou, Y. Guan, P. Gao, T. 92. Treanor, J.J., E.L. Tierney, W.T. London, and Ito, Y. Kawaoka, S. Kodihalli, S. Krauss, D. B.R. Murphy. 1991. Characterization of the atten- Markwell, K.G. Murti, M. Norwood, D. Senne, uating M and NP gene segments of the avian L. Sims, A. Takada, and R.G. Webster. 1998. infl uenza A/mallard/78 virus during in vitro pro- Characterization of avian H5N1 infl uenza viruses duction of avian-human reassortant vaccine from poultry in Hong Kong. Virology 252:331– viruses and after replication in humans and pri- 342. mates. Vaccine 9:495–501. 83. Skehel, J.J., T. Bizebard, P.A. Bullough, F.M. 93. Tumpey, T.M., C.F. Basler, P.V. Aguilar, H. Hughson, M. Knossow, D.A. Steinhauer, S.A. Zeng, A. Solorzano, D.E. Swayne, N.J. Cox, J.M. Wharton, and D.C. Wiley. 1995. Membrane Katz, J.K. Taubenberger, P. Palese, and A. fusion by infl uenza hemagglutinin. Cold Spring Garcia-Sastre. 2005. Characterization of the Harbor Symposium on Quantitative Biology reconstructed 1918 Spanish infl uenza pandemic 60:573–580. virus. Science 310:77–80. 84. Skehel, J.J., K. Cross, D. Steinhauer, and D.C. 94. Tumpey, T.M., D.L. Suarez, L.E. Perkins, D.A. Wiley. 2001. Infl uenza fusion peptides. Bioche- Senne, J.G. Lee, Y.J. Lee, I.P. Mo, H.W. Sung, mical Society Transactions 29(Pt 4):623–626. and D.E. Swayne. 2002. Characterization of a 85. Smith, G.J., T.S. Naipospos, T.D. Nguyen, J. de, highly pathogenic H5N1 avian infl uenza A virus D. Vijaykrishna, T.B. Usman, S.S. Hassan, T.V. isolated from duck meat. Journal of Virology Nguyen, T.V. Dao, N.A. Bui, Y.H. Leung, C.L. 76:6344–6355. Cheung, J.M. Rayner, J.X. Zhang, L.J. Zhang, 95. Tweed, S.A., D.M. Skowronski, S.T. David, A. L.L. Poon, K.S. Li, V.C. Nguyen, T.T. Hien, J. Larder, M. Petric, W. Lees, Y. Li, J. Katz, M. Farrar, R.G. Webster, H. Chen, J.S. Peiris, and Y. Krajden, R. Tellier, C. Halpert, M. Hirst, C. Guan. 2006. Evolution and adaptation of H5N1 Astell, D. Lawrence, and A. Mak. 2004. Human infl uenza virus in avian and human hosts in Indo- illness from avian infl uenza H7N3, British nesia and Vietnam. Virology 350:258–268. Columbia. Emerging Infectious Diseases 86. Steinhauer, D.A., E. Domingo, and J.J. Holland. 10:2196–2199. 1992. Lack of evidence for proofreading mecha- 96. Vaccaro, L., K.J. Cross, J. Kleinjung, S.K. Straus, nisms associated with an RNA virus polymerase. D.J. Thomas, S.A. Wharton, J.J. Skehel, and F. Gene 122:281–288. Fraternali. 2005. Plasticity of infl uenza haemag- 87. Suarez, D.L., M.L. Perdue, N. Cox, T. Rowe, C. glutinin fusion peptides and their interaction with Bender, J. Huang, and D.E. Swayne. 1998. Com- lipid bilayers. Biophysical Journal 88:25–36. parisons of highly virulent H5N1 infl uenza A 97. Wainright, P.O., M.L. Perdue, M. Brugh, and viruses isolated from humans and chickens from C.W. Beard. 1991. Amantadine resistance among Hong Kong. Journal of Virology 72:6678–6688. hemagglutinin subtype 5 strains of avian infl u- 88. Suarez, D.L., D.A. Senne, J. Banks, I.H. Brown, enza virus. Avian Diseases 35:31–39. S.C. Essen, C.W. Lee, R.J. Manvell, C. Mathieu- 98. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. Benson, V. Moreno, J.C. Pedersen, B. Panigrahy, Chambers, and Y. Kawaoka. 1992. Evolution and H. Rojas, E. Spackman, and D.J. Alexander. ecology of infl uenza A viruses. Microbiological 2004. Recombination resulting in virulence shift Reviews 56:152–179. in avian infl uenza outbreak, Chile. Emerging 99. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. Infectious Diseases 10:693–699. Chambers, and Y. Kawaoka. 1992. Evolution and 89. Subbarao, K., and J. Katz. 2000. Avian infl uenza ecology of infl uenza A viruses. Microbiological viruses infecting humans. Cellular and Molecular Reviews 56:152–179. Life Sciences 57:1770–1784. 100. Webster, R.G., Y. Guan, L. Poon, S. Krauss, R. 90. Subbarao, K., A. Klimov, J. Katz, H. Regnery, Webby, E. Govorkovai, and M. Peiris. 2005. The W. Lim, H. Hall, M. Perdue, D. Swayne, C. spread of the H5N1 bird fl u epidemic in Asia in Bender, J. Huang, M. Hemphill, T. Rowe, M. 2004. Archives of Virology Supplementum:117– Shaw, X. Xu, K. Fukuda, and N. Cox. 1998. 129. Characterization of an avian infl uenza A (H5N1) 101. Webster, R.G. and D.J. Hulse. 2004. Microbial virus isolated from a child with a fatal respiratory adaptation and change: avian infl uenza. Revue illness. Science 279:393–396. Scientifi que et Technique 23:453–465. 2 / Molecular Determinants of Pathogenicity for Avian Influenza Viruses 41

102. Webster, R.G., Y. Kawaoka, and W.J. Bean, Jr. Norfolk, England. Archives of Virology 134:185– 1986. Molecular changes in A/chicken/Pennsyl- 194. vania/83 (H5N2) infl uenza virus associated with 106. Wood, G.W., J.W. McCauley, J.B. Bashiruddin, acquisition of virulence. Virology 149:165–173. and D.J. Alexander. 1993. Deduced amino acid 103. Webster, R.G., M. Peiris, H. Chen, and Y. Guan. sequences at the haemagglutinin cleavage site of 2006. H5N1 outbreaks and enzootic infl uenza. avian infl uenza A viruses of H5 and H7 subtypes. Emerging Infectious Diseases 12:3–8. Archives of Virology 130:209–217. 104. Wiley, D.C., and J.J. Skehel. 1987. The structure 107. Wright, P.F., and R.G. Webster. 2001. Ortho- and function of the hemagglutinin membrane gly- myxoviruses. In: D.M. Knipe and P.M. Howley coprotein of infl uenza virus. Annual Review of (eds.). Fields Virology, 4th ed. Lippincott Wil- Biochemistry 56:365–394. liams and Wilkins: Philadelphia, pp. 1533–1579. 105. Wood, G.W., J. Banks, J.W. McCauley, and D.J. 108. Zhou, H., M. Jin, H. Chen, Q. Huag, and Z. Yu. Alexander. 1994. Deduced amino acid sequences 2006. Genome-sequence analysis of the patho- of the haemagglutinin of H5N1 avian infl uenza genic H5N1 avian infl uenza A virus isolated in virus isolates from an outbreak in turkeys in China in 2004. Virus Genes 32:85–95. 3 Ecology of Avian Influenza in Wild Birds

David E. Stallknecht and Justin D. Brown

INTRODUCTION free-living birds were frequently exposed to these The emergence of highly pathogenic avian infl uenza viruses but also demonstrated that this potential res- (HPAI) H5N1 in Asia and the subsequent spillover ervoir involved a wide diversity of wild avian species into wild bird populations have brought unprece- (8, 25, 60, 123). This was confi rmed by the isolation dented attention to the epidemiology of avian infl u- of AI viruses from wedge-tailed shearwaters (Puffi - enza (AI) viruses in wild birds. This is a unique nus pacifi cus) in Australia (23) and from ducks in situation, but there is an extensive body of knowl- California (98). Since 1970, there were more than edge related to AI viruses in wild birds that may 50 published reports of AI viruses from free living provide direction for understanding the potential avian species in Australia, Africa, Asia, Europe, and ramifi cations associated with these events. The North America (73, 104), and to date, with the fi rst primary goal of this chapter is to review our current isolation from a wild bird in South America (102), understanding of the complex natural history of it is recognized that AI viruses have a global distri- these viruses in free-living birds. It is our hope that bution within free-living avian populations. such a review will not only identify additional research needs associated with the distribution, HOST RANGE maintenance, and transmission of AI viruses in free- Several reviews provide detailed information on the living avian reservoirs but also provide a base of host range of AI viruses (37, 73, 104); species from information to understand potential risks associated which AI viruses have been isolated are shown in with introductions of “new” AI viruses or HPAI Table 3.1. viruses into these populations. Overall, AI viruses have been reported from free- living birds representing more than 100 species in HISTORY OF AVIAN INFLUENZA VIRUSES 12 avian orders and all of the known HA (H1–H16) IN WILD BIRDS and NA (N1–N9) subtypes of AI viruses have been The fi rst isolation of AI virus from a free-living bird isolated from wild birds (44, 45, 73, 111). As a was made from a common tern (Sterna hirundo) in general trend, most of the species from which AI South Africa during 1961 (12). Prior to the detection viruses have been isolated are associated with of the Asian strains of H5N1 HPAI in wild birds, aquatic habitats and most species are in two avian A/tern/South Africa/61 (H5N3) represented the only orders, the Anseriformes (ducks, geese, and swans) HPAI virus ever isolated from free-living wild birds. and the Charadriiformes (gulls, terns, and shore- This event established that wild birds could be birds). Species within Anseriformes and Charadri- infected with type A infl uenza viruses and was fol- iformes are diverse, and the distribution of reported lowed from 1968 to 1972 by the detection of AI AI viruses within these groups is associated with virus antibodies in 21 species of free-living birds. specifi c families and, in some cases, individual These serological results not only confi rmed that species. Within Anseriformes, most AI virus

Avian Influenza Edited by David E. Swayne 43 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 Table 3.1. Free-living species from which avian inﬂ uenza viruses have been isolated. Taxonomic group Species References

Anseriformes, American black duck (Anas rubripes), American wigeon 1, 6, 9, 10, 13, 19, Anatidae (Anas americana), Australian shelduck (Tadorna 21, 27, 31, 32, Anatinae tadornoides), Blue-winged teal (Anas discors), 36, 38, 39, 42, Bufﬂ ehead (Bucephala albeola), Canvasback (Aytha 44, 45, 46, 47, valisineria), Cinnamon teal (Anas cyanoptera), Eurasian 50, 52, 53, 56, wigeon (Anas penelope), Falcated teal (Anas falcata), 57, 62, 66, 70, Gadwall (Anas strepera), Garganey (Anas querquedula), 72, 74, 76, 77, Common teal (Anas crecca), Long-tailed duck (Clangula 81, 83, 83, 86, hyemalis), Mallard (Anas platyrhynchos), Mottled duck 87, 95, 97, 98, (Anas fulvigula), Northern pintail (Anas acuta), Northern 99, 100, 105, 108, shoveler (Anas clypeata), Paciﬁ c black duck (Anas 109, 111, 112, uperciliosa), Redhead (Aythya americana), Ring-necked 114, 115, 118, duck (Athya collaris), Ruddy duck (Oxyura 120, 125 jamaicensis), Spot-billed duck (Anas poecilorhyncha), Sunda teal (Anas gibberifrons), Tufted duck (Aythya fuligula), White-winged scoter (Melanitta fusca), Wood duck (Aix sponsa), Yellow-billed duck Anseriformes Brent goose (Branta bernicla), Canada goose (Branta 13, 27, 31, 47, 54, Anatidae canadensis), Egyptian goose (Alopochen aegyptiacus), 74, 75, 81, 85, Anserinae Graylag goose (Anser anser), Greater white-fronted 95, 97, 111, 114 goose (Anser albifrons), Mute swan (Cygnus olor), Tundra swan (Cygnus columbianus) Anseriformes Common shelduck (Tadorna tadorna), South African 35, 36, 81 Anatidae shelduck (Tadorna cana) Tadorinae Charadriiformes Dunlin (Calidris alpina), Eurasian woodcock (Scolopax 38, 49, 53, 55, 67, Scolopacidae rusticola), Least sandpiper (Calidris minutilla), Red knot 896, 89, 127 (Calidris canutus), Ruddy turnstone (Arenaria interpres), Sanderling (Calidris alba), Semipalmated sandpiper (Calidris pusilla), Spur-winged lapwing (Vanellus spinosus), Temmick’s stint (Calidris temminckii) Charadriiformes Arctic tern (Sterna paradisaea), Black-headed gull (Larus 12, 16, 27, 31, 45, Laridae ridibundus), Black-tailed gull (Larus crassirostris), 64, 66, 72, 86, Common tern (Sterna hirundo), Franklin’s gull (Larus 95, 114, 127 pipixcan), Great black-backed gull (Larus marinus), Herring gull (Larus argentatus), Laughing gull (Larus atricilla), Lesser noddy (Anous tenuirostris), Ring-billed gull (Larus delawarensis), Sandwich tern (Sterna sandvicensis), Slender-billed gull (Larus genei), Sooty tern (Sterna fuscata), White-winged tern (Chlidonias leucoptera) Charadriiformes Common murre (Uria aalge), Guillemot (Cepphus spp.) 27, 91 Alcidae Ciconiiformes Glossy ibis (Plegadis falcinellus), Gray heron (Ardea 52, 81, 86, 87 cinerea), Hadeda ibis (Bostrychia hagedash), Squacco heron (Ardeola ralloides)

44 3 / Ecology of Avian Influenza in Wild Birds 45

Table 3.1. Continued Taxonomic group Species References

Columbiformes Collard dove (Streptopelia decaocto) 82 Galliformes Ring-necked pheasant (Phasianus colchicus) Rock 62, 83 partridge (Alectoris graeca) Gaviiformes Arctic loon (Gavia arctica), Red-throated loon (Gavia 52, 127 stellata) Gruiformes American coot (Fulica americana), Eurasian coot (Fulica 13, 62, 66, 77, 83, atra) 97, 111 Passeriformes American redstart (Setophaga ruticilla), Barn swallow 7, 13, 51, 53, 61, 87 (Hirundo rustica), Black-faced bunting (Emberiza spodocephala), Carrion crow (Corvus corone), Common jackdaw (Corvus monedula), Common redstart (Phoenicurus phoenicurus), Common whitethroat (Sylvia communis), Dark-eyed junco (Junco hyemalis), European starling (Sturnus vulgaris), Garden warbler (Sylvia borin), Hermit thrush (Catharus guttatus), House sparrow (Passer domesticus), Icterine warbler (Hippolais icterina), Purple fi nch (Carpodacus purpureus), Red- backed shrike (Lanius collurio), Song sparrow (Melospiza melodia), Spotted fl ycatcher (Musicapa striata), Swainson’s thrush (Catharus ustulatus), Tennessee warbler (Vermivora peregrina), Willow fl ycatcher (Empidonax traillii), Willow warbler (Phylloscopus trochilus), Yellow vented bulbul (Pycnonotus goiaver personatus), Yellow wagtail (Motacilla fl ava), Yellow warbler (Dendroica petechia), Yellow-breasted bunting (Emberiza aureola), Yellow- rumped warbler (Dendroica coronata), Yellow-throated warbler (Dendroica dominica) Pelecaniformes Great cormorant (Phalacrocorax carbo) 52, 111 Piciformes Great-spotted woodpecker (Dendrocopos major) 86 Podicipediformes Pied-billed grebe (Podilymbus podiceps) 13 Procellariiformes Wedge-tailed shearwater (Puffi nus pacifi cus) 23, 24, 66

isolations have been reported from the subfamily orders Ciconiiformes, Gaviiformes, Gruiformes, Anatinae (dabbling and diving ducks), and more Pelecaniformes, Podicipediformes, and Procellari- isolations of AI viruses have been reported from iformes. Collectively, these include relatively few mallard than any other species. Within the Chardri- positive species (Table 3.1). Although there are iformes, AI viruses have been isolated from three reports of AI viruses from species in the orders families but most have been associated with species Columbiformes, Piciformes, and Passeriformes that in the Scolopacidae (sandpipers and turnstones) and are commonly associated with terrestrial habitats, Laridae (gulls and terns). there are few reports of isolations, and most isola- Isolations have been reported from other species tion attempts from species within these groups have that utilize aquatic habitats, including birds in the proved unsuccessful (21, 45, 72). 46 Avian Influenza

HOST SUSCEPTIBILITY and H16), that gulls (Laridae) represent a separate Host range represents the combined effects of AI AI virus reservoir for at least some virus subtypes. virus contact, related to the behavior and population This may not be the case with other AI virus sub- dynamics, and species susceptibility. There are rela- types or with the Scolopacidae, where molecular tively few studies that have investigated host sus- studies involving numerous AI virus genes have not ceptibility or response to LPAI AI virus. In general, shown genetic differences between shorebird and it has been demonstrated that it is possible to infect duck AI viruses (101, 122). Another consideration a broad diversity of taxonomic groups with AI associated with this group relates to the very low viruses but species-related differences exist for indi- prevalence of infection that has been documented vidual AI virus strains (3, 4, 48, 55, 96, 124). This globally from species in this family (73). Most AI is supported by numerous reported AI virus isola- virus isolations worldwide have been associated tions from species in taxonomic groups, such as with one species (ruddy turnstones) at one site (Del- Passeriformes, Psittaciformes, and Galliformes, that aware Bay) at one time of year (May/June). Based are not normally associated with infection under on these isolation results alone, it is diffi cult to deter- natural conditions (2, 3, 93). A similar situation has mine if this group (Scolopacidae), or even the ruddy been reported with H5N1 HPAI under experimental turnstone, represents signifi cant contributors to the conditions (78–80). The recognition that these overall AI virus reservoir or simply represent local- viruses have the potential to infect diverse avian ized spillover hosts for viruses that are maintained species is important from the standpoint of prevent- in duck and gulls. ing introduction into domestic animal populations, Are there other avian reservoirs outside of the especially when considering that potential transmis- Anseriformes and Charadriiformes? This is a recur- sion may not directly involve a species that repre- ring question that deserves additional study. sents an AI virus reservoir under natural Although there is little indication that unique AI conditions. virus subtypes are maintained outside of the recognized Anseriformes and Charadriiformes reservoirs, WILD BIRD RESERVOIRS it should be understood that of the more than 8,600 A reservoir is defi ned as “any animate or inanimate unique avian species, relatively few have been tested object or any combination of these serving as a for AI viruses. In addition, there are some AI virus habitat of a pathogen that reproduces itself in such subtypes that appear to be underrepresented in duck, a way as to be transmitted to a susceptible host” gull, and shorebird populations tested to date (58, (113). With AI viruses and wild birds, it may be 94). Defi ning AI virus reservoirs and understanding inappropriate to assign reservoir status to individual the potential interactive species and population con- species, and virus isolation should not be used solely tributions within these reservoirs are probably the as evidence of an AI virus reservoir. The collective most important components of defi ning transmission information on AI viruses in wild birds clearly iden- risks and in determining the potential for introduced tifi es two broad taxonomic groups that represent viruses to persist and move within wild avian overlapping and in some cases unique AI virus res- populations. ervoirs, the Anseriformes and the Charadriiformes. However, this is not to say that all species within SUBTYPE AND GENETIC DIVERSITY IN these groups contribute equally to maintaining these WILD AVIAN POPULATIONS viruses. For example, although mallards are recog- Avian infl uenza virus subtypes are not equally rep- nized as an important component of the wild duck resented among wild bird populations and variation AI virus reservoir worldwide, this species often can occur between hosts, locations, and years. Most cohabits with other duck species. These species AI viruses isolated from ducks are represented by assemblages may be important to AI virus mainte- viruses in the H3, H4, and H6 subtypes (58, 94, 105) nance and probably more realistically defi ne the but the H11 subtype also can be common (77, 99, reservoir. Within the Charadriiformes, most AI 105). Viruses representing the H5, H7, H8, and H9 viruses have been isolated from species in two fam- are generally reported at low prevalence rates from ilies, the Scolopacidae and Laridae. In this case, ducks (58, 104), but these virus subtypes can be there is evidence, based on unique subtypes (H13 more common at specifi c locations or years (38, 71). 3 / Ecology of Avian Influenza in Wild Birds 47

Subtype diversity varies between years, and the pre- ing on juvenile birds during this time period. The dominant subtypes (H3, H4, and H6) are reported to temporal patterns observed in ducks correspond to follow a 2-year cycle (58). consistent spatial patterns. In general, AI virus prev- In shorebirds and gulls, subtype diversity is not alence in North America is highest in waterfowl as well understood, but differences exist between the staging areas in Canada and the northern United predominant AI viruses observed in Charadriiformes States. During migration, prevalence rapidly de- species compared to ducks (55, 58). This is espe- creases, and on wintering areas AI virus prevalence cially true with the H13 and possibly the H16 sub- often is lower than 1% to 2% (104); low AI virus types, which appear to be gull associated (73). To prevalence estimates have also been reported at this date, nine subtypes of AI viruses have been shown time in ducks in Europe (27, 73). to occur more often in Chardriiformes than in ducks, including the H5, H7, and H9 AI viruses (58). Shorebirds However, the prevalence of H5 and H7 viruses It has been reported that peak AI virus prevalence reported from Charadriiformes, like Anseriformes, in Charadriiformes occurs in the spring, with a lesser is generally low. It is important to understand that peak occurring in the fall (55). This seasonality, existing data on subtype diversity in shorebirds are however, is largely based on observations from limited in scope, with most isolates recovered from ruddy turnstones at Delaware Bay, and to date, ruddy turnstone at Delaware Bay in the United strong seasonal peaks in AI virus prevalence have States. not been detected in other species in this order. From genetic studies of AI viruses isolated from Spatial patterns, as with temporal patterns, in Char- wild birds, it has been shown that free-living wild adriiformes are more difﬁ cult to understand, but a birds maintain a diverse genetic assortment of strong spatial relationship has been observed with viruses. Although it was initially viewed that AI ruddy turnstones during spring migration stopovers viruses within wild bird populations were in a state at Delaware Bay (37, 55, 58). This is the only site, of evolutionary stasis (29), more recent studies have worldwide, where consistent AI virus isolations demonstrated a much more complicated picture with from shorebirds have been reported, and in general, a high degree of genetic diversity and the simultane- reported prevalence rates from these species, outside ous circulation of multiple lineages of a given of Delaware Bay, are either very low or negative subtype even within the same host population (101). (27, 37, 103). These viruses freely reassort within the wild bird reservoir (40), and genetic studies also have clearly MECHANISMS FOR AVIAN INFLUENZA demonstrated distinct North American and Eurasian VIRUS MAINTENANCE AND avian lineages (22, 28, 92, 101). These broad-scale TRANSMISSION geographic differences suggest some global isolation, but infrequent mixing of Eurasian and North The Transmission Cycle American viruses does occur (68,101) as would be Transmission of AI viruses in wild bird populations expected with the migratory behavior of many of the is dependent on a fecal/oral route (43, 90, 95). In species that can be infected with these viruses. ducks, replication occurs primarily in the intestinal tract (97) with high concentrations of infectious SPATIAL AND TEMPORAL VARIATION IN virus shed in feces (45, 119). It has been reported AVIAN INFLUENZA VIRUS INFECTION that experimentally infected Muscovy ducks (Cairina moschata) can shed an estimated 1 × 1010

Ducks mean embryo infectious doses (EID50) of AI viruses The prevalence of AI viruses in duck populations within a 24-hour period (119). Prolonged viral shed- peaks in late summer/early fall in North America ding also has been demonstrated; Hinshaw et al. (44) and is associated with concentration of susceptible reported that infected Pekin ducks were capable of hatching-year birds during premigration staging shedding AI viruses for more than 28 days. Avian (46). At this time, AI virus infection rates can exceed inﬂ uenza viruses also have been isolated from 30% in this age group. For this reason, AI virus surface water in Canada (44), Minnesota (33), and surveillance can be greatly enhanced by concentrat- Alaska (54), and with regard to viral transmission 48 Avian Influenza from wild to domestic fowl, contaminated surface and ground water both have been suggested as long- and short-term sources of AI viruses for domestic turkeys (34).

Environmental Persistence Despite the recognized importance of fecal/oral transmission of these viruses in wild bird populations, existing data on AI virus persistence in water are extremely limited. The subject of environmental persistence of AI viruses was initially investigated by Webster et al. (119) using A/duck/Memphis/546/74 H3N2 in both fecal material and nonchlorinated 6.8 river water. An initial dose of 10 EID50 (feces) and 8.1 10 EID50 (water) remained infective for at least 32 days. Subsequent to this work, AI virus persistence was evaluated in feces (11, 65) and allantoic fl uid (65). Other than the original work by Webster et al. (119), only three studies (14, 106, 107) have evaluated the persistence of wild-type AI viruses in water. These studies demonstrated that wild-type AI viruses can persist for extended periods of time in water at 4º C, 17º C, and 28º C and that water temperature, pH, and salinity, within ranges normally encoun- Figure 3.1. Proposed avian infl uenza virus maintenance cycle in wild duck populations tered in the fi eld, can greatly affect AI virus persis- may involve multiple species and different tence. Because individual viruses vary in their species assemblages on breeding and response to these variables, it is possible that the wintering habitats. physical and chemical attributes of the water associated with wild bird habitats may infl uence transmission dynamics. To date, information on AI virus persistence is restricted to relatively few viruses rep- the isolation of AI viruses from resident mottled resenting the H3, H4, H5, H6, H7, H10, and H12 ducks in North America (105) and the detection of (14, 106), and many of the physical and environ- seroconversion in ducks wintering in Italy (19). A mental factors that may potentially limit persistence potentially interesting and poorly understood portion in water have not been investigated. of this proposed cycle relates to individual species contributions which may be related to migratory The Avian Infl uenza Virus Maintenance Cycle behavior. This has been suggested for blue-winged It is currently unclear how these viruses are main- teal, which are early migrants that are not present in tained in nature, but existing evidence from ducks northern areas when AI virus prevalence rates peak supports a maintenance cycle based on the combined in other duck species (105), and for their ecological effects of continual bird-to-bird transmission and equivalent, the garganey teal in Europe and Africa environmental persistence. A representation of the (20). This migratory behavior may provide a suscep- proposed AI virus maintenance cycle is shown on tible population for virus maintenance on wintering Figure 3.1. grounds. AI viruses have been detected in this and This proposed cycle is based on AI virus trans- other duck species into the late winter and early mission on both the breeding and wintering areas. spring (39), indicating that AI viruses are present in Transmission on breeding and staging areas is well duck populations prior to spring migration. A very established, as is the movement of AI viruses south- low prevalence of AI viruses (0.3%) also has been ward during fall migration. The transmission of AI reported from ducks on breeding grounds in April viruses on wintering areas has been documented by (94). This same multispecies relationship may exist 3 / Ecology of Avian Influenza in Wild Birds 49 with ducks, gulls, and shorebirds, which share sus- result in the introduction of “novel” viruses into ceptibility to some AI viruses (55); each group may wildlife populations. play a related but different role in the movement and In viewing the wild bird species from which maintenance of these viruses (58). H5N1 HPAI viruses have been isolated, it is inter- As for environmental persistence, the isolation of esting that most of these species fall into two catego- these viruses from unconcentrated lake water and ries. The fi rst involves aquatic birds, and this group the demonstration of very long-term persistence of is predominated by members of the Anseriformes, AI viruses in water under experimental conditions which is consistent with the known natural history provide the basis for this hypothesis. Although it is of AI viruses. The second group includes raptors and possible that these viruses are maintained in water- other species (e.g., crows), which potentially either fowl breeding habitats from year to year, it is more predate or scavenge other birds (wild or domestic). probable that environmental persistence serves to This is not known to occur with naturally occurring increase seasonal transmission by providing a resid- AI viruses in wild birds, but this possibility has not ual source of virus following the departure of been adequately investigated. Infection in this latter infected birds from the immediate habitat. This group probably is related to increased exposure to potential contributory role in AI virus maintenance the H5N1 HPAI viruses from contact with domestic requires additional study and may be important in and wild bird morbidity and mortality. defi ning how viruses move between migratory and Another general trend that may be worth noting locally mobile species and populations that may is the differences in wild bird mortality (species have limited direct contact. diversity) observed in Asia and Europe. Avian species affected in Asia are much more taxonomi- THE EMERGENCE OF H5N1 HPAI IN WILD cally diverse, which probably relates to increased BIRDS exposure to infected domestic birds. In Europe, most mortality (and H5N1 HPAI virus isolations) was Natural Infections associated with Anseriformes and with certain Prior to 2002, there was only a single report of an species such as the mute swan and tufted duck, HPAI virus isolation from free-living wild birds that which were overrepresented in mortality reports were not associated with infected domestic fowl. (88). This virus (an H5N3) infected common terns in South Africa in 1961 (12) but did not persist in this Experimental Infections species or population. The host range for HPAI There have been numerous experimental infections viruses in wild birds has recently increased as a of wild bird species with H5N1 HPAI (14, 78–80, result of the H5N1 HPAI outbreak in Eurasia. The 110). Results from these studies consistently have fi rst indication of potential wild bird involvement in shown that viral shedding is primarily associated this outbreak occurred during 2002 and 2003 when with the respiratory rather than the alimentary tract Asian lineages of H5N1 HPAI viruses were isolated infections. Clinical response for a given species is from both captive and free living birds in Hong related to the lineage of the H5N1 HPAI virus. This Kong (26). Isolations of H5N1 HPAI viruses from was demonstrated with mallards where clinical wild birds have been reported since 2002 from more response varied from asymptomatic to fatal depend- than 50 species of wild birds in both Asia and eastern ing on viral stain (110). Variation in clinical response Europe (117), and it appears that these viruses may to different H5N1 HPAI strains also have been have been transported throughout Eurasia during observed in laughing gulls infected with a 1997 (79) wild bird migrations during 2005. Reports of Asian and later 2001 and 2005 isolates (14). In these H5N1 HPAI in free-living wild birds are shown in studies, no clinical disease was observed with the Table 3.2. 1997 isolate, whereas the 2001 and 2005 isolates These results refl ect a wide host range for the resulted in mortality. Species differences in suscep- Eurasian H5N1 HPAI viruses but offer little insight tibility also are apparent even between closely taxo- into potential wildlife reservoirs for these viruses. nomically related species. This was demonstrated by They do provide evidence of AI virus susceptibility Brown et al. (14) using fi ve species of North Amer- and an example of how anthropogenic activities can ican ducks (mallard, blue-winged teal, redhead, 50 Avian Influenza

Table 3.2. Free-living species from which highly pathogenic H5N1 viruses have been isolated (17, 26, 59, 63, 69, 117). Taxononomic Group Species

Anseriformes Bar-headed goose (Anser indicus), Common pochard (Aythya ferina), Gadwall, Greater white-fronted goose (Anser albicans), Greylag goose, Mute swan, Red-breasted goose (Branta ruﬁ collis), Ruddy shelduck (Tadorna ferruginea), Smew (Mergus albellus), Tufted duck, Whooper swan (Cygnus cygnus) Charadriiformes Black-headed gull, Brown-headed gull (Larus atricilla), Great black-backed gull, Green sandpiper (Tringa ochropus) Ciconiiformes Chinese pond heron (Ardeola bacchus), Grey heron, Little egret (Egretta garzetta), Open-billed stork (Anastomus oscitans), White stork (Ciconia ciconia) Columbiformes Red-collared dove (Streptopelia tranquebarica), Rock pigeon (Columba livia) Falconiformes Crested hawk-eagle (Spizaetus nipalensis), Eurasian buzzard (Buteo buteo), Northern Goshawk (Accipiter gentiles), Peregrine falcon (Falco peregrinus), Rough-legged buzzard (Buteo lagopus) Galliformes Kalij pheasant (Lophura leucomelanos), White Indian peafowl (Pavo cristatus) Gruiformes Brown crake (Amaurornis akool), Common moorhen (Gallinula chloropus), Eurasian coot, Purple swamphen (Porphyrio porohyrio) Passeriformes Black drongo (Dicrurus macrocercus), Crested Mynah (Acidotheres cristatellus), Eurasian tree sparrow (Passer domesitcus), House crow (Corvus splendens), Japanese white-eye (Zosterops japonicus), Jungle crow (Corvus macrorhynchos), Korean magpie (Pica pica, sericea), Oriental magpie robin (Copsychus saularis), Scaly-breasted munia (Oriolus chinensis), White- rumped munia (Lonchura stiata) Pelecaniformes Great cormorant, Little cormorant (Phalocrocorax niger) Podicipediformes Great crested grebe (Podicps cristatus), Little grebe (Tachybaptus ruﬁ collis)

wood duck, and Northern pintail). With the excep- viruses are established in wild bird populations. The tion of the wood duck, clinical disease was not proposed movement of H5N1 HPAI from southeast apparent; viral titers were generally very low and Asia to Europe involved the movement of the virus viral shedding was of limited duration. Observed with wild bird populations to China (May 2005) and viral titers appear to be related to clinical response Mongolia (August 2005), subsequent introduction with the highest titers associated with birds with the of H5N1 HPAI in wild birds in Siberia, and intro- most severe clinical response (14, 110). duction to Europe (Turkey) (October 2005) via fall migrants that presumably had contact with infected Maintenance and Transmission of H5N1 High birds in Siberia (88). Wild bird mortality was Pathogenicity Avian Inﬂ uenza Viruses in Wild detected along this proposed route, but the presence Bird Populations of these viruses in healthy birds was not documented. There is very strong ﬁ eld evidence that these viruses Because of this, it is unclear what species actually were transmitted over long distances by the migra- moved this virus, and it is possible that the move- tory movement of wild birds and that transmission ment within Europe primarily resulted from the occurred within wild bird populations. However, the weather-dependent movement of mute swans (88). risk factors associated with such transmission cur- Results of experimental studies of wild bird rently are not clear and there is little evidence to species provide an interesting perspective for support or refute the possibility that H5N1 HPAI viewing past events and extrapolating to the future. 3 / Ecology of Avian Influenza in Wild Birds 51

Based on the maintenance cycle for LPAI AI viruses, there are no realistic options for reducing AI virus three factors should control transmission effi ciency: prevalence in wild bird populations, prevention (1) signifi cant viral shedding both in terms of source, through decreased direct or environmental contact is viral titer, and duration; (2) environmental stability the primary defense and such prevention should be in water; and (3) a low infective dose. Based on centered on wild bird–domestic animal interface. experimental studies to date, viral shedding patterns Effective prevention can only be achieved with a for H5N1 HPAI in ducks appear to relatively inef- complete understanding of AI virus natural history fi cient compared with LPAI AI viruses. In general, and the existing connections between wild and unless clinical disease is evident, viral titers are low, domestic animals populations. they are of short duration, and viral shedding is Surveillance is the second critical component for associated with the oral cavity shedding following reducing potential domestic animal and public health respiratory tract infection rather than the cloaca impacts. The potential risk of AI virus contact from shedding from alimentary tract infections. Although wild birds is location, time, and species dependent. transmission of H5N1 HPAI has been demonstrated Understanding these risks will allow for rational between infected and contact mallards under penned allocation of prevention and education related conditions (110), a respiratory infection would prob- resources to clearly defi ned high-risk areas and pop- ably not result in the same environmental contami- ulations. The overall goal of any surveillance effort nation levels as fecal shedding. Transmission also should be to better defi ne the natural history of these may be restrained by decreased survival in the envi- viruses, and to effectively do this, much more infor- ronment, and it has been demonstrated experimen- mation on host range, transmission cycles, and tally that these viruses may not be as environmentally potential wild avian reservoirs is needed globally. fi t as wild-type H5 and H7 LPAI viruses (15). With such information it will not only be possible Although these viruses seem to fall short with regard to defi ne risks and develop appropriate preventive to viral shedding and environmental persistence, measures, but it will also provide a much needed these factors may be offset by transmission via a low perspective to anticipate the course of emerging AI infective dose. This has been demonstrated with virus problems related to domestic animal, wildlife, wood ducks, which can be infected with as little as and public health. 10 1.5 median egg infective doses of H5N1 HPAI Researchable questions related to AI viruses in virus (J.D. Brown, unpublished data). wild avian populations are too numerous to list. From an ornithological perspective, the role of RECOMMENDATIONS FOR LIVING WITH migration in AI virus movement and maintenance AVIAN INFLUENZA VIRUSES IN WILD has not received adequate attention; basic studies on BIRD POPULATIONS: PREVENTION, bird migration, especially related to temporal and SURVEILLANCE, AND RESEARCH spatial patterns, are needed. Wildlife surveillance Wild birds represent the historic source of all type has and continues to provide a wealth of wild-type A infl uenza viruses that infect domestic animals and AI viruses for genetic studies; the scope of such humans, and transmission of these viruses to domes- work needs to be increased to fully understand AI tic animals (especially poultry) is well established. virus genetic diversity and the extent of interspecifi c With regard to direct wild bird to human transmis- exchange. The epidemiology and ecology of these sion, there is circumstantial evidence that this can viruses are complex and require additional study; at occur in association with extensive contact with wild present, the host range of AI viruses is not complete, ducks (28) or through contact with birds (mute maintenance cycles and the potential for interconti- swans) that die as a result of H5N1 HPAI (121); nental movement are not completely understood, however, the risks associated with such contact cur- and potential impacts associated with introduced rently are undefi ned. Based on our current knowl- HPAI viruses in these populations are undefi ned. edge and the extent of close contact between humans Finally, additional research is needed to clearly and domestic as opposed to wild animals, infected defi ne risk factors associated with the movement of domestic animals probably represent a more likely these viruses between wild birds, domestic animals, source of human infection. This is consistent with and humans. These broad research questions are all the current situation with H5N1 HPAI. Because linked, and efforts to answer them will require a 52 Avian Influenza diversity of disciplines ranging from basic fi eld orni- wild bird–domestic animal interface. This can only thology and virology to applied disciplines in vet- be achieved with a complete understanding of AI erinary and public health. virus natural history and potential connections between wild and domestic animal populations. CONCLUSIONS There is an extensive body of knowledge related to REFERENCES the epidemiology of AI viruses in wild birds that 1. Abenes, G.B., K. Okazaki, H. Fukushi, H. Kida, may provide insight in understanding recent events E. Honda, K. Yagyu, M. Tsuji, H. Sato, E. Ono, associated with the spillover of H5N1 HPAI virus R. Yanagawa, and N. Yamauchi. 1982. Isolation into wild bird populations in Eurasia. Two primary of orthomyxoviruses and paramyxoviruses from feral birds in Hokkaido, Japan 1980 and 1981. avian reservoirs of AI viruses have been identifi ed Japanese Journal of Veterinary Science 44:703– including the Anseriformes (ducks, geese, and 708. swans) and the Charadriiformes (shorebirds and 2. Alexander, D.J., W.W. Allan, and J.W. Harkness. gulls). Subtype and genetic diversity of AI viruses 1974. Isolation of infl uenza virus from psitta- that circulate within these reservoirs is extensive and cines. Research in Veterinary Science 17:125– these viruses exist within these populations on a 127. global scale. The epidemiology of AI viruses in wild 3. Alexander, D.J., W.H. Allan, D. Parsons, and G. bird populations is best understood in ducks where Parsons. 1978. The pathogenicity of four avian predictable temporal and spatial patterns of infection infl uenza viruses for fowls, turkeys, and ducks. have been described. In ducks, transmission occurs Research in Veterinary Science 24:242–247. through a fecal/oral cycle involving contaminated 4. Alexander, D.J., G. Parsons, and R.J. Manvell. 1986. Experimental assessment of the pathoge- water and these viruses are adapted to persist in this nicity of eight avian infl uenza viruses of H5 medium for extended periods of time. The epidemi- subtype for chickens, turkeys, ducks, and quail. ology of AI viruses in Charadriiformes is less well Avian Pathology 15:647–662. understood and may primarily involve gulls (Laridae) 5. Alexander, D.J. 2000. A review of avian infl u- which have been infected with some unique AI virus enza in different bird species. Veterinary Micro- subtypes (i.e., H13 and H16). With shorebirds (Scol- biology 74:3–13. opacidae), a high prevalence of infection has only 6. Alfonso, C.P., B.S. Cowen, and H. Van Campen. been consistently reported from Delaware Bay, USA 1995. Infl uenza A viruses isolated from water- and the reservoir status of this group is unclear. fowl in two wildlife management areas of Penn- The emergence of H5N1 HPAI virus in wildlife sylvania. Journal of Wildlife Diseases was fi rst detected in 2002 in Asia. Subsequent to this 31:179–185. 7. Amin, A., M.A. Shalaby, and I.Z. Imam. 1980. event, H5N1 HPAI viruses have been detected in Studies on infl uenza virus isolated from migrat- numerous wild bird species, and during 2005–2006, ing birds in Egypt. Comparative Immunology the virus spread throughout Eurasia, presumably Microbiology and Infectious Diseases 3:241– through the movement of infected wild birds. 246. However, it is currently unknown if these viruses 8. Asplin, F.D. 1970. Examination of sera from can persist in wild bird populations or if such events wildfowl for antibodies against the viruses of represent temporary spillover events from infected duck plague, duck hepatitis, and duck infl uenza. domestic poultry. Veterinary Record 87:182–183. Wild birds are the reservoir for LPAI viruses and 9. Bahl, A.K., B.S. Pomeroy, B.C. Easterday, and S. these viruses represent the precursors to those infl u- Mamgundimedjo. 1975. Isolation of type A infl u- enza viruses, such as the H5N1 HPAI viruses, that enza viruses from migratory waterfowl along the Mississippi fl yway. Journal of Wildlife Diseases can represent signifi cant potential problems to both 11:360–363. domestic animal and human health. Because there 10. Bahl, A.K., B.S. Pomeroy, S. Mangundimedjo, are no realistic options for reducing AI virus preva- and B.C. Easterday. 1977. Isolation of type A lence in wild bird populations, prevention of AI infl uenza and Newcastle disease viruses from virus infection through decreased contact between migratory waterfowl in Mississippi fl yway. domestic and wild bird population is the primary Journal of the American Veterinary Medical defense and such prevention should be centered on Association 171:949–951. 3 / Ecology of Avian Influenza in Wild Birds 53

11. Beard, C.W., M. Brugh, and D. Johnson. 1984. 21. Deibel, R., D.E. Emord, W. Dukelow, V.S. Laboratory studies with the Pennsylvanian avian Hinshaw, and J.M. Wood. 1985. Infl uenza viruses infl uenza virus (H5N2). In: R.W. Bankowski and paramyxoviruses in ducks in the Atlantic (ed.). Proceedings of the First International Sym- Flyway, 1977–1983, including an H5N2 isolate posium on Avian Infl uenza, April 1981, Belts- related to the virulent chicken virus. Avian Dis- ville, Maryland. Symposium on Avian Infl uenza, eases 29:970–985. US Animal Health Association: Richmond, VA, 22. Donis, R.O., W.J. Bean, Y. Kawaoka, and R.G. pp. 462–473. Webster. 1989. Distinct lineages of infl uenza 12. Becker, W.B. 1966. Isolation and classifi cation of virus H4 hemagglutinin genes in different regions tern virus: infl uenza virus A/tern/South Africa/ of the world. Virology 169:408–417. 1961. Journal of Hygiene 64:309–320. 23. Downie, J.C., and W.G. Laver. 1973. Isolation of 13. Boudreault, A., J. Lecomte, and V.S. Hinshaw. a type A infl uenza virus from an Australian 1980. Antigenic characterization of infl uenza A pelagic bird. Virology 51:259–269. viruses isolated from avian species in Ontario, 24. Downie, J.C., V. Hinshaw, and W.G. Laver. Quebec and Maritimes during the 1977 season. 1977. Ecology of infl uenza: isolation of type A Revue Canadienne de Biologie 39:107–114. infl uenza viruses from Australian pelagic birds. 14. Brown, J.D., D.E. Stallknecht, J.R. Beck, D.L. Australian Journal of Experimental Biology and Suarez, and D.E. Swayne. 2006. Susceptibility of Medical Science 55:635–643. North American Ducks and Gulls to H5N1 highly 25. Easterday, B.C., D.O. Trainer, B. Tumova, and pathogenic avian infl uenza viruses. Emerging H.G. Pereira. 1968. Evidence of infection with Infectious Diseases 12(11):1663–1670. infl uenza viruses in migratory waterfowl. Nature 15. Brown, J.D., D.E. Swayne, R.J. Cooper, R.E. 219:523–524. Burnes, and D.E. Stallknecht. 2007. Persistence 26. Ellis, T.M., R.B. Bousfi eld, L.A. Bisset, K.C. of H5 and H7 infl uenza viruses in water. Avian Dyrting, G. Luk, S.T. Tsim. K. Sturm-Ramirez, Diseases 51(Suppl):285–289. R.G. Webster, Y. Guan, and J.S. Peiris. 2004. 16. Campbell, D.G. 1999. Gull mortality. Kitchener Investigation of outbreaks of highly pathogenic Wildlife Health Centre Newsletter 6:8–9. H5N1 avian infl uenza in waterfowl and wild birds 17. Chen, H., G.J.D. Smith., S.Y. Zhang, K. Oin, J. in Hong Kong in late 2002. Avian Pathology Wang, K.S. Li, R.G. Webster, J.S.M. Peris, and 33:492–505. Y. Guan. 2005. H5N1 outbreak in migratory 27. Fouchier, R.A.M., B. Olsen, S. Bestebroer, S. waterfowl. Nature 436(7048):191–192. Herfst, L. Van der Kemp, G.F. Rimmelzwaan, 18. Cross, G.M. 1987. The status of avian infl uenza and A.D.M.E. Osterhaus. 2003. Infl uenza A virus in poultry in Australia. In: B.C. Easterday and surveillance in wild birds in northern Europe in C.W. Beard (ed.). Proceedings of the Second 1999 and 2000. Avian Diseases 47(3 Suppl):857– International Symposium on Avian Infl uenza, 860. Georgia Center for Continuing Education, the 28. Gill J.S., R. Webby, M.J.R. Gilchrist, and G.C. University of Georgia, Athens, Georgia, USA, Gray. 2006. Avian infl uenza among waterfowl September 3–5, 1986. Symposium on Avian hunters and wildlife professionals. Emerging Infl uenza, US Animal Health Association: Rich- Infectious Diseases 12:1284–1286. mond, VA, pp. 96–103. 29. Gorman, O.T., W.J. Bean, Y. Kawaoka, and R.G. 19. De Marco, M.A., G.E. Foni, L. Campitelli, E. Webster. 1990. Evolution of the nucleoprotein Raffi ni, L. Di Trani, M. Delogu, V. Guberti, G. gene of infl uenza A virus. Journal of Virology Barigazzi, and W. Di Donatelli. 2003. Circulation 64:1487–1497. of infl uenza viruses in wild waterfowl wintering 30. Gorman, O.T., W.J. Bean, and R.G. Webster. in Italy during the 1993–99 period: Evidence of 1992. Evolutionary processes in infl uenza viruses: virus shedding and seroconversion in wild ducks. divergence, rapid evolution, and stasis. Current Avian Diseases 47(3 Suppl):861–866. Topics in Microbiology and Immunology 176:75– 20. De Marco, M.A., E. Foni, L. Campitelli, M. 97. Delogu, E. Raffi ni, C. Chiapponi, G. Barigazi, P. 31. Graves, I.L. 1992. Infl uenza viruses in birds of Cordioli, L. Di Trani, and I. Donatelli. 2005. the Atlantic fl yway. Avian Diseases 36:1–10. Infl uenza virus circulation in wild aquatic birds 32. Gresikova, M., B. Tumova, A. Stumpa, and M. in Italy during H5N2 and H7N1 poultry epidemic Sekeyova. 1978. Isolation of infl uenza virus from periods (1998 to 2000). Avian Pathology 34:480– wild ducks (Anas platyrhynchos). Acta Virolog- 485. ica 22:296–301. 54 Avian Influenza

33. Halvorson, D.A., D. Karunakaran, D. Senne, C. and genetic characterization of a novel hemag- Kelleher, C. Bailey, A. Abraham, V. Hinshaw, glutinin subtype of infl uenza A viruses from and J. Newman. 1983. Simultaneous monitoring gulls. Journal of Virology 42:865–872. of sentinel ducks and turkeys in Minnesota. Avian 46. Hinshaw, V.S., J.M. Wood, R.G. Webster, R. Diseases 27:77–85. Deibel, and B. Turner. 1985. Circulation of infl u- 34. Halvorson, D.A., C.J. Kelleher, and D.A. Senne. enza viruses and paramyxoviruses in waterfowl 1985. Epizootiology of avian infl uenza: effect of originating from two different areas of North season on incidence in sentinel ducks and domes- America. Bulletin of the World Health Organiza- tic turkeys in Minnesota. Applied and Environ- tion 63:711–719. mental Microbiology 49:914–919. 47. Hinshaw, V.S., V.F. Nettles, L.F. Schorr, J.M. 35. Hannoun, C. 1977. Isolation from birds of Wood, and R.G. Webster. 1986. Infl uenza virus infl uenza viruses with human neuraminidase. surveillance in waterfowl in Pennsylvania after Developments in Biological Standardization the H5N2 avian outbreak. Avian Diseases 39:469–472. 30:207–212. 36. Hannoun, C., and J.M. Devayx. 1980. Circulation 48. Homme, P.J., and B.C. Easterday. 1970. Avian of infl uenza viruses in the bay of the Somme infl uenza virus infections. IV. Response of pheas- River. Comparative Immunology Microbiology ants, ducks, and geese to infl uenza A/turkey/ and Infectious Diseases 3:177–183. Wisconsin/1966 virus. Avian Diseases 14:285– 37. Hanson, B.A. 2002. Temporal, spatial and species 290. patterns of avian infl uenza viruses among wild 49. Honda, E., H. Kida, R. Yanagawa, Y. Matsuura, birds. MS dissertation, The University of Georgia: K. Yagyu, M. Tsuji, K. Ueno, N. Yamauchi, S. Athens, Georgia. Mishima, H. Ogi, and K. Shimazaki. 1981. Survey 38. Hanson, B.A., D.E. Stallknecht, D.E. Swayne, of infl uenza viruses in feral birds in 1979 and L.A. Lewis, and D.A. Senne. 2003. Avian infl u- isolation of a strain possessing Hav6Nav5 from enza viruses in Minnesota ducks during 1998– cloaca of an Eastern Dunlin. Japanese Journal of 2000. Avian Diseases 47(3 Suppl):867–871. Veterinary Research 29:83–87. 39. Hanson, B.A., D.E. Swayne, D.A. Senne, D.S. 50. Hua, Y.-P., H.-L. Chai, S.-Y. Yang, X.-W. Zeng, Lobpries, J. Hurst, and D.E. Stallknecht. 2005. and Y. Sun. 2005. Primary survey of avian infl u- Avian infl uenza and paramyxoviruses in winter- enza virus and Newcastle disease virus infection ing and resident ducks in Texas. Journal of Wild- in wild birds in some areas of Heilongjiang Prov- life Diseases 41:624–629. ince, China. Journal of Veterinary Science 6:311– 40. Hatchette, T.F., D. Walker, C. Johnson, A. 315. Backer, S.P. Pryor, and R.G. Webster. 2004. 51. Ibrahim, H.M., I.P.R. Awamg, D.J. Alexander, Infl uenza A viruses in feral Canadian ducks; R.J. Manvell, I. Aini, and A.L. Ibrahim. 1990. extensive reassortment in nature. Journal of Isolations of infl uenza A viruses from passerine General Virology 85:2327–2337. birds in Malaysia. Veterinary Record 127:528– 41. Hinshaw, V.S., and R.G. Webster. 1982. The 528. natural history of infl uenza A viruses. In: A.S. 52. Iftimovici, R., V. Iacobescu, A.L. Petrescu, A. Beare (ed.). Basic and Applied Infl uenza Research. Mutiu, and M. Chelaru. 1980. Isolation of infl u- CRC Press: Boca Raton, FL, pp. 79–104. enza virus A/USSR 90/77 (H1N1) from wild 42. Hinshaw, V.S., R.G. Webster, and B. Turner. birds. Revue Roumaine De Médicine, Virologie 1978. Novel infl uenza A viruses isolated from 31:243. Canadian feral ducks: Including strains antigeni- 53. Isachenko, V.A., L.Y. Zakstelskaya, I.G. Roslaya, cally related to swine infl uenza (Hsw1N1) viruses. L.D. Odinok, E.V. Molibog, and D.K. Lvov. Journal of General Virology 41:115–127. 1974. Strains similar to Hong-Kong variant of 43. Hinshaw, V.S., R.G. Webster, and B. Turner. infl uenza virus isolated from synanthropic and 1979. Water-borne transmission of infl uenza A wild migrating birds (in Russian). Ivanovsky viruses. Intervirology 11:66–69. Institute of Virology 2:156–165. 44. Hinshaw, V.S., R.G. Webster, and B. Turner. 54. Ito, T., K. Okazaki, Y. Kawaoka, A. Takada, R.G. 1980. The perpetuation of orthomyxoviruses and Webster, and H. Kida. 1995. Perpetuation of paramyxoviruses in Canadian waterfowl. Cana- infl uenza A viruses in Alaskan waterfowl reser- dian Journal of Microbiology 26:622–629. voirs. Archives of Virology 140:1163–1172. 45. Hinshaw, V.S., G.M. Air, A.J. Gibbs, L. Graves, 55. Kawaoka, Y., T.M. Chambers, W.L. Sladen, and B. Prescott, and D. Karunakaran. 1982. Antigenic R.G. Webster. 1988. Is the gene pool of infl uenza 3 / Ecology of Avian Influenza in Wild Birds 55

viruses in shorebirds and gulls different from that chickens and their environments. Avian Diseases in wild ducks? Virology 163:247–250. 47(3 Suppl):1015–1021. 56. Kida, H., and R. Yanagawa. 1979. Isolation and 66. Mackenzie, J.S., E.C. Edwards, R.M. Holmes, characterization of infl uenza A viruses from and V.S. Hinshaw. 1984. Isolation of orthovi- wild free-fl ying ducks in Hokkaido, Japan. ruses and paramyxovirus from wild birds in Zentralblatt Fur Bakteriologie Mikrobiologie Western Australia and the characterization of Und Hygiene Series A: Medical Microbiology novel infl uenza A viruses. Australian Journal of Infectious Diseases Virology Parasitology 244: Experimental Biology and Medical Science 135–143. 62:89–99. 57. Kocan, A.A., V.S. Hinshaw, and G.A. Daubney. 67. Manjunath, L.H., and B.B. Mallick. 1981. Preva- 1980. Infl uenza A viruses isolated from migrating lence of myxo- and paramyxoviruses in birds of ducks in Oklahoma. Journal of Wildlife Diseases Himalayan region. Indian Journal of Animal Sci- 16:281–285. ences 51:1139–1143. 58. Krauss, S., D. Walker, S. Paul-Pryor, L. Niles, L. 68. Makarova, N.V., N.V. Kaverin, S. Krauss, D. Chenghong, V.S. Hinshaw, and R.G. Webster. Senne, and R.G. Webster. 1999. Transmission of 2004. Infl uenza A viruses in migrating wild Eurasian avian H2 infl uenza virus to shorebirds aquatic birds in North America. Vector-borne and in North America. Journal of General Virology Zoonotic Diseases 4:177–189. 80:3167–3171. 59. Kwon, Y.-K., S.-J. Joh, M.-C. Kim, Y.-J. Lee, 69. Mase, M., K. Tsukamoto, T. Imada, K. Imai, N. J.-G. Choi, E.-K. Lee, S.-H. Wee, H.-W. Sung, Tanimura, K. Nakamura, Y. Yamoto, T. Hitomi, J.-H. Kwon, M.-I. Kang, and J.-H. Kim. 2005. T. Kira, T, Nakai, M.Hrimoto, Y. Kawaoka, and Highly pathogenic avian infl uenza in magpies S. Yamaguchi. 2005. Characterization of H5N1 (Pica pica sericea) in South Korea. Journal of highly pathogenic infl uenza viruses isolated Wildlife Diseases 41:618–623. during the 2003–2004 infl uenza outbreak in 60. Laver, W.G., and R.G. Webster. 1972. Antibod- Japan. Virology 332:167–176. ies to human infl uenza virus neuraminidase (the 70. Mikami, T., M. Kawamura, T. Kondo, T. Murai, A/Asian/57 H2N2 strain) in sera from Australian M. Horiuchi, H. Kodama, H. Izawa, and H. Kida. pelagic birds. Bulletin World Health Organiza- 1987. Isolation of ortho- and paramyxoviruses tion 47:535–541. from migrating feral ducks in Japan. Veterinary 61. Lipkind, M.A., Y. Weisman, E. Shihmanter, and Record 120:417–418. D. Shoham. 1979. The fi rst isolation of animal 71. Munster, V.J., A. Wallensten, C. Baas, G. F. Rim- infl uenza virus in Israel. Veterinary Record melzaann, M. Schutten, B. Olsen, A.D.N.E. 105:510–511. Osterhaus, and R.A.M. Foucier. 2005. Mallards 62. Lipkind, M., Y. Weisman, E. Shihmanter, and D. and highly pathogenic avian infl uenza ancestral Shoham. 1981. Review of the three-year studies viruses, Northern Europe. Emerging Infectious on the ecology of avian infl uenza viruses in Israel. Diseases 11(10):1545–1551. In: R.W. Bankowski (ed.). Proceedings of the 72. Nettles, V.F., J.M. Wood, and R.G. Webster. First International Symposium on Avian Infl u- 1985. Wildlife surveillance associated with an enza, April 1981, Beltsville, Maryland. Sympo- outbreak of lethal H5N2 avian infl uenza in sium on Avian Infl uenza, US Animal Health domestic poultry. Avian Diseases 29:733–741. Association: Richmond, VA, pp. 69–78. 73. Olsen, B., V.J. Munster, A. Wallensten, J. Wal- 63. Liu, J., H. Xiao, F. Lei, Q. Zhu, K. Qin, X. Zhang, denstrom, A.D.M.E. Osterhaus, and R.A.M. Fou- D. Zhao, G. Wang. Y. Feng, J. Ma, W. Liu, J. chier. 2006. Global patterns of infl uenza A virus Wang, and F. Gao. 2005. Highly pathogenic in wild birds. Science 312:384–388. H5N1 infl uenza virus infection in migratory 74. Otsuki, K., O. Takemoto, R. Fujimoto, K. birds. Science 309(5738):1206. www.science. Yamazaki, T. Kubota, H. Hosaki, T. Mitani, Y. org/cgi/content/full/1115273/DCI. Kawaoka, and M. Tsubokura. 1984. Isolation of 64. L’vov, D.K. 1978. Circulation of infl uenza viruses H5 infl uenza viruses from whistling swans in in natural biocoenosis. In: E. Kurstak and K. Mar- western Japan in November 1983. Acta Viro- morosch (eds.). Viruses and Environment. Aca- logica 28:524–524. demic Press: New York, pp. 351–380. 75. Otsuki, K., O. Takemoto, R. Fujimoto, K. 65. Lu, H., A.E. Castro, K. Pennick, J. Liu, Q. Yang, Yamazaki, N. Kubota, H. Hosaki, Y. Kawaoka, P. Dunn, D. Weinstock, and D. Henzler. 2003. and M. Tsubokura. 1987. Isolation of infl uenza A Survival of avian infl uenza virus H7N2 in SPF viruses from migratory waterfowls in San-in Dis- 56 Avian Influenza

trict, Western Japan, in the winter of 1982–1983. Trop. 1974. Circulation of arbo- and myxoviruses Acta Virologica 31:439–442. in populations of waterfowl and shore birds of the 76. Ottis, K., and P.A. Bachmann. 1980. Occurrence lower Amur (in Russian). Ivanovsky Institute of of Hsw1N1 subtype infl uenza A viruses in wild Virology 2:148–156. ducks in Europe. Archives of Virology 63:185– 88. Sabirovic, M., J. Wilesmith, S. Hall, N. Coulson, 190. and F. Landdg. 2006. Outbreaks of HPAI H5N1 77. Ottis, K., and P.A. Bachmann. 1983. Isolation virus in Europe during 2005/2006. International and characterization of ortho- and paramyxovi- Animal Health Division: London. Version 1. ruses from feral birds in Europe. Zentralblatt Fur 89. Saito, T., T. Horimoto, Y. Kakaoka, D.A. Senne, Veterinarimedizin Riehe B 30:22–35. and R.G. Webster. 1994. Emergence of a poten- 78. Perkins, L.E.L., and D.E. Swayne. 2002. Patho- tially pathogenic H5N2 infl uenza virus in chick- genicity of a Hong Kong-origin H5N1 highly ens. Virology 201:277–284. pathogenic avian infl uenza virus for emus, geese, 90. Sandhu, T., and V.S. Hinshaw. 1981. Infl uenza A ducks, and pigeons. Avian Diseases 46:53–63. virus infection in domestic ducks. In: R.W. 79. Perkins, L.E.L., and D.E. Swayne. 2002. Suscep- Bankowski (ed.). Proceedings of the First Inter- tibility of Laughing Gulls (Larus atricilla) to national Symposium on Avian Infl uenza, April H5N1 and H5N3 highly pathogenic avian infl u- 1981, Beltsville, Maryland. Symposium on Avian enza viruses. Avian Diseases 46:877–885. Infl uenza, US Animal Health Association: Rich- 80. Perkins, L.E.L., and D.E. Swayne. 2003. Varied mond, VA, pp. 93–99. pathogenicity of a Hong Kong-origin H5N1 avian 91. Sazonov, A.A., D.K. L’vov, R.G. Webster, T.V. infl uenza virus in four passerine species and bud- Sokolova, N. A. Braude, and N.V. Portyanko. gerigars. Veterinary Pathology 40:14–24. 1977. Isolation of an infl uenza virus, similar to 81. Pfi tzer, S., D.J. Verwoerd, G.H. Gerdes, A.E. A/Port Chalmers/1/73 (H3N2) from a common Labuschagne, A. Erasmus, R.J. Manvell, and C. murre at Sakhalin Island in U.S.S.R (Strain A/ Grund. 2000. Newcastle disease and avian infl u- common murre/Sakhalin/1/74). Archives of enza A virus in wild waterfowl in South Africa. Virology 53:1–7. Avian Diseases 44:655–660. 92. Schafer, J.R., Y. Kawaoka, W.J. Bean, J. Suss, D. 82. Romvary, J., and J. Tanyi. 1975. Occurrence of Senne, and R.G. Webster. 1993. Origin of the Hong Kong infl uenza A (H3N2) virus infection pandemic 1957 H2 infl uenza A virus and the per- in Budapest Zoo. Acta Veterinaria Academiae sistence of its possible progenitors in the avian Scientiarum Hungaricae 25:251–254. reservoir. Virology 194:781–788. 83. Romvary, J., J. Meszaros, J. Tanyi, J. Rozsa, and 93. Senne, D.A., J.E. Pearson, L.D. Miller, and G.A. L. Fabian. 1976. Infl uenza infectedness of cap- Gustafson. 1983. Virus isolations from pet birds tured and shot wild birds on northeastern and submitted for importation into the United States. southeastern parts of Hungary. Acta Veterinaria Avian Diseases 27:731–741. Academiae Scientiarum Hungaricae 26:363–368. 94. Sharp, G.B., Y. Kawaoka, S.M. Wright, B. 84. Romvary, J., J. Meszaros, J. Tanyi, J. Rozsa, and Turner, V. Hinshaw, and R.G. Webster. 1993. L. Fabian. 1976. Spreading of virus infection Wild ducks are the reservoir for only a limited among wild birds and monkeys during infl uenza number of infl uenza A subtypes. Epidemiology epidemic caused by Victoria75 variant of a and Infection 110:161–176. (H3N2) virus. Acta Veterinaria Academiae Sci- 95. Sinnecker, R., H. Sinnecker, E. Zilske, and D. entiarum Hungaricae 26:369–376. Kohler. 1983. Surveillance of pelagic birds 85. Rosenberger, J.K., W.C. Krauss, and R.D. for infl uenza A viruses. Acta Virologica 27:75– Slemons. 1974. Isolation of Newcastle-disease 79. and type-A infl uenza viruses from migratory 96. Slemons, R.D., and B.C. Easterday. 1972. Host waterfowl in Atlantic Flyway. Avian Diseases response differences among fi ve avian species 18:610–613. to infl uenza virus A/turkey/Ontario/7732/66 86. Roslaya, I.G., G.E. Roslyakov, and D.K. Lvov. (Hav5N?). Bulletin World Health Organization 1975. Isolation of infl uenza A viruses and detec- 47:521–525. tion of antibodies in common herons (Ardea 97. Slemons, R.D., and B.C. Easterday. 1975. The cinera) nesting in the lower Amur. Ekologiia natural history of type A infl uenza viruses in wild Virusov 3:138–142. waterfowl. In: L.A. Page (ed.). Third Interna- 87. Roslaya, I.G., G.E. Roslyakov, D.K. L’vov, V.A. tional Wildlife Disease Conference. Plenum Isacenko, L.Y. Zakstelskaya, L. Ya, and I.T. Press: New York. 3 / Ecology of Avian Influenza in Wild Birds 57

98. Slemons, R.D., D.C. Johnson, J.S. Osborn, and F. 109. Stunzner, D., W. Thiel, F. Potsch, and W. Sixl. Hayes. 1974. Type A infl uenza viruses isolated 1980. Isolation of infl uenza viruses from exotic from wild free-fl ying ducks in California. Avian and central European birds. Zentralblatt fur Bak- Diseases 18:119–124. teriologie Mikrobiologie und Hygiene Series A: 99. Slemons, R.D., M.C. Shieldcastle, L.D. Heyman, Medical Microbiology Infectious Diseases Virol- K.E. Bednarik, and D.A. Senne. 1991. Type A ogy Parasitology 247:8–17. infl uenza viruses in waterfowl in Ohio and impli- 110. Sturm-Ramirez, K.M., T. Ellis, B. Bousfi eld, L. cations for domestic turkeys. Avian Diseases Bissett, K. Dyrting, J.E. Rehg, L. Poon, Y. Guan, 35:165–173. M. Peiris, and R.G. Webster. 2004. Reemerging 100. Smitka, C.W., and H.F. Massab. 1981. Ortho- and H5N1 infl uenza viruses in Hong Kong in 2002 paramyxoviruses in the migratory waterfowl of are highly pathogenic to ducks. Journal of Virol- Michigan. Journal of Wildlife Diseases 17:147– ogy 78:4892–4901. 151. 111. Suss, J., J. Schafer, H. Sinnecker, and R.G. 101. Spackman, E., D.E. Stallknecht, R.D. Slemons, Webster. 1994. Infl uenza virus subtypes in aquatic K. Winker, D.L. Suarez, M. Scott, and D.E. birds of eastern Germany. Archives of Virology Swayne. 2005. Phylogenetic analysis of type A 135:101–114. infl uenza genes in natural reservoir species in 112. Thorsen, J., I.K. Barker, and V.S. Hinshaw. 1980. North America reveals genetic variation. Virus Infl uenza viruses isolated from waterfowl in Research 114:89–100. southern Ontario, 1976–1978. Canadian Journal 102. Spackman, E., K. Winker, K. McCracken, and D. of Microbiology 26:1511–1514. Swayne. 2007. An avian infl uenza virus from 113. Toma, B., J.-P. Vaillancourt, B. Dufour, M. Eloit, waterfowl in South America contains genes from F. Moutou, W. Marsh, J.-J. Bénet, M. Sanaa, and North American avian and equine lineages. Avian P. Michel. 1999. Dictionary of Veterinary Epide- Disease 51(1 Suppl):273–274. miology. Iowa State University Press: Ames, 103. Stallknecht, D.E. 1998. Ecology and epidemi- IA. ology of avian infl uenza viruses in wild bird 114. Tsubokura, M., K. Otsuki, Y. Kawaoka, and R. populations: Waterfowl, shorebirds, pelicans, Yanagawa. 1981. Isolation of infl uenza A viruses cormorants, etc. In: D.E. Swayne and R.D. from migratory waterfowls in San-in District, Slemons (eds.). Proceedings of the Fourth Inter- Western Japan in 1979–1980. Zentralblatt Fur national Symposium on Avian Infl uenza, May Bakteriologie Mikrobiologie Und Hygiene Serie 29–31, 1997, Athens, Georgia. Symposium on B-Umwelthygiene Krankenhaushygiene Arbeits- Avian Infl uenza, US Animal Health Association: hygiene Praventive Medizin 173:494–500. Richmond, VA, pp. 61–69. 115. Tsubokura, M., K. Otsuki, H. Yamamoto, Y. 104. Stallknecht, D.E., and S.M. Shane. 1988. Host Kawaoka, and K. Nerome. 1981. Isolation of an range of avian infl uenza virus in free-living birds. Hswl Nav4 Infl uenza Virus from a Tufted Duck Veterinary Research Communications 12:125– (Aythya fuligula) in Japan. Microbiology and 141. Immunology 25:819–825. 105. Stallknecht, D.E., S.M. Shane, P.J. Zwank, D.A. 116. Turek, R., B. Tumova, V. Mucha, and A. Stumpa. Senne, and M.T. Kearney. 1990. Avian infl uenza 1983. Type A infl uenza virus strains isolated viruses from migratory and resident ducks of from free living ducks in Czechoslovakia during coastal Louisiana. Avian Diseases 34:398–405. 1978–1981. Acta Virologica 27:523–527. 106. Stallknecht, D.E., S.M. Shane, M.T. Kearney, 117. U.S. Geological Survey. 2006. Referenced reports and P.J. Zwank. 1990. Persistence of avian infl u- of highly pathogenic avian infl uenza H5N1 in enza virus in water. Avian Diseases 34:406–411. wildlife and domestic animals. Available at http:// 107. Stallknecht, D.E., M.T. Kearney, S.M. Shane, www.usgs.gov. Accessed April 2006. and P.J. Zwank. 1990. Effects of pH, temperature, 118. Webster, R.G., M. Morita, C. Pridgen, and B. and salinity on persistence of avian infl uenza Tumova. 1976. Orthoviruses and paramyxovi- viruses in water. Avian Diseases 34:412–418. ruses from migrating feral ducks: characterization 108. Stanislawek, W.L., C.R. Wilks, J. Meers, G.W. of a new group of infl uenza A viruses. Journal of Horner, D.J. Alexander, R.J. Manvell, J.A. Kat- General Virology 32:217–225. tenbelt, and A.R. Gould. 2002. Avian paramyxo- 119. Webster, R.G., M. Yakhno, V.S. Hinshaw, W.R. viruses and infl uenza viruses isolated from Bean, Jr., and K.G. Murti. 1978. Intestinal infl u- mallard ducks (Anas platyrhynchos) in New enza; replication and characterization of infl uenza Zealand. Archives of Virology 147:1287–1302. viruses in ducks. Virology 84:268–278. 58 Avian Influenza

120. Webster, R.G., V.S. Hinshaw, W.J. Bean, K.L. 125. Yamane, N., T. Odagiri, J. Arikawa, and N. Van Wyke, J.R. Geraci, D.J. St. Aubin, and G. Ishida. 1979. Isolation and characterization of Petursson. 1981. Characterization of infl uenza A infl uenza A viruses from wild ducks in northern virus from seals. Virology 113:712–724. Japan: appearance of Hsw1 antigens in the Japa- 121. Widjaja, L., S.L. Krauss, R.J. Webby, T. Xie, and nese duck population. Acta Virologica 23:375– R.G. Webster. 2004. Matrix gene of infl uenza A 384. viruses from wild aquatic birds: ecology and 126. Yamane, N., T. Odagiri, J. Arikawa, H. Morita, emergence of infl uenza viruses. Journal of Virol- N. Sukeno, and N. Ishida. 1978. Isolation of ogy 78:8771–8779. orthomyxovirus from migrating and domestic 122. World Health Organization. 2006. Human avian ducks in northern Japan in 1976–1977. Japanese infl uenza in Azerbijan, February-March 2006. Journal of Medical Science and Biology 31:407– Weekly Epidemiology Report 81:183–188. 415. 123. Winkler, W.G., D.O. Trainer, and B.C. Easter- 127. Zakstelskaya, L.Y., M.A. Yakhno, V.A. Isa- day. 1972. Infl uenza in Canada geese. Bulletin chenko, S.M. Klimenko, E.V. Molibog, V.P. World Health Organization 47:507–513. Andreev, D.K. Lvov, and S.S. Yamnikova. 1974. 124. Wood, J.M., R.G. Webster, and V.F. Nettles. 1985. Isolation and peculiarities of infl uenza virus (tern) Host range of A/chicken/Pennsylvania/83 (H5N2) (Turkmenistan 18) 73. Virologiia Virusov 2:93– infl uenza virus. Avian Diseases 29:198–207. 98. 4 Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems

David E. Swayne

HISTORY OF HUMANS AND BIRDS (120). The Egyptians were the fi rst to focus on the To understand the ecology and epidemiology of chicken as an important agricultural commodity and avian infl uenza (AI), a review of human activities as developed the fi rst artifi cial-heat egg incubators. related to domestication of birds, and agricultural Some of these incubators held 10,000 to 15,000 eggs production are necessary. As stated by Edmund Saul and would have required large fl ocks of chickens to Dixon, “Next to the dog, the fowl has been the con- support such agricultural endeavors. Chickens were stant attention upon man in his migration and occu- widespread in Greek civilization, but it was the pation of strange lands” (cited in Smith and Daniel Romans who brought them into the center of life of [120]). We must also understand the role many bird Western civilization and developed much of the species have occupied in human life, from religious science and husbandry for use in everyday life. The symbol to pet to entertainment to food, with each husbandry principles established by the Romans culture emphasizing different aspects that have were used until well into the 19th century. affected captivity and raising parameters. The use of chickens varied with each culture: the Egyptians were the fi rst to report importance of Chicken chickens as an agricultural commodity; the Persians Domestic chicken (Gallus gallus domesticus) has idealized them in cock fi ghting; the Greeks devel- origins in various species of wild jungle fowl from oped various breeds of chickens; and the Romans southeast Asia, but no living wild ancestor exists elevated them to a religious symbol (120). In modern today and debate continues as to the exact ancestral European culture, the keeping of pure breeds arose origin (28). The major contributor is believed to be in the 18th century and peaked in the 1800s both in the red jungle fowl (Gallus gallus), but there may Europe and North America, where poultry rearing be additional contributions by the other three jungle became a craze for exhibition, for personal amuse- fowl species. The earliest archeological evidence of ment, and as pets. This period has been called domesticated chickens came from 6000 bc in China, “chickenmania” or “poultrymania” with widespread but the original date and site of domestication were rearing of chickens by everyone from common probably earlier in southeast Asia with subsequent people to nobility. During this time, national, migration of humans north, moving the domesti- regional, and international poultry exhibits became cated chickens with them. By 2500 bc, chickens popular, eliminating geographic isolation of poultry, were found in Europe and western Asia. Chickens and subsequently their diseases. Although a byprod- were fi rst used for religion and were not used as a uct of the “poultrymania” was eggs and sometimes food source until later. A primary early activity of meat, the primary functions were entertainment and bird rearing was for entertainment in cock fi ghting showmanship.

Roman Period type chickens with increased feed effi ciency, and Aldrovandi, a 16th-century professor at the Univer- rapid growth through crossing Cornish and White sity of Bologna, wrote extensively on chickens, Plymouth Rock stocks (28, 120). In the United States compiling information from Greek and Roman civ- and Europe in the mid 1900s, a large fl ock of egg- ilizations (120). He cites 2nd-century bc and 1st- laying or meat chickens was 3000 to 4000 birds. century ad authors Varro and Columella, respectively, Around the world, chickens are the primary poultry on poultry husbandry items such as house design species reared for food, for both eggs and meat. In and fl ock sizes. Flocks sizes at this time varied from 2005, the world production of broiler meat was 59 50 to 200 birds, with poultry houses having doors million metric tons (mt) with the top fi ve producers that could be closed to protect the birds at night from being the United States (15.9 million mt), China thieves and animals. These poultry houses were also (10.2 million mt), Brazil (9.4 million mt), European plastered to keep out snakes and cats. These were Union-25 (EU-25) (7.7 million mt), and Mexico (2.6 the same housing designs and number of chickens million mt) (151). The top fi ve exporters of chicken raised per premise at the time of Aldrovandi. meat were Brazil (2.7 million mt), United States (2.3 million mt), EU-25 (0.76 million mt), China (0.33 Commercial Era million mt), and Thailand (0.24 million mt), while The organization of poultry for food production in the top importers were Russian Federation (1.2 the modern era began in the late 1800s in Europe, million mt), Japan (0.75 million mt), EU-25 (0.52 fi rst to provide eggs and later to provide meat (28, million mt), Saudi Arabia (0.48 million mt), and 120). These early endeavors were very modest by Mexico (0.37 million mt). Consumption of broiler today’s standards. The increasing importance of meat is highest in the United States with 102 lb per poultry as a food source for all people is evident in capita, which in 2005 was supplied from 8.8 billion the U.S. census of 1910, which reported 280 million broilers raised for a $20.9 billion market value. In the chickens on 5.5 million farms, averaging 51 birds United States during 2005, 90 billion eggs were proper farm; in addition, 80.4% of all farms had poultry duced by 343 million layers for a value of $4 billion (120). At this time, most fl ocks were small, averag- and 255 eggs were consumed per capita. ing 50 to 200 birds, and were reared with outdoor access. In the United States, the trend toward indoor Turkeys production began in the late 1800s in California with Domestication of turkeys occurred in Central development of the fi rst commercial hatchery for America, presumably in Mexico between 200 bc egg-laying chickens, which shipped White Leghorn and 700 ad, and arose initially from Mexican sub- chicks up to 400 miles throughout California. This species (Meleagris gallapavo gallopavo) (28). These was followed by the formation of the Petaluma small domesticated turkeys were carried from the Poultry Association, with the goal of increasing New World to Europe by the Spanish beginning in poultry production and consumption, which at the the early 16th century and returned back to the New time averaged 500 chickens per egg-laying fl ock in World as early as 1607 in the northeastern United California. By 1905, the value of poultry in the States, where they were hybridized with the larger United States was $500 million, and in 1910, poultry eastern wild turkey (Meleagridis gallopavo silves- was second only to corn as a revenue crop. In 1913, tris) to produce a larger, more vigorous bird. Selec- the Peteluma area shipped 100 million table eggs per tion for the broad-breasted trait was initiated by year and the area had over 1 million chickens. Pro- Jesse Throssel in Canada and further developed in duction reached 450 million eggs in 1918. One the United States in the early 1920s. Initially, pro- hatchery produced 150,000 chicks in 3 weeks. In duction used traditional outdoor rearing methods 1940, the largest chicken farm was a 250,000-bird with birds primarily being raised for seasonal Christ- layer farm in Petaluma, California. mas markets. Beginning in the late 1950s to early Meat production lagged behind egg production, 1960s, concentrated production began following the with meat-producing birds fi rst being a byproduct of development of controlled-environment houses and the laying industry (i.e., culls, young cockerels and chemotherapeutic methods to control the protozoal capons) (120). However, genetic and nutritional disease blackhead. In recent periods, most industrial efforts in the 1950s led to the development of meat- production has been in developed countries with 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 61 consumption being highest in the United States derived from the wild graylag goose (Anser anser) (16.9 lb per capita in 2005) (151). For 2005, world in Egypt (1500 bc) and possibly Germany (28). The production of turkey meat was 4.9 million mt with Romans raised geese as a barnyard alarm and for the top producing countries including the United eggs, meat, and fatty livers. The goose is primarily States (2.5 million mt), EU-25 (1.9 million mt), raised in China and northern climates, with most Brazil (0.28 million mt), and Canada (0.16 million being raised in semicommercial and village settings mt). The major exporting countries were the United and some industrial production. Major goose meat– States (0.26 million mt), EU-25 (0.19 million mt), consuming countries include Russia, China, and Brazil (0.16 million mt), and Canada (24,000 mt). eastern Europe. In the remaining developed coun- Major importing countries were Mexico (0.18 tries, goose production is a minor industry and few million mt), EU-25 (0.1 million mt), and Russian geese are raised in developing nations. Federation (0.1 million mt). Duck and goose production accounts for 7.5% of the world poultry production (95). China is the Waterfowl number one producer of both ducks (2.1 million tons Domestic ducks comprise two different species: (1) in 2002) and geese (1.9 million tons in 2002), domestic or mallard-type ducks including Pekin and accounting for 66% and 93% of the world produc- Indian Runner types (Anas platyrhynchos) origi- tion of these species, respectively. Leading countries nated in Asia and Europe and (2) the Muscovy duck in duck production after China include France (0.24 (Cairina moschata), which originated in the tropics million tons in 2002), India (0.14 million tons in and subtropics of central and northern South America 2002), Thailand (0.11 million tons in 2002), Vietnam (28). The mallard was domesticated in two indepen- (74,000 tons in 2002), and Taiwan (73,000 tons in dent events, fi rst in southeast Asia a very long time 2002). The primary goose-producing countries after ago and again in Europe during the Middle Ages. China are the Ukraine (97,000 tons in 2002), Domestic ducks are a minor poultry species in Hungary (49,000 tons in 2002), Egypt (49,000 tons Europe but are the predominant poultry species in in 2002), Taiwan (30,000 tons in 2002), and Poland much of southeast Asia, where they are grown for (18,000 tons). In addition to meat, duck eggs, duck both meat and eggs. Most of the production is of liver, and duck and goose feathers are major exported semicommercial type, but industrial production does products. The latter is used for stuffi ng coats, sleep- exist as specialty meats, mostly in Europe and North ing bags, pillows, etc. America. The Pekin duck was developed on Long Island, United States, in the mid 1800s. Minor Other Poultry The Muscovy duck was taken from the Americas Various other species of poultry are raised for meat, to Europe, primarily France, and to Africa by the eggs, feathers, and hides throughout the world but Spanish and Portuguese explorers, but it was also are minor contributors to agricultural production. transported to Asia, where it adapted well to the hot However, in some countries with wet market systems, climates (28). The Muscovy duck has remained a minor species have been very important contributors minor poultry species, with the greatest commercial to livelihoods. Such minor species include ratites production located in France, eastern European (ostrich [Struthio camelus] and emu [Dromaius countries, Taiwan, and southeast Asia as a meat novaehollandiae]), Japanese quail (Coturnix cotur- source. Production in other parts of the world, espe- nix japonicus), bobwhite quail (Colinus virginia- cially Africa and South America, is as village poultry nus), ring-necked pheasant (Phasianus colchicus), and in sustenance farming for eggs and meat. A chukar partridge (Alectoris chukar), guinea fowl sterile hybrid of the domestic and Muscovy duck, (Numida meleagris), and pigeons (Columba livia). the mulard is commercially important in southeast Asia, especially in Taiwan and in France. GENERAL ECOLOGY AND Two types of domestic geese exist: (1) Eastern EPIDEMIOLOGY OF INFLUENZA breeds such as Chinese and African, which were A VIRUSES derived from the wild swan goose (Anser cygnoides) The ecology and epidemiology of infl uenza A in China around 4500 years ago, and (2) Western viruses are very complex, involving various free- breeds, such as Embden and Toulouse, which were living, captive-raised, and domestic bird hosts, as 62 Avian Influenza well as various wild and domesticated mammalian gene segments (26). Infl uenza A viruses are classi- hosts within diverse environments (Fig. 4.1). Avian fi ed by their surface glycoproteins into 16 different infl uenza viruses, like all swine and equine infl uenza subtypes of hemagglutinin (H1–H16) and nine dif- viruses and most human infl uenza viruses, are nega- ferent subtypes of neuraminidase (N1–N9). The tive-sense RNA viruses of the family Orthomyxo- greatest diversity of hemagglutinin and neuramini- viridae, genus Infl uenzavirus A, and contain eight dase subtypes is contained in AI viruses.

Figure 4.1. Diagrammatic representation of the source and movement of inﬂ uenza A viruses or their genes within avian and mammalian ecological and epidemiological situations (modiﬁ ed from Stallknecht et al. (126) and Swayne (135). H = hemagglutinin subtype; () = subtype previously common but no longer circulating. Source: K. Carter, University of Georgia, and D. Swayne, U.S. Department of Agriculture, Agricultural Research Service, Athens, GA. 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 63

Free-Living Aquatic Birds as Primordial bird species is most likely greater than the 105 Reservoirs reported species (3). Free-living birds should not be viewed as a single The majority of the LPAI viruses have been iso- entity of “wild birds” occupying one ecosystem with lated from aquatic birds from the orders Anseri- equal risk for low pathogenicity AI (LPAI) virus formes (ducks, geese, and swans) and Charadriiformes infection. Rather, birds are a diverse group of (e.g., shorebirds [turnstones and sandpipers], gulls, animals with different degrees of relatedness and a terns, murre, and guillemots) (126). Such birds are variety of habitats and ecosystems. Speciﬁ cally, the considered the main LPAI virus reservoirs with most class Aves contains 29 orders, 187 families, over frequent AI viruses isolated from Anseriformes are 2000 genera, and over 9600 species based on ana- of the H3, H4, and H6 subtypes and from Charadri- tomical, behavioral, ecological, physiological, and iformes are of the H3, H9, H11, and H13 subtypes genetic differences (42). LPAI viruses have been (Fig. 4.1 and Table 4.1) (2–4, 77, 127). In addition, shown to naturally infect more than 105 free-living LPAI viruses have been infrequently isolated from bird species representing 12 avian orders (87, 124, other aquatic birds in the orders Ciconiiformes 126, 156). However, the number of naturally infected (herons and ibis), Gaviiformes (loons), Gruiformes

Table 4.1. Hemagglutinin subtype distributiona between different birds (class: Aves) and mammals (class: Mammalia). Host of Origin Aves Charadriiformes and Anseriformes Procellariiformes Galliformes Mammalia (e.g., Dabbling (e.g., Shorebirds, (Domestic HA Subtype Humans Swine Equine Ducks) Gulls, Seabirds) Poultry)

H1 +++ + + ++d H2 (++)b +++ H3 ++ ++ ++ ++ ++ ++d H4 ±++++ H5 ±± + + ++c H6 ++ + + H7 ±±(++)b ++++c H8 ±± H9 ±± + ++++ H10 +++ H11 ++++ H12 ++± H13 ++++ H14d ± H15d ±± H16d + a ± = sporadic, + = multiple reports, ++ = most common. b () = Previously common but now not reported. c Both LP and HP viruses. d Primarily swine inﬂ uenza virus infections of domestic turkeys. Adapted from Stallknecht (124). 64 Avian Influenza

(coots), Pelecaniformes (cormorant), Podicipedi- of AI and free-living birds is presented in Chapter 3 formes (grebe), and Procellariiformes (shearwater). (Ecology of Avian Infl uenza in Wild Birds). On rare occasions, LPAI viruses have been isolated from free-living nonaquatic birds in the orders Infl uenza A Viruses in Mammals Piciformes (woodpecker), Passeriformes (perching Infl uenza A viruses have caused infections in a birds; e.g., sparrows, starlings, mynahs, fi nches, and variety of mammalian species and either these weaverbirds), Columbiformes (doves and pigeons), viruses or some of their genes have their origins and Galliformes (pheasant and partridge) (124, 126). from AI viruses maintained in the free-living aquatic However, nonaquatic species have not been consid- bird reservoirs (Fig. 4.1). Typically, infl uenza A ered reservoirs of LPAI viruses, and generally infec- viruses are not promiscuous as are many enteric tion in these species is thought to occur via spillover bacteria, that is, jumping easily between host species; from infected domestic poultry (124). Infections by instead they exhibit some host adaptation thus LPAI viruses in free-living birds have typically pro- requiring long periods of time, such as years or duced asymptomatic infections, and such viruses are decades, to infect, circulate, and adapt to a new host passed within and between species of birds that species and become endemic. Infl uenza A viruses occupy the same ecosystem (140). Most free-living are responsible for infections in mammals under bird species are not exposed to or infected with three broad situations: (1) endemic infections with LPAI viruses, especially upland game or terrestrial established, host-adapted viruses such as swine birds of the order Galliformes (jungle fowl, wild infl uenza, equine infl uenza, most human infl uenza turkeys, Bobwhite quail, etc.) because of habitat uti- strains, and, recently, canine infl uenza A viruses; (2) lization and behavior (90). sporadic, limited to epizootic infections such as in In contrast to LPAI viruses, high pathogenicity AI mink, seals, whales, and some pig and some human (HPAI) virus have been less frequently isolated from cases with LPAI viruses of free-living bird or poultry free-living birds (152) and the existence of a wild origin; and (3) recent sporadic infections with H5N1 bird reservoir has not been demonstrated (101). HPAI virus such as has occurred with tigers and However, since 2002, the H5N1 HPAI virus has leopards, house cats, dogs, civets, stone martins, been isolated from fatalities in various dead captive pigs, and some human cases. and free-living birds or live birds spatially associ- First, endemic infl uenza A virus infections have ated with an outbreak. In addition to birds in most been established in pigs, horses, and humans causing of the orders listed above, H5N1 HPAI has been common upper respiratory infections and frequent isolated from birds of the orders Falconiformes disease (Fig. 4.1). These host-specifi c viruses (falcons, eagles, hawks, buzzards, and Old World became established by reassortment of gene seg- vultures), Phoenicopteriformes (fl amingoes), and ments from AI viruses and host-adapted infl uenza A Strigiformes (owls) (152). Most often, these wild viruses to producing a hybrid or reassortant virus. bird infections have resulted from exposure to For equine, the classic infl uenza A virus strain has infected poultry and two-thirds of these free-living been the H7N7 subtype, fi rst described in the early birds had scavenging or carnivorous feeding habits, 1950s, which has been displaced by the H3N8 strain which could have resulted in transmission from con- that today is the most common (Table 4.1) (147). sumption of infected poultry carcasses. However, in Furthermore, in the early 2000s, equine H3N8 strain some cases, poultry exposure was not evident, sug- was identifi ed in racing greyhounds in Florida gesting some wild bird involvement in dissemina- exhibiting severe respiratory disease, and subse- tion. However, a reservoir status of this H5N1 HPAI quently, the H3N8 infl uenza has become a common virus lineage in free-living aquatic birds has not etiological agent of kennel cough for dogs in the been demonstrated, with domestic ducks in Asia and United States (Fig. 4.1) (17, 27). For swine, the fi rst Africa being the major reservoir and propagators of reports of infl uenza-associated respiratory disease the virus within these regions (58, 130). Historically, occurred during August 1918 in Illinois, which fol- HPAI viruses have arisen from LPAI viruses after lowed the spring wave of H1N1 Spanish Flu in circulation in gallinaceous poultry and are the result humans of 1918, suggesting an initial human-to- of mutations at the proteolytic cleavage site of the swine transmission of the H1N1 virus (32, 145) An hemagglutinin protein (142). A detailed discussion H1N1 infl uenza A virus was isolated in 1931 from 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 65 pigs with respiratory disease and was determined to Third, a few HPAI viruses have caused infection be the etiologic agent of swine infl uenza (112). This and deaths in mammals including humans, but these H1N1 virus is the source of the classic H1N1 swine viruses have not become established as endemic infl uenza present in North America today (Figure viruses and mammal-to-mammal transmission has 4.1) (64, 65). In 1979, an H1N1 AI virus appeared been limited. Since 1997, the H5N1 HPAI virus has in European swine and is the predominant infl uenza caused sporadic cases of infection and death in large A virus in swine of Europe (32). In the mid 1980s felines (tigers and leopards), house cats, dogs, in Europe and mid 1990s in the United States, H3N2 Owston’s palm civets, a stone martin, and domestic infl uenza appeared in swine and these viruses are pigs (Fig. 4.1) (22, 63, 68, 100, 121, 162). An H7N7 also common. Reassortant strains between H1N1 HPAI virus has caused infections without disease in and H3N2 have been isolated from swine, including pigs during the 2003 Dutch outbreak (75), and a single the H1N2 and H3N1 subtypes (149). In humans, case of H5N2 virus infection has been reported in pigs endemic infl uenza A produces a self-limiting respi- during the 1983–1984 outbreak in the United States ratory disease that has resulted in signifi cant mortal- (14). Most of these H5N1 and H7N7 cases have ity in elderly and immunocompromised individuals. involved close contact with infected birds or con- Periodically, new subtypes have emerged resulting sumption of infected birds or their raw products. Some in pandemics and displacement of prior endemic cases of natural infections by H5N1, H7N3, and H7N7 infl uenza A subtypes. For example, the 1918 pan- HPAI viruses have occurred in humans (18, 40). demic resulted from either introduction and slow For details of human infections by AI viruses see adaptation of an H1N1 virus of avian-origin or Chapter 20 (Public Health Implications of Avian reassortment of genes between an AI virus and an Infl uenza Viruses). existing human infl uenza virus (98, 99, 144). From sequence analysis of infl uenza A viral genes, the Poultry and Captive Birds 1957 (H2N2) and 1968 (H3N2) human pandemic Humans have created new niches for birds outside infl uenza A viruses resulted from reassortment of of the natural ecology through captivity and domes- three (HA, NA, and PB1) and two (HA and PB1) AI tication (135). Such new environments have favored viral genes with fi ve and six human infl uenza viral transmission, adaptation, and perpetuation of AI genes, respectively (62, 97, 104, 105). The H2N2 viruses outside the free-living aquatic bird reservoirs displaced the H1N1 subtype in 1957 and the H3N2 to other bird species, including galliforme poultry, displaced the H2N2 subtype in 1968, but the which are not naturally infected by AI viruses (133). H1N1 reappeared in 1977 and, currently, H1N1 and Outdoor rearing and lack of biosecurity favor the H3N2 strains are co-circulating worldwide, although introduction, adaptation, maintenance, and spread of the H3N2 predominates. AI viruses in captive birds and domestic poultry. Second, some LPAI viruses have caused sporadic Infections of domestic poultry have been reported respiratory disease in mink (H10N4 and H10N7) with H1–H13 subtypes of LPAI viruses and H5 and (35), seals (H7N7, H4N5, and H3N?) (15, 41, 53), H7 HPAI viruses, but the most frequently reported and whales (H1N1, H13N2, and H13N9) (52, 76), infections have been from H1, H5, H7, and H9 sub- but infections have been limited individual infec- types (Table 4.1). tions to small epidemics and there has been a lack In general, LPAI viruses have been isolated from of evidence for these to become endemic (Fig. domestic poultry, most frequently (in decreasing 4.1 and Table 4.1) (15, 36, 41, 52, 70, 76, 158). order) turkeys, ducks, and chickens. These viruses Self-limiting, sporadic infections have been repor- have also been isolated, although less frequently, ted in swine with H1N7, H4N6, and H9N2 LPAI from captive wild birds held as caged pets, or in viruses (11, 61, 85, 160). There have also been a quarantine stations, private collections/reserves, and few sporadic infections of LPAI viruses in humans zoological parks (2,3). However, incidence and dis- with complete recovery. Most of the strains in- tribution vary greatly with geographic region, volved have not become established, but sporadi- species, age of bird, time of year, and the environ- cally some H7N2 and H9N2 LPAI viruses have mental or agricultural system occupied (140). The caused limited numbers of individual human cases remainder of this chapter focuses on epidemiology (18, 88). of AI in domestic poultry and captive birds. 66 Avian Influenza

CONCEPTS FOR UNDERSTANDING process, but control of virus exposure can prevent PATHOBIOLOGY infection. For example, exposure is prevented for Defi nitions of several pathobiological terms and animals within an AI-free country, zone, or compart- explanation of some disease concepts are essential ment (CZC), or if the animals are in an AI-affected to understand the complex ecology and epidemiol- CZC when biosecurity is adequate to keep the virus ogy of AI viruses (Fig. 4.2 and Table 4.2) (136). off a farm or premises. However, if virus exposure Exposure to an AI virus can initiate the infection does occurs, infection will not result if the exposure route is inappropriate, the exposure dose is below the infection threshold, adequate immunity is present in the host, or the AI virus strain is not adapted to No the specifi c host species. Infection will occur follow- Exposure Exposure Severe Disease ing exposure if the virus has some adaptation to the • Improper & Death exposure route, • Low to high virus dose host, the host is exposed to a dose of virus above the • Low virus dose, • Low to high degree of minimum infectious dose, and the host has incom- • Inadequate host adaptation, adaptation, and/or • Incomplete immunity, and/or plete or total lack of immunity. The outcome of such • Complete • Secondary diseases immunity infections may vary from infection without clinical signs to mild disease to severe disease with high Infection, mortality. For AI viruses with low adaptation to the no clinical No infection Mild Disease signs host, infection may require a high exposure or secondary factors to increase host susceptibility, and even then infection may only result in virus replication and shedding without disease. By contrast, AI Figure 4.2. Pathobiology concepts for virus strains with high adaptation to a host species understanding avian infl uenza in poultry. usually require low exposure doses to produce infec- Source: D. Swayne, U.S. Department of tion. Generally, an AI virus strain is optimally Agriculture, Agricultural Research Service, adapted for a single host species. However, closely Athens, GA. related host species may also be susceptible and

Table 4.2. Pathobiological terms. Term Deﬁ nition

Adaptation Progressive genetic changes in a virus, resulting in increasing effi ciency of replication Exposure Access of the host to the virus Fomite Inanimate objects such as clothes, shoes, equipment, and supplies than can be contaminated with avian infl uenza virus and serve in their transmission Incubation period Time from exposure to appearance of clinical signs Infectious or patent period Time from fi rst detection of the virus from excretions or secretions to when the virus is no longer detected Infectivity Ability of the virus to bind, replicate, and release the virus from host cells; i.e., ability to produce infection Pathobiological changes Abnormal physiological and anatomic changes that occur as a result of virus replication within the cell, tissue, and/or organ Pathogenicity or virulence Disease-producing capacity of the avian infl uenza virus Prepatent period Time from exposure to the virus to when virus is produced and detected in excretions or secretions Transmissibility Natural host-to-host spread 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 67 become infected when exposed, but the AI virus strain will exhibit a lesser degree of adaptation as evident by lower replication titers and inconsistent disease. In most free-living aquatic birds, optimally adapted LPAI viruses replicate in the alimentary tract and are shed in the feces. Such infections are not associated with pathobiological changes (i.e., no disease). However, when these viruses are passed to domestic poultry through exposure and progressive adaptation, the results can vary. Typically in poultry, as the LPAI virus replication titers increase, so does the severity of pathobiological changes such as gross and microscopic lesions in respiratory, alimentary, and reproductive systems. The most pathogenic virus strains cause major cell damage and death. Figure 4.3. Five means or modes by which With HPAI viruses, the replication is systemic and avian infl uenza viruses are introduced into can result in severe damage to critical organs with poultry. Source: K. Carter, University of resulting high death rates within the affected popula- Georgia, and D. Swayne, U.S. Department of tion. Generally, an AI virus that is transmissible Agriculture, Agricultural Research Service, within a given species population implies suffi cient Athens, GA. adaptation to allow natural host-to-host spread from infected-to-naïve, susceptible host of the same or closely related species. Transmission is dependent secretions, feces, or dust from infected birds; the on multiple complex factors, including the follow- materials act as mechanical vectors. Humans, who ing: magnitude, route, and duration of virus shed- are highly mobile, have been a source of indirect ding; host species, population density, and exposure to AI virus through viral contaminated confi nement methods; environmental conditions shoes, clothing, and hands when such persons have that increase virus survival; and opportunities for been in direct contact with infected birds or their mechanical spread of the virus via humans, birds, secretions/excretions; the humans act as mechanical and equipment (14). vectors. In addition, birds have been exposed to AI viruses through contact with contaminated water; Exposure and Transmission such exposed birds have included wild aquatic birds, Exposure is the fi rst step in the transmission and domestic ducklings, and possibly turkeys (55, 78). initiation of infection. Conceptually, an AI virus can Some evidence exists for exposure to AI viruses be introduced into a poultry fl ock via fi ve different through contaminated dust particles or water drop- means (Fig. 4.3): (1) direct exposure to AI infected lets within air and movement by wind. High-volume birds (2) exposure to equipment or materials that are air sampling outside of affected poultry during the contaminated with AI virus in respiratory secretions 1983–1984 H5N2 HPAI outbreak in the United or feces (3) movement of people with AI virus on States detected AI virus in samples taken up to 45 shoes or clothing (4) AI virus contaminated water, meters from houses but not at further distances (14). and (5) AI virus moved in air. The most effi cient During the Canadian H7N3 HPAI outbreak, 102.4 3 means of introduction is through direct contact with mean tissue culture infectious doses (TCID50)/m infected birds that shed large quantities of the AI was detected in low-volume air sampling inside virus into the common environment through their barns with infected poultry, and low concentrations respiratory secretions and the feces, that is, birds of AI virus nucleic acids were found outside the acting as biological vectors. The infected birds can affected barns (96). The wind dispersion of AI virus be free-living birds or domestic poultry. However, through aerosols, dust, and feathers was proposed as birds can be exposed to the AI virus through indirect a means of transmission from affected farm in this means such as via equipment or materials (fomites) outbreak, especially during depopulation activities that have been surface contaminated by respiratory that generated dust and aerosols that were dispersed 68 Avian Influenza by wind (10, 86). Also, a few fl ocks in the 1983– mallard duck (order Anseriformes)–to–turkey (order 1984 H5N2 HPAI outbreak in the United States Galliformes), but such transfers have been less apparently were infected by spreading of noncom- common than between closely related host species posted contaminated litter or manure on adjacent (135). Rarely, interspecies transmission has occurred fi elds (14). However, in other outbreaks, such as the between species from different phylogenetic classes H7N2 LPAI outbreaks in Virginia, United States, such as chicken–(class Aves)–to–human (class during 2002, the geographic distribution of effected Mammalia) (135). An exception to the rare inter- farms was random, suggesting the movement of class transmission has been the ease and frequency fomites by people, and not wind dispersion, was the of transfer of swine H1 and H3 infl uenza A viruses primary means of farm-to-farm spread (1). Experi- to turkeys when the two species were raised on the mental studies have suggested the airborne route is same farm or within close geographic proximity (79, not a primary mode of virus transmission (8, 39, 56, 134, 135, 143). Finally, other factors increase AI 81, 82). virus cross-species transmission and increase the While proof for spread of AI viruses by horizon- frequency of infections including intermixing of tal means is common as discussed earlier, demon- species on the same premises (e.g., domestic ducks stration of vertical transmission is lacking (7, 14, and geese with chickens and turkeys); the presence 31). However, HPAI viruses do produce systemic of young birds, which are more susceptible to infec- infection in hens, and eggs from such infected hens tion; high density of birds, which increases oppor- have HPAI virus on the eggshell surface and within tunities for exposure; and humid weather and cool the internal contents of the eggs for the last few eggs temperatures, which increase environmental sur- laid before death (7, 16). Because HPAI viruses kill vival of the virus (135). embryos, incubation of infected eggs has not yielded An example of adaptation of AI virus strain and viable young (7). However, cleaning fecal material impact on transmission was the outbreak of H7N2 off the eggshell surfaces and disinfection may be LPAI in Virginia during 2002. In the outbreak zone, necessary to prevent hatchery-associated dissemina- a higher proportion of turkey farms were affected by tion of AI viruses should the eggs come from LPAI the H7N2 LPAI than chicken farms. This is partially virus–infected fl ocks (14). Most LPAI and HPAI explained by experimental data that showed turkeys viruses cause reduction or cessation, respectively, of required 100 to 250 times less virus to cause infec- egg production, further limiting the potential for ver- tion than chickens, indicating this H7N2 LPAI virus tical transmission of AI virus. was better adapted to, and more contagious for, turkeys than chickens (111). Similarly, a greater sus- Adaptation and Transmissibility ceptibility for turkeys compared with chickens has Infl uenza A viruses exhibit varying degrees of host also been noted for LPAI viruses from free-living adaptation, which affects infectivity and transmis- aquatic birds, which explains why turkey fl ocks in sibility. The ease of transmission for individual AI North America have been more frequently infected virus strains is affected by how close the hosts are with AI viruses from free-living aquatic birds than genetically and the degree of host adaptation have chickens. Japanese quail and pheasants also expressed by individual virus strains. Following may have increased susceptibility to LPAI viruses exposure, transmission of infl uenza A viruses is from free-living birds compared with chickens (59, most frequent and easy between individuals of the 69, 91). The mechanism of species adaptation is same host species for which the virus strain is highly poorly understood but is probably associated with host adapted (135) (i.e., intraspecies transmission). multiple genetic and biochemical factors such as Interspecies transmission has occurred and has been hemagglutinin receptor binding affi nity, effi ciency most frequent between individuals of closely related of release by neuraminidase, and effi ciency with species, especially within the same taxonomic which AI viral polymerase genes are expressed and family, such as between chickens, turkeys, quail, their ability to take over the host metabolic machin- pheasants, and guinea fowl (order Galliformes, ery to produce the AI virus (67). family Phasianidae). However, interspecies trans- Species adaptation of AI viruses has been shown mission has occurred between less closely related to be a multistep process when transferring between birds (i.e., different orders) such as free-living free-living aquatic birds and domestic gallinaceous 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 69 poultry (46, 90). For example, in Minnesota during infected chicken fl ocks can be up to 14 days (29, the 1980s and 1990s, the index case of AI virus in 31). However, excretion of the virus occurs earlier turkeys began as an asymptomatic infection in range- than the appearance of clinical signs and may last raised birds during the early fall with detection of longer than the apparent disease. The infectious infection by seroconversion in a few birds at slaugh- period extends from the time when the virus is fi rst ter. The turkey infections were preceded by 6 to 8 detected in a bird to the time when the virus is no weeks by AI virus infections in sentinel ducks placed longer detected in the oropharyngeal and cloacal among free-living ducks (44). Such newly intro- swabs (140). The prepatent or preinfectious period duced AI viruses were passed through multiple indicates the lag in time between exposure to when turkey fl ocks over several months before being opti- virus can be detected in the birds or their environ- mally adapted to turkeys. The turkey-adapted AI ment. Therefore, the use of the infectious period for viruses produced infection in a high percentage of preventing transmission, and initiating disease birds within the affected fl ock and produced clinical control is better than using the incubation period signs such as respiratory disease, drops in egg pro- because many species may not show illness when duction, and mortality; and interfl ock transmission infected but shed the virus, such as domestic ducks of the viruses became much easier (46, 90) (David infected with H5N1 HPAI virus. Halvorson, personal communication). However, the The lengths of the incubation and infectious transfer of AI viruses from free-living waterfowl periods are dependent on the dose of virus, the route (order Anseriformes) to domestic ducks and geese of exposure, the species exposed, various environ- has been much easier than to turkeys, requiring mental factors, and the ability to detect clinical signs minimal adaptation because of the close genetic rela- or infection, respectively (31). tionship of the wild and domestic duck species, and the more frequent contact between outdoor-reared Maintenance of Avian Infl uenza Viruses in domestic ducks and geese, and free-living aquatic Populations birds. However, many AI viruses from free-living Typically, the majority of individual birds secrete shorebirds and gulls (order Charadriiformes) do not virus for only 7 to 10 days, and AI viruses do not replicate in intranasally inoculated domestic ducks persist or produce latent infections in individual (order Anseriformes), indicating that transfer of AI birds, as has been shown to occur with some avian viruses between some wild aquatic bird species is viruses such as the herpesvirus of infectious laryn- more diffi cult and may require multiple steps for gotracheitis. However, AI virus can be maintained adaptation (51, 124, 156). This information indicates for much longer time periods within large popula- AI viruses express various degrees of host adaptation tions of birds such as in village production, live for different bird species and that labeling such infl u- poultry markets, or commercial operations because enza A viruses from different birds as “AI viruses” the initial exposure and acute phase do not cause an with equal ability to infect all birds is a misnomer immediate 100% infection rate. However, as the with the same inaccuracy as categorizing equine, number of susceptible birds decreases in the popula- human, swine, seal, etc. infl uenza A viruses as “mam- tion, the viral transmission slows. AI viruses have malian infl uenza A viruses,” which clearly have dis- reemerged from previous infected fl ocks after a sig- parate abilities to infect different mammalian species. nifi cantly stressful event or following the introduc- The relative rarity of successful species jumps of AI tion of naïve susceptible birds. In some studies, on suggests that adaptive responses are complex and rare occasions, few birds have been shown to excrete affected by factors other than exposure (67). virus longer within the population, especially when the exact exposure time is unclear, when birds are Incubation and Infectious Periods added and removed from the population, or when With HPAI viruses in chickens, the incubation the population was environmentally stressed. LPAI period (i.e., time from exposure to appearance of virus has been recovered for up to 36 days after clinical signs) ranges from a few hours in intrave- known exposure time from a chicken (tracheal nously inoculated birds to 24 hours in intranasally sample) (6) and 22 days from a turkey (71), but inoculated chickens to 3 days in naturally infected in turkeys, when the time of exposure was not individual birds. The incubation period for naturally certain, the virus was recovered up to 72 days after 70 Avian Influenza beginning of the fl oor pen experiment (57). As a the virus particles from physical and chemical inac- fi eld example, H7N2 LPAI virus in Pennsylvania tivation (31). In addition, specifi c environmental during 1997–1998 was recovered from the daily conditions such as cool and moist conditions increase mortality of a clinically normal fl ock 6 months after survival times, having a profound impact on trans- recovery from the acute LPAI clinical disease and mission. For example, H5N2 and H5N1 HPAI from another fl ock 8 weeks after the acute LPAI viruses have remained viable in liquid poultry clinical disease but following the induction of a molt manure for 105 days in the winter under freezing (164). Therefore, once a fl ock is diagnosed as AI conditions, 30 to 35 days at 4º C, 7 days at 20º C, virus infected, it should be considered a potential and 4 days at 25 to 32º C when kept out of direct source of virus for the life of the fl ock until those sunlight (7, 38, 122, 159). In experimental studies birds are eliminated and the farm properly cleaned with H5N1 HPAI virus added to poultry manure, no and disinfected and repopulated with AI-free stock. virus was recovered after 24 hours at 25º C and after Vaccination has been shown to be effective at reduc- 15 minutes when held at 40º C. However, ultraviolet ing virus shed into the environment and stopping (UV) light exposure to manure was not effective at virus transmission but may not eliminate all virus killing the HPAI virus, probably because of inade- shedding (154, 155). quate penetration of the UV light into the manure (24). For AI viruses in water, two H5N1 HPAI 1 Environmental Persistence viruses had a 10 EID50 decrease in infectious titer In infected animals, AI viruses are excreted from the after 4 to 5 days at 28º C (pH 7.2; salinity 0 parts per nares and mouth (respiratory secretions), conjunc- thousand), and no virus was detected after 30 days, tiva, and cloaca (feces) into the environment. Envi- but at 17º C, under the same pH and salinity, the two ronmental virus sources are responsible for most viruses persisted until up to 94 and 158 days, respec- exposures, but birds can also be exposed to AI tively (12). These H5N1 HPAI viruses had shorter viruses through predation or cannibalization of environmental survival times compared to H5 LPAI infected carcasses of dead birds. From experimental viruses obtained from wild waterfowl. For example, studies in chickens, HPAI viruses were shed in the LPAI viruses from free-living waterfowl were greatest quantity from the oropharynx (104.2–7.7 mean shown to remain infective at 17º C for up to 207 days chicken embryo infective doses [EID50]/mL of respi- and at 28º C for up to 102 days. Increasing water ratory secretions) and in a slightly lower quantity salinity or pH shortened the AI virus survival times 2.5–4.5 from the cloaca (10 EID50/g of feces) (138, at both temperatures (125, 128). These studies and 139). For LPAI viruses in chickens, the environmen- fi eld observations suggest AI viruses could remain tal shedding was lower for oropharyngeal (swabs: infective in water or in moist organic materials held 1.1–5.5 1.0–4.3 10 EID50/mL) and cloacal (swabs: 10 at cool temperatures, such as over wintering condi-

EID50/mL) samples (138). In experimental studies, tions, to be infective for long periods of time to these higher shedding titers for HPAI viruses have free-living birds or poultry. Recently, a study translated into greater environmental contamination reported the detection of inﬂ uenza A virus H1 genes and greater transmissibility than comparable LPAI in ice and water from high-latitude lakes in Siberia viruses (153). Titers in carcasses, such as meat, vary that were visited by large numbers of migratory with virus strain, bird species, and clinical stage of birds (163). However, attempts were not made to infection: (1) titers from dead chickens infected with determine if any viable viruses were present. 1983 H5N2 HPAI Pennsylvania virus had 102.2–3.2

EID50/g of meat, whereas 2003 H5N1 HPAI South Inactivation 5.5–8.0 Korean virus had 10 EID50/g of meat (2) H5N1 AI virus shed into the environment must be properly HPAI virus in heart of chickens has been reported eliminated and/or inactivated in order to prevent 10.6 as high as 10 EID50/g, and (3) H5N1 HPAI viruses transmission and to control fi eld infections. AI produced different titers in clinically normal (102.0–3.4 viruses are very labile and thus susceptible to heat 4.0–6.0 EID50/g) or sick (10 EID50/g) domestic ducks and various disinfectants. For buildings, the follow- (136, 138, 146). ing has been suggested as an effective program to Environmentally, the excreted AI viruses are pro- eliminate AI virus from infected premises: heating tected by accompanying organic material that shields to 90–100º F for 1 week, followed by removal and 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 71 proper disposal of manure and litter, cleaning and made systems were identifi ed that could affect AI disinfection of buildings and equipment, and a 2- to virus ecology and epidemiology (135): (1) bird col- 3-week vacancy period before restocking (45). AI lection and trading systems, including captive wild viruses in poultry carcasses or litter and manure are birds and zoological collections; (2) village, back- effectively killed in less than 10 days when properly yard, and hobby fl ocks, including fi ghting cocks and composted or can be buried or incinerated (110). A exhibition poultry; (3) live poultry marketing (LPM) variety of disinfectants are effective at inactivating systems with rural-to-urban movement of poultry AI viruses on clean surfaces such as sodium hypo- for sale and slaughter; (4) outdoor-raised commer- chlorite (household bleach), quaternary ammonium cial poultry, including organic poultry, free-range compounds, sodium hydroxide (lye), phenolic com- turkeys, and game birds; and (5) integrated indoor pounds, acidifi ed ionophor compounds, chlorine commercial poultry. Specifi cally for poultry, the dioxide disinfectants, strong oxidizing agents, and Food and Agriculture Organization (FAO) of the sodium carbonate/sodium silicate combinations United Nations has defi ned four sectors (37): (1) (25). However, organic material must be removed sector 1—industrial integrated system with high- by dry or wet cleaning with detergents before disin- level biosecurity and birds or products marketed fectants can work properly. commercially (e.g., farms that are part of an integrated broiler production enterprise with clearly Virulence and Pathogenicity defi ned and implemented standard operating proce- Confusion has arisen from confl icting use of the dures for biosecurity); (2) sector 2—commercial terms “virulence” or “pathogenicity,” which are poultry production system with moderate to high related to the disease-producing capacity of the AI biosecurity and birds or products that are usually virus as measured by production of clinical signs, or marketed commercially (e.g., farms with birds kept gross, microscopic, and/or ultrastructural lesions indoors continuously; strictly preventing contact (13). Offi cial reporting of AI viruses as LP or HP is with other poultry or wildlife); (3) sector 3—com- based on illness and mortality outcomes following mercial poultry production system with low to experimental inoculation into chickens, on the target minimal biosecurity and birds or products entering species, or on the sequence of the hemagglutinin LPM systems (e.g., a caged layer farm with birds in proteolytic cleavage site (see Chapter 1, Infl uenza A open sheds; a farm with poultry spending time Virus). This offi cial pathotype classifi cation is only outside the shed; a farm producing chickens and specifi c for the chicken but has pathobiological waterfowl); and (4) sector 4—village or backyard application to related galliforme species. However, production with minimal biosecurity and birds and/ the chicken pathotype is not predictive of the AI or products consumed locally. However, consistent virus’s pathogenicity potential in other unrelated categorization of poultry by any single scheme is not birds and humans or other mammals. possible because of country-to-country variations in levels of biosecurity, variations in marketing INFECTIONS WITHIN AGRICULTURAL schemes, partial modernization of production AND OTHER MAN-MADE SYSTEMS systems, and variable fi nancial resources. Chickens, turkeys, and other gallinaceous birds in From a historical perspective, the defi nition of a their wild state have not had AI viruses isolated and commercial farm has changed with the cultural they are not natural reservoirs of AI viruses (90, period. For example, from the Roman era to the mid 133). Humankind has changed the natural ecosys- 1800s, most poultry farms had 50 to 100 chickens. tems of birds through captivity, domestication, agri- A large “commercial” farm in the late 1800s in culture, and commerce beginning thousands of years northern Italy had 1000 chickens, which were used ago (135). Thus, new niches have been created for primarily for egg production (see Chapter 7). Cur- AI viruses, which has changed the incidence and rently, in developed countries, a 1000-chicken farm distribution of AI viruses and the infections they would be considered a small or hobby farm, while cause. Various man-made systems have been devel- a large commercial farm in the United States could oped, and classifi cation schemes vary depending on have over 1 million layers. the perspective of the author. For example, in one The sources for AI virus introduction into poultry classifi cation scheme, fi ve broad categories of man- operations vary with host species, virus strain, 72 Avian Influenza husbandry system, and biosecurity practices or lack outdoor-raised domestic turkeys (2) AI virus–con- thereof. For example, AI virus has been introduced taminated drinking water to indoor-raised turkeys into turkey fl ocks in the United States over the past when the water was derived from ponds or lakes four decades from a variety of sources including the containing AI virus–infected free-living aquatic following (Fig. 4.4): (1) free-living aquatic birds to birds (3) exposure to fomites from the LPM, and (4) infection to swine H1 and H3 infl uenza A viruses when pigs were raised on the same or nearby farms (Fig. 4.4). Similarly, the predominance of AI virus infections in turkeys during the fall in northern Italy coincided with staging of free-living waterfowl prior to southern migration for the winter (94).

Introduction of Avian Inﬂ uenza Viruses into Poultry from Free-Living Birds LPAI viruses from free-living aquatic birds have been introduced into domesticated gallinaceous poultry (primarily chickens and turkeys but also Japanese quail, guinea fowl, pheasants, partridges, and other species) and domestic waterfowl (primar- Figure 4.4. Four proven sources responsible ily ducks and geese), resulting in infections through for introduction of LPAI virus into commercial a two-step process: (1) exposure to an infected host turkeys within the United States from 1960s and (2) adaptation to the new host (136) (Fig. 4.5). to 2000. Source: K. Carter, University of Typically, exposure to the LPAI viruses of free- Georgia, and D. Swayne, U.S. Department of living birds has resulted from direct contact with Agriculture, Agricultural Research Service, infected birds or indirect contact with fomites. Such Athens, GA. exposures have transmitted AI viruses from free-

LPAIV (H1-16) Re-adaptation Exposure Exposure Adaptation X X Most Asian H5N1 HPAIV HPAIV

LPAIV HA HPAIV (H1-16) Mutation (H5/H7)

Figure 4.5. Epidemiology of LPAI and HPAI viruses between free-living aquatic birds and poultry. Source: D. Swayne, U.S. Department of Agriculture, Agricultural Research Service, Athens, GA. 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 73

effi cient replication if the LPAI viruses are suffi - ciently adapted for the poultry host. Poor virus adaptation to the host will result in one of the following: (1) sporadic, ineffi cient transmission within the population until progressive adaptation of the virus to the new poultry species results in emergence of a clinically signifi cant problem with sustained poultry- to-poultry transmission or (2) sporadic individual infections with limited poultry-to-poultry transmission and infection, and eventually dying out of the LPAI virus. When LPAI viruses adapt to the new gallinaceous poultry host, these AI viruses have rarely been transmitted back into a free-living aquatic bird population because they are now de-adapted to Figure 4.6. Relative importance of different the original wild bird host (Fig. 4.5). Furthermore, means for introduction of avian infl uenza once LPAI viruses have been introduced and adapted viruses from free-living birds to poultry. to domestic poultry, free-living aquatic birds have Source: K. Carter, University of Georgia, and had a very limited or no role in secondary dissemina- D. Swayne, U.S. Department of Agriculture, tion or farm-to-farm spread (54, 83). Agricultural Research Service, Athens, GA. When circulating in gallinaceous poultry, some H5 and H7 LPAI viruses have abruptly changed to HPAI viruses through changes in the cleavage site living aquatic birds to outdoor-raised village poultry of the hemagglutinin protein (89). Historically, these more frequently than to indoor commercially reared new HPAI viruses that originated from LPAI viruses poultry. The highest risk activity for exposure were already adapted to gallinaceous poultry and leading to infection has been through direct contact have typically not gone back into free-living bird with infected free-living aquatic birds (Fig. 4.6), HPAI viruses. Similarly, these HPAI viruses have which can be prevented by indoor rearing, by tem- had limited or no infectivity for domestic ducks (5, porary confi nement from outdoor access when risk 136). Except for H5N3 HPAI in common terns of exposure to infected wild birds is high, or by (Sterna hirundo) in South Africa in 1961, only a few providing poultry outdoor access but in specially isolated cases of HPAI virus infections have been constructed areas where wild birds are excluded detected in wild birds before 2002, thus indicating through netting. Slightly lower risk for exposure that HPAI viruses have traditionally not been estab- leading to infections is through use of untreated AI lished in wild birds (101, 131, 133). However, the virus–contaminated surface water from ponds or ecological situation has changed for HPAI with the lakes occupied by AI virus–infected wild aquatic H5N1 strain, which was fi rst described in geese of birds or via wind transmission when such ponds or Guangdong, China, in 1996, followed by infections lakes were located close to poultry houses. The in chickens in Hong Kong in 1997 and followed by former risk can be mitigated by treating the water to repeated reports of infection in poultry in China and kill all viruses or using untreated water from deep Hong Kong over the next 5 years (19, 117, 161). wells. Construction of houses away from waterfowl Signifi cant infection and mortality in nonpoultry habitats can reduce the latter risk. Contaminated species were fi rst reported in captive waterfowl, and clothing and shoes are lower risks because clothing spatially associated free-living aquatic birds, in two and shoes contaminated from hunting or sightseeing parks in Hong Kong during 2002. These outbreaks excursions to waterfowl refuges are unlikely to be were followed by individual reports of mortality in worn into poultry fl ocks. Similarly, no equipment or free-living birds in Cambodia and Thailand and supplies would be used for hunting, sightseeing, and reports of signifi cant mortality in 2005 in free-living working in a poultry fl ock. waterfowl at Qinghai, China, and Lake Erhel, Mon- However, exposure to AI viruses of free-living golia (20, 33, 73, 73) (Fig. 4.5). Experimental infec- birds will only result in sustained transmission and tivity trials parallel the changes in H5N1 HPAI 74 Avian Influenza infectivity for wild birds under natural fi eld condi- rate) or other gallinaceous birds (113, 114). Simi- tions: a 1997 H5N1 HPAI virus strain from Hong larly, in the United States, outdoor duck production Kong was poorly infectious for domestic ducks (93), systems on Long Island, New York, during 1979– many of the 1999–2002 viruses were very infectious 1980 had a 23% positive rate of virus isolation in but produced asymptomatic infections in domestic 2- to 5-week-old ducklings but the lack of clinical ducks (19), many 2002–2004 viruses produced disease (102). severe illness with some deaths, and recent strains have caused high death rates in young domestic Introduction of Avian Infl uenza Viruses from ducklings (60, 136). These fi eld and experimental Village to Commercial Poultry data suggest the H5N1 HPAI virus has readapted The greatest risk for introduction of AI into com- back to some free-living aquatic bird species. Paral- mercial poultry is from an AI virus–infected village lel to the readaptation to free-living aquatic birds poultry or LPM system. For example, the H7N2 was the introduction and adaptation of the virus to LPAI viruses that were endemic in LPM systems of domestic ducks, causing infection, disease, and the northeast United States from 1994 through 2006 eventually death. Both events resulted in a changed were introduced into the index commercial farm and ecologically and epidemiologically situation from were disseminated to cause outbreaks in commercial previous HPAI viruses. However, the establishment poultry as follows: (1) layers in Pennsylvania during of a free-living aquatic bird reservoir for H5N1 1996–1998; (2) broiler breeders in Pennsylvania HPAI viruses, like those existing for LPAI viruses, during 2001–2002; (3) commercial poultry in Vir- has not been demonstrated. ginia, West Virginia, and North Carolina during H5N1 HPAI infections in wild birds have been 2002; (4) a large layer company in Connecticut documented and they could serve a role in fl ock-to- during 2003; (5) a small layer farm in Rhode Island fl ock spread in village or rural poultry systems as during 2003, and (6) three broiler farms in Delmarva either biological or mechanical vectors. For example, during 2004 (1, 30, 34, 108, 123, 132, 137, 164). H5N1 HPAI virus infections of tree sparrows (Passer The primary risk activity for introduction of AI virus montanus), which are epidemiologically closely from infected village poultry to commercial poultry associated with human habitation and agricultural is through direct contact with infected birds or indi- buildings, have been reported in China, which raises rect contact with contaminated fomites such as concern that this species could serve as a vector for transport cages and shoes and clothing of employees spreading of the virus and thus creating a need to (Fig. 4.7). For example, the fi rst case of H7N2 LPAI bird-proof poultry barns (66). However, most impor- in Pennsylvania during December 1996 occurred on tant, the domestic duck has become the major reser- the premises of an LPM dealer who had 50 birds at voir of H5N1 HPAI virus in Asia and Africa (58, any time in the facility and the dealer had made 405 118). pick-ups from neighboring farms over the prior 3 Historically, some domestic waterfowl produc- months (50). The second case was in a commercial tion systems have had high infection rates for AI layer fl ock located within 1.5 miles of the fi rst virus resulting from close interactions and cohabita- premise, and the third case was in a small layer farm. tion with free-living aquatic birds. For example, Four LPM dealers from Pennsylvania and New York the Pearl River Delta of south China has countless had made multiple load-outs from each fl ock. Such duck ponds with intermingling of vast numbers of risks can be mitigated by education of employees on domestic duck populations and wild aquatic birds the risk of AI introduction and prohibition of other (115). This environment favors fecal-oral trans- birds or equipment onto the farm and prohibition on mission through ingestion of contaminated water raising or having contact with village or LPM birds serving as a source of virus for outdoor-reared by employees. If commercial poultry are to be pro- poultry in the region, especially domestic waterfowl vided to the LPM, extra biosecurity precautions (114). In the late 1970s and early 1980s, AI viruses must be taken to avoid introduction of pathogens were detected in the markets of Hong Kong, primar- onto source fl ocks by only allowing clean and dis- ily in domestic ducks (6.5% of samples were AI infected cages and vehicles on the farm and requir- virus positive), as well as in domestic geese (1.1% ing personnel to wear disposable outerwear and positive rate), but less in chickens (0.4% positive clean boots. Transport of AI virus via air or wind 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 75

Figure 4.8. Relative importance of different means for secondary dissemination of avian Figure 4.7. Relative importance of different inﬂ uenza viruses with poultry. Source: K. means for introduction of avian inﬂ uenza Carter, University of Georgia, and D. Swayne, viruses from village to commercial poultry. U.S. Department of Agriculture, Agricultural Source: K. Carter, University of Georgia, and Research Service, Athens, GA. D. Swayne, U.S. Department of Agriculture, Agricultural Research Service, Athens, GA.

78% of poultry farms (667 farms) remained free of H7N2 LPAI (107, 109). Recently, in two H5 HPAI can occur if village poultry are housed on the farm outbreaks, HPAI virus was isolated from blow fl ies, or close to the commercial layer operation. However, house fl ies, black garbage fl ies, and small dung fl ies, water has the lowest possibility as a mode for intro- which suggests they could serve as mechanical duction of AI viruses from village poultry or LPM vectors of AI virus spread between farms (14, 103). into commercial poultry. However, mechanical transmission of AI viruses can occur between farms by anything that can walk, Secondary Spread of Avian Infl uenza Virus crawl, or fl y such as insects, humans and other mam- between Poultry Flocks mals, and wild birds, but confi rmation is diffi cult (14). The primary risk activities for secondary spread of AI virus is through one of the following (Fig. 4.8): Risk Factors for Introduction and Maintenance (1) direct contact by moving of infected birds such as of Avian Infl uenza Viruses pullets into a multiage egg production farm and (2) The frequency of AI infections within different man- movement of infected fomites such as AI virus–con- made systems varies with the structure of animal taminated equipment, materials, clothing, or shoes agriculture within the area, density of poultry pro- onto a nonaffected farm. Transmission by water is duction in the area, the specifi c production sector, unlikely, but air transmission has occurred espe- the level of biosecurity practices used, the country, cially with wind dispersion of virus during high- and the market system. In most developed countries, risk depopulation activities such as dust generation HPAI has been rare and LPAI sporadic in the 25 to during removal of noncomposted contaminated litter 30 billion chickens raised annually within the inte- or grinding of carcasses before composting (10, 86). grated commercial poultry systems. However, AI Practicing high-level biosecurity on farms will miti- virus infections have been more frequently reported gate the risk of farm-to-farm spread as was evident in poultry within LPM systems or village settings in Southern California during the 2002–2003 velo- (140, 150). In the industrial sector of developed genic NDV outbreak, where 81 of 103 commercial countries, LPAI outbreaks have been most fre- poultry farms remained NDV free, and in the Shenan- quently reported in turkeys, slightly less frequently doah Valley of Virginia during 2002, where over in laying chickens, and even less frequently in other 76 Avian Influenza domesticated poultry (14, 69). By contrast, various outdoor production and slaughter still exist today as HPAI and LPAI infections have been commonly the LPM system in developed countries and are the reported in village poultry and commercial poultry major production system for poultry in the develop- in some developing countries. For example, H9N2 ing world. Outdoor-raised village and rural poultry LPAI virus became endemic in commercial chick- and LPM systems have higher AI virus infection ens in some developing countries of Asia and the rates than industrial poultry produced in the devel- Middle East during the mid to late 1990s (21, 23, oped world (116). 72, 74, 80, 84, 92). Similarly, since 2003, H5N1 HPAI has become endemic in many Asian countries, Species Susceptibility being maintained in village or rural poultry, espe- The LPAI viruses from free-living aquatic birds are cially domestic ducks. When AI virus infections most closely host adapted to domestic waterfowl, have occurred in the industrial or live market system making them an easier transfer host. In addition, sectors, they have spread rapidly throughout the some studies suggest that turkeys, pheasants, and systems from farm-to-farm when biosecurity prac- Japanese quail are more susceptible to AI viruses tices have been inadequate, resulting in epidemics from free-living aquatic birds than are chickens (59, of HPAI or LPAI. 91, 140). The co-mingling of different species in the LPM systems, especially with domestic waterfowl, Outdoor Rearing favors the transmission of AI viruses between The rearing of poultry outdoors or on range in areas species and initiation of infections (148). Although with access to AI virus–infected free-living birds is chickens are resistant to direct transfer of AI viruses a major risk factor for transmission from wild birds from free-living waterfowl, after passage through an to agricultural systems. For example, in Minnesota intermediate host (such as turkeys, Japanese quail, between the 1970s and 1990s, up to 5% of com- or pheasants) and subsequent adaptation to the mercial turkeys were raised on range each year to chickens, AI viruses can infect chickens and be provide extra birds for the Thanksgiving and Christ- transmitted effi ciently. There has been one published mas holidays (140). Such range rearing in the early fi eld report of some strains of native chickens being fall during staging of wild ducks in lakes in Min- resistant to H5N1 HPAI virus (9), but experimental nesota before migration south for the winter exposed studies have not been published to substantiate the turkeys to migrating free-living waterfowl infected claim. One experimental study has reported more with LPAI viruses, which resulted in infections (49). severe disease in commercial White Leghorns chick- The number of fl ocks with infected turkeys varied ens than broiler chickens following inoculation with from year to year, with a minimum of two affected H4N8 LPAI virus (141). fl ocks (1983) and peak affected fl ocks of 141 (1978), 258 (1988), and 178 (1995) (48). In 1998, the Min- Lack of Movement Controls and Biosecurity nesota turkey industry eliminated range rearing of Historically, the lack of movement controls, includ- turkeys, and as a result, only 33 fl ocks were infected ing quarantines, and poor biosecurity were associ- from 1996 to 2000, and most of these were infected ated with the spread of HPAI in Europe between with H1N1 swine infl uenza (47). This outdoor access 1900 and 1930 and were responsible for the estab- is especially prominent in production systems that lishment of sustained endemic infections (129). supply the LPM systems. Typically, such poultry Since the 1980s, surveys of poultry in LPM systems supplies include small operations where birds are of Hong Kong, New York, and other large cities raised in backyards and in other outdoor settings, have identifi ed infections by HPAI and LPAI viruses especially for domestic waterfowl (106, 148). (111, 114, 116, 148, 157). The implementation of Prior to the development of the modern vertically movement controls, cleaning and disinfection proto- integrated commercial poultry production and refrig- cols, market rest days, and other measures have led eration for storage and shipping of perishable prod- to the eradication of H5N1 HPAI in Hong Kong ucts in the 1950s, most meat and egg type stock were markets (119), and similar measures have eliminated raised outdoors and locally in backyard and hobby the H7N2 LPAI virus from LPM systems of the fl ocks or on small commercial farms with immediate northeastern United States (S. Trock, personal com- slaughter and consumption (43). Such small local munication, July 18, 2007). 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 77

Prevention of spread on commercial farms readapted to both wild and domestic waterfowl with quires practice of the highest level of biosecurity. production of infection, disease, and, in some situa- High-risk activities must be reduced and controlled. tions, death. For example, a high-risk activity leading to the Transfer of LPAI viruses from free-living aquatic transmission of H7N2 LPAI virus in Virginia in birds requires a complex, multistep process that 2002 was movement of dead infected birds from includes exposure and adaptation of the viruses to a farms through a shared rendering system or from the new host species. Such transmissibility implies farm for burial without adequate sealing and decon- exposure, host adaptation, and effi cient virus repli- tamination of transport vehicles (10). Such risks cation in the host species with easy transfer between must be controlled by proper biosecurity procedures individuals. and practices. Also, to minimize the risk of introduc- At high risk for introduction of AI viruses from tion and dissemination of AI viruses, producers free-living aquatic birds are outdoor-reared domes- should raise only one species of bird in an individual tic poultry, especially domestic waterfowl (ducks, operation, have an all-in-all-out production system, geese, and swans). Additional risk factors include or add new birds only after testing and quarantine intermixing of poultry species on the same premises and practicing a high degree of biosecurity. and lack of biosecurity and movement controls. There are fi ve means or ways of introducing an AI CONCLUSIONS virus onto a premises: (1) direct contact with infected Humankind has changed the natural ecosystems of wild or domestic birds; (2) exposure to contaminated birds through captivity, domestication, agriculture, fomites such as agricultural equipment, vehicles, and commerce that began thousands of years ago and materials; (3) through human movement with and continues through today. This has profoundly AI virus contained on shoes, clothing, hair, hands, changed the existence of LPAI viruses from being a and skin; (4) contaminated water; and (5) airborne diverse group of viruses circulating asymptomati- contaminated dust or water droplets. The importance cally in certain free-living aquatic birds to also of each of these methods depends on the individual becoming a less diverse group of infl uenza A viruses, farm circumstances. which have arisen from reassortment or adaptation of whole viruses, causing endemic respiratory REFERENCES disease in horses, pigs, humans, and some domestic 1. Akey, B.L. 2003. Low pathogenicity H7N2 avian poultry. In addition, the diverse LPAI viruses of infl uenza outbreak in Virginia during 2002. Avian free-living aquatic birds have caused sporadic infec- Diseases 47(Special Issue):1099–1103. tions in a variety of wild and domestic mammals and 2. Alexander, D.J. 1982. Avian infl uenza. Recent developments. Veterinary Bulletin 52:341–359. poultry as these viruses attempt to establish new 3. Alexander, D.J. 1993. Orthomyxovirus infec- niches. The man-made systems are very diverse and tions. In: J.B. McFerran and M.S. McNulty (eds.). include hobby, village, and rural poultry; fi ghting Virus Infections of Birds. Elsevier Science: cocks; captive wild birds; outdoor-reared noncom- London, pp. 287–316. mercial and commercial poultry; and industrial 4. Alexander, D.J., and R.E. Gough. 1986. Isola- indoor-reared poultry. The defi nition of a commer- tions of avian infl uenza virus from birds in Great cial farm has changed dramatically since the 1800s, Britain. Veterinary Record 118:537–538. and the development of indoor commercial produc- 5. Alexander, D.J., G. Parsons, and R.J. Manvell. tion has accelerated since the 1950s. 1986. Experimental assessment of the pathoge- HPAI viruses are not maintained in a wild bird nicity of eight infl uenza A viruses of H5 subtype reservoir like LPAI viruses. HPAI viruses have for chickens, turkeys, ducks and quail. Avian Pathology 15:647–662. arisen through the mutation of H5 and H7 LPAI 6. Bankowski, R.A., and R.D. Conrad. 1966. A new viruses following uncontrolled circulation of the respiratory disease of turkeys caused by virus. In: viruses in susceptible gallinaceous poultry. Histori- E.A. Duyunov, G. Kopylovskaya, I.T. Masliyev, cally, HPAI viruses have not been very infectious G.K. Otryganyev, E.E. Penionzhkevich, N.V. for domestic or free-living waterfowl (geese and Pigarev, and A.P. Valdman (eds.). Proceedings of ducks), but over the past two decades, the H5N1 Thirteenth World’s Poultry Congress, Kiev, HPAI virus that originated in southern China has USSR, pp. 371–379. 78 Avian Influenza

7. Beard, C.W., M. Brugh, and D.C. Johnson. 1984. 17. Castleman, W.L., P.C. Crawford, E.P.J. Gibbs, Laboratory studies with the Pennsylvania avian E.J. Dubovi, R. Donis, and J.R. Powe. 2006. infl uenza viruses (H5N2). Proceedings of the Pathologic fi ndings in dogs infected with newly United States Animal Health Association 88:462– emerged canine H3N8 infl uenza virus. FASEB 473. Journal 20:A214. 8. Beaudette, F.R. 1925. Observations upon fowl 18. CDC. 2005. Avian infl uenza infection in humans. plague in New Jersey. Journal of the American Available at http://www.cdc.gov/fl u/avian/gen- Veterinary Medical Association 20:186–194. info/avian-fl u-humans.htm. Accessed on April 7, 9. Boonyanuwat, K., S. Thummabutra, N. Sookma- 2007. nee, V. Vatchavalkhu, and V. Siripholvat. 2006. 19. Chen, H., G. Deng, Z. Li, G. Tian, Y. Li, P. Jiao, Infl uences of major histocompatibility complex L. Zhang, Z. Liu, R.G. Webster, and K. Yu. 2004. class I haplotypes on avian infl uenza virus disease The evolution of H5N1 infl uenza viruses in ducks traits in Thai indigenous chickens. Animal in southern China. Proceedings of the National Science Journal 285–289. Academy of Sciences U S A 101:10452–10457. 10. Bowes, V.A., S.J. Ritchie, S. Byrne, K. Sojonky, 20. Chen, H., G.J.D. Smith, S.Y. Zhang, K. Qin, J. J.J. Bidulka, and J.H. Robinson. 2004. Virus Wang, K.S. Li, R.G. Webster, J.S.M. Peiris, and characterization, clinical presentation, and pathol- Y. Guan. 2005. H5N1 virus outbreak in migratory ogy associated with H7N3 avian infl uenza in waterfowl: a worrying development could help to British Columbia broiler breeder chickens in spread this dangerous virus beyond its stronghold 2004. Avian Diseases 48:928–934. in southeast Asia. Nature (London) 436:191– 11. Brown, I.H., D.J. Alexander, P. Chakraverty, 192. P.A. Harris, and R.J. Manvell. 1994. Isolation of 21. Choi, Y.K., H. Ozaki, R.J. Webby, R.G. Webster, an infl uenza A virus of unusual subtype (H1N7) J.S. Peiris, L. Poon, C. Butt, Y.H. Leung, and Y. from pigs in England, and the subsequent experi- Guan. 2004. Continuing evolution of H9N2 infl u- mental transmission from pig to pig. Veterinary enza viruses in Southeastern China. Journal of Microbiology 39:125–134. Virology 78:8609–8614. 12. Brown, J.D., D.E. Swayne, R.J. Cooper, R.E. 22. Choi, Y.K., T.D. Nguyen, H. Ozaki, R.J. Webby, Burns, and D.E. Stallknecht. 2007. Persistence of P. Puthavathana, C. Buranathal, A. Chaisingh, P. H5 and H7 avian infl uenza viruses in water. Auewarakul, N.T.H. Hanh, S.K. Ma, P.Y. Hui, Y. Avian Diseases 51:285–289. Guan, J. Peiris, Sr., and R.G. Webster. 2005. 13. Brugh, M. 1998. Re-examination of pathogenic- Studies of H5N1 infl uenza virus infection of pigs ity, virulence and lethality. In: D.E. Swayne and by using viruses isolated in Vietnam and Thailand R.D. Slemons (eds.). Proceedings of the Fourth in 2004. Journal of Virology 79:10821–10825. International Symposium on Avian Infl uenza, 23. Choi, Y.K., S.H. Seo, J.A. Kim, R.J. Webby, and May 29–31, 1997, Athens, Georgia. Symposium R.G. Webster. 2005. Avian infl uenza viruses in on Avian Infl uenza, US Animal Health Associa- Korean live poultry markets and their pathogenic tion: Richmond, VA, pp. 129–132. potential. Virology 332:529–537. 14. Brugh, M., and D.C. Johnson. 1987. Epidemiol- 24. Chumpolbanchorn, K., N. Suemanotham, N. ogy of avian infl uenza in domestic poultry. In: Siripara, B. Puyati, and K. Chaichoune. 2006. B.C. Easterday and C.W. Beard (eds.). Proceed- The effect of temperature and UV light on infec- ings of the Second International Symposium on tivity of avian infl uenza virus (H5N1, Thai fi eld Avian Infl uenza, Georgia Center for Continuing strain) in chicken fecal manure. Southeast Asian Education, the University of Georgia, Athens, Journal of Tropical Medicine and Public Health Georgia, USA, September 3–5, 1986. Sympo- 37:102–105. sium on Avian Infl uenza, US Animal Health 25. Committee on Foreign Animal Diseases. 1998. Association: Richmond, VA, pp. 177–186. Appendix 3: Cleaning and disinfection. In: 15. Callan, R.J., G. Early, H. Kida, and V.S. Hinshaw. Foreign Animal Diseases. U.S. Animal Health 1995. The appearance of H3 infl uenza viruses in Association: Richmond, VA, pp. 445–448. seals. Journal of General Virology 76:199–203. 26. Cox, N.J., F. Fuller, N. Kaverin, H.D. Klenk, 16. Cappucci, D.T., Jr., D.C. Johnson, M. Brugh, R.A. Lamb, B.W. Mahy, J.W. McCauley, K. T.M. Smith, C.F. Jackson, J.E. Pearson, and D.A. Nakamura, P. Palese, and R.G. Webster. 2000. Senne. 1985. Isolation of avian infl uenza virus Orthomyxoviridae. In: M.H. Van Regenmortel, (subtype H5N2) from chicken eggs during a C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, natural outbreak. Avian Diseases 29:1195–1200. M.K. Estes, S.M. Lemon, J. Maniloff, M.A. 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 79

Mayo, D.J. McGeoch, C.R. Pringle, and R.B. break of contagious interstitial pneumonia in Wickner (eds.). Virus taxonomy. Seventh report mink. Acta Veterinarius Scandinavia 27:497–504. of the International Committee on Taxonomy of 37. United Nations Food and Agriculture Organiza- Viruses. Academic Press: San Diego, pp. 585– tion. 2006. Production system characteristics. 597. Available at http://www.fao.org/docs/eims/ 27. Crawford, P.C., E.J. Dubovi, W.L. Castleman, I. upload//214190/ProductionSystemsCharacteris- Stephenson, E.P. Gibbs, L. Chen, C. Smith, R.C. tics.pdf. Food and Agriculture Organization: Hill, P. Ferro, J. Pompey, R.A. Bright, M.J. Rome. Medina, C.M. Johnson, C.W. Olsen, N.J. Cox, 38. Fichtner, G.J. 1987. The Pennsylvania/Virginia A.I. Klimov, J.M. Katz, and R.O. Donis. 2005. experience in eradication of avian infl uenza Transmission of equine infl uenza virus to dogs. (H5N2). In: B.C. Easterday and C.W. Beard Science 310:482–485. (ed.). Proceedings of the Second International 28. Crawford, R.D. 1995. Origin, history, and dis- Symposium on Avian Infl uenza, Georgia Center tribution of commercial poultry. In: P. Hunton for Continuing Education, the University of (ed.). Poultry Production. Elsevier: Amsterdam, Georgia, Athens, Georgia, USA, September 3–5, pp. 1–21. 1986. Symposium on Avian Infl uenza, US Animal 29. Das, A.M., E. Spackman, C. Thomas, D.E. Health Association: Richmond, VA, pp. 33–38. Swayne, and D.L. Suarez. 2007. Detection of 39. Forman, A.J., I.M. Parsonson, and W.J. Doughty. H5N1 high pathogenicity avian infl uenza virus in 1986. The pathogenicity of an avian infl uenza meat and tracheal samples from experimentally virus isolated in Victoria. Australian Veterinary infected chickens. Avian Diseases, in press. Journal 63:294–296. 30. Dunn, P.A., E.A. Wallner-Pendleton, H. Lu, D.P. 40. Fouchier, R.A.M., P.M. Schneeberger, F.W. Shaw, D. Kradel, D.J. Henzler, P. Miller, D.W. Rozendaal, J.M. Broekman, S.A.G. Kemink, V. Key, M. Ruano, and S. Davison. 2003. Summary Munstert, T. Kuiken, G.F. Rimmelzwaan, M. of the 2001–02 Pennsylvania H7N2 low pathoge- Schutten, G.J.J. van Doornum, G. Koch, A. nicity avian infl uenza outbreak in meat type Bosman, M. Koopmans, and A.D.M.E. Oster- chickens. Avian Diseases 47(Suppl):812–816. haus. 2004. Avian infl uenza A virus (H7N7) asso- 31. Easterday, B.C., V.S. Hinshaw, and D.A. Halvor- ciated with human conjunctivitis and a fatal case son. 1997. Infl uenza. In: B.W. Calnek, H.J. of acute respiratory distress syndrome. Proceed- Barnes, C.W. Beard, L.R. McDougald, and Y.M. ings of the National Academy of Sciences U S A Saif (eds.). Diseases of Poultry, 10th ed. Iowa 101:1356–1361. State University Press: Ames, IA, pp. 583–605. 41. Geraci, J.R., D.J. St.Aubin, I.K. Barker, R.G. 32. Easterday, B.C., and K. Van Reeth. 1999. Swine Webster, V.S. Hinshaw, W.J. Bean, H.L. Ruhnke, infl uenza. In: B.E. Straw, S. D’Allaire, W.L. J.H. Prescott, G. Early, A.S. Baker, S. Madoff, Mengeling, and D.J. Taylor (eds.). Diseases of and R.T. Schooley. 1982. Mass mortality of Swine. Iowa State University Press: Ames, IA, harbor seals: pneumonia associated with infl u- pp. 277–290. enza A virus. Science 215:1129–1131. 33. Ellis, T.M., B.R. Barry, L.A. Bissett, K.C. 42. Gill, F.B. 1995. Ornithology, 2nd ed. W.H. Dyrting, G.S.M. Luk, S.T. Tsim, K. Sturm- Freeman: New York, pp. 3–20. Ramirez, R.G. Webster, Y. Guan, and J.S.M. 43. Gordy, J.F. 1974. Broilers. In: O.A. Hanke, J.L. Peiris. 2004. Investigation of outbreaks of highly Skinner, and J.H. Florea (eds.). American Poultry pathogenic H5N1 avian infl uenza in waterfowl History: 1823–1973. American Poultry Historical and wild birds in Hong Kong in late 2002. Avian Society: Madison, WI, pp. 370–432. Pathology 33:492–505. 44. Halvorson, D., D. Karunakaran, D. Senne, C. 34. Enck, J. 1998. Update on avian infl uenza situa- Kelleher, C. Bailey, A. Abraham, V. Hinshaw, tion in Pennsylvania. In: Proceedings of the and J. Newman. 1983. Epizootiology of avian 102nd Annual Meeting of the United States infl uenza—simultaneous monitoring of sentinel Animal Health Association. U.S. Animal Health ducks and turkeys in Minnesota. Avian Diseases Association: Richmond, VA, pp. 102:632–633. 27:77–85. 35. Englund, L. 2000. Studies on infl uenza viruses 45. Halvorson, D.A. 1987. A Minnesota cooperative H10N4 and H10N7 of avian origin in mink. Vet- control program. In: B.C. Easterday and C. W. erinary Microbiology 74:101–107. Beard (ed.). Proceedings of the Second Interna- 36. Englund, L., B. Klingeborn, and T. Mejerland. tional Symposium on Avian Infl uenza, Georgia 1986. Avian infl uenza A virus causing an out- Center for Continuing Education, the University 80 Avian Influenza

of Georgia, Athens, Georgia, USA, September 56. Homme, P.J., B.C. Easterday, and D.P. Ander- 3–5, 1986. Symposium on Avian Infl uenza, US son. 1970. Avian infl uenza virus infections II. Animal Health Association: Richmond, VA, pp. Experimental epizootology of infl uenza A/turkey/ 327–336. Wisconsin/1966 virus in turkeys. Avian Diseases 46. Halvorson, D.A. 1995. Avian infl uenza control in 14:240–247. Minnesota. Poultry Digest 54:12–19. 57. Homme, P.J., B.C. Easterday, and D.P. Ander- 47. Halvorson, D.A. 2002. Twenty-fi ve years of son. 1970. Avian infl uenza virus infections. II. avian infl uenza in Minnesota. In: Proceedings of Experimental epizootiology of infl uenza A- the 53rd North Central Avian Disease Confer- turkey-Wisconsin-1966 virus in turkeys. Avian ence. NCADC: Minneapolis, pp. 65–69. Diseases 14:240–247. 48. Halvorson, D.A., D.D. Frame, K.A.J. Friendshuh, 58. Hulse-Post, D.J., K.M. Sturm-Ramirez, J. and D.P. Shaw. 1998. Outbreaks of low patho- Humberd, P. Seiler, E.A. Govorkova, S. Krauss, genicity avian infl uenza in U.S.A. In: D.E. C. Scholtissek, P. Puthavathana, C. Buranathai, Swayne and R.D. Slemons (eds.). Proceedings of T.D. Nguyen, H.T. Long, T.S.P. Naipospos, H. the Fourth International Symposium on Avian Chen, T.M. Ellis, Y. Guan, J.S.M. Peiris, and R. Infl uenza, May 29–31, 1997, Athens, Georgia. G. Webster. 2005. Role of domestic ducks in the Symposium on Avian Infl uenza, US Animal propagation and biological evolution of highly Health Association: Richmond, VA, pp. 36– pathogenic H5N1 infl uenza viruses in Asia. Pro- 46. ceedings of the National Academy of Sciences 49. Halvorson, D.A., C.J. Kelleher, and D.A. Senne. U S A 102:10682–10687. 1985. Epizootiology of avian infl uenza: effect of 59. Humberd, J., Y. Guan, and R.G. Webster. 2006. season on incidence in sentinel ducks and domes- Comparison of the replication of infl uenza A tic turkeys in Minnesota. Applied and Environ- viruses in Chinese ring-necked pheasants and mental Microbiology 49:914–919. chukar partridges. Journal of Virology 80:2151– 50. Henzler, D.J., D.C. Kradel, S. Davison, A.F. 2161. Ziegler, D. Singletary, P. DeBok, A.E. Castro, H. 60. Jackwood, M.J.P., and D.E. Swayne. 2007. Lu, R. Eckroade, D. Swayne, W. Lagoda, B. Pathobiology of Asian H5N1 avian infl uenza Schmucker, and A. Nesselrodt. 2003. Epidemiol- virus infections in ducks. Avian Diseases ogy, production losses, and control measures 51(Suppl):250–259. associated with an outbreak of avian infl uenza 61. Karasin, A.I., I.H. Brown, S. Carman, and C.W. subtype H7N2 in Pennsylvania (1996–98). Avian Olsen. 2000. Isolation and characterization of Diseases 47:1022–1036. H4N6 avian infl uenza viruses from pigs with 51. Hinshaw, V.S., G.M. Air, A.J. Gibbs, L. Graves, pneumonia in Canada. Journal of Virology B. Prescott, and D. Karunakaran. 1982. Antigenic 74:9322–9327. and genetic characterization of a novel hemag- 62. Kawaoka, Y., S. Krauss, and R.G. Webster. 1989. glutinin subtype of infl uenza A viruses from Avian-to-human transmission of the PB1 gene of gulls. Journal of Virology 42:865–872. infl uenza A viruses in the 1957 and 1968 pan- 52. Hinshaw, V.S., W.J. Bean, J. Geraci, P. Fiorelli, demics. Journal of Virology 63:4603–4608. G. Early, and R.G. Webster. 1986. Characteriza- 63. Keawcharoen, J., K. Oraveerakul, T. Kuiken, tion of two infl uenza A viruses from a pilot whale. A.M. Fouchier Ron, A. Amonsin, S. Payungporn, Journal of Virology 58:655–656. S. Noppornpanth, S. Wattanodorn, A. Theamboo- 53. Hinshaw, V.S., W.J. Bean, R.G. Webster, J.E. niers, R. Tantilertcharoen, R. Pattanarangsan, N. Rehg, P. Fiorelli, G. Early, J.R. Geraci, D.J.S. Arya, P. Ratanakorn, D.M.E. Osterhaus, and Y. Aubin, and D.J. St. Aubin. 1984. Are seals fre- Poovorawan. 2004. Avian infl uenza H5N1 in quently infected with avian infl uenza viruses? tigers and leopards. Emerging Infectious Diseases Journal of Virology 51:863–865. 10:2189–2191. 54. Hinshaw, V.S., V.F. Nettles, L.F. Schorr, J.M. 64. Koen, J. 1919. A practical method for fi eld diag- Wood, and R.G. Webster. 1986. Infl uenza virus nosis of swine diseases. Journal of the American surveillance in waterfowl in Pennsylvania after Veterinary Medical Association 14:468–470. the H5N2 avian outbreak. Avian Diseases 65. Kothalawala, H., M.J. Toussaint, and E. Gruys. 30:207–212. 2006. An overview of swine infl uenza. Veteri- 55. Hinshaw, V.S., R.G. Webster, and B. Turner. nary Quarterly 28:46–53. 1979. Water-bone transmission of infl uenza A 66. Kou, Z., F.M. Lei, J. Yu, Z.J. Fan, Z.H. Yin, C. viruses? Intervirology 11:66–68. Jia, X, K.J. Xiong, Y.H. Sun, X.W. Zhang, X.M. 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 81

Wu, X.B. Gao, and T. Li, X. 2005. New genotype nikova, R.Y. Podcernjaeva, S.M. Klimenko, V.P. of avian infl uenza H5N1 viruses isolated from Andrejev, and M.A. Yakhno. 1978. Comparison tree sparrows in China. Journal of Virology of infl uenza viruses isolated from man and from 79:15460–15466. whales. Bulletin of the World Health Organiza- 67. Kuiken, T., E.C. Holmes, J. McCauley, G.F. Rim- tion 56:923–930. melzwaan, C.S. Williams, and B.T. Grenfell. 77. Manvell, R.J., P. McKinney, U. Wernery, and 2006. Host species barriers to infl uenza virus K.M. Frost. 2000. Isolation of a highly patho- infections. Science (Washington) 312:394–397. genic infl uenza A virus of subtype H7N3 from a 68. Kuiken, T., G. Rimmelzwaan, D. van Riel, G. van peregrine falcon (Falco peregrinus). Avian Amerongen, M. Baars, R. Fouchier, and A. Oster- Pathology 29:635–637. haus. 2004. Avian H5N1 infl uenza in cats. Science 78. Markwell, D.D., and K.F. Shortridge. 1982. Pos- 306:241. sible waterborne transmission and maintenance 69. Lang, G. 1981. A review of infl uenza in Canadian of infl uenza viruses in domestic ducks. Applied domestic and wild birds. In: R.W. Bankowski and Environmental Microbiology 43:110–115. (ed.). Proceedings of the First International Sym- 79. Mohan, R., Y.M. Saif, G.A. Erickson, G.A. Gus- posium on Avian Infl uenza, April 1981, Belts- tafson, and B.C. Easterday. 1981. Serologic and ville, Maryland. Symposium on Avian Infl uenza, epidemiologic evidence of infection in turkeys US Animal Health Association: Richmond, VA, with an agent related to the swine infl uenza virus. pp. 21–27. Avian Diseases 25:11–16. 70. Lang, G., A. Gagnon, and J.R. Geraci. 1981. Iso- 80. Naeem, K., A. Ullah, R.J. Manvell, and D.J. lation of an infl uenza A virus from seals. Archives Alexander. 1999. Avian infl uenza A subtype of Virology 68:189–195. H9N2 in poultry in Pakistan. Veterinary Record 71. Lang, G., B.T. Rouse, O. Narayan, A.E. Fergu- 145:560. son, and M.C. Connell. 1968. A new infl uenza 81. Narayan, O., G. Lang, and B.T. Rouse. 1969. A virus infection in turkeys. I. Isolation and charac- new infl uenza A virus infection in turkeys. IV. terization of virus 6213. Canadian Veterinary Experimental susceptibility of domestic birds to Journal 9:22–29. virus strain turkey-Ontario 7732–1966. Archiv 72. Li, C., K. Yu, G. Tian, D. Yu, L. Liu, B. Jing, J. fur die Gesamte Virusforschung 26:149–165. Ping, and H. Chen. 2005. Evolution of H9N2 82. Narayan, O., G. Lang, and B.T. Rouse. 1969. A infl uenza viruses from domestic poultry in main- new infl uenza A virus infection in turkeys. V. land China. Virology 340:70–83. Pathology of the experimental disease by strain 73. Liu, J., H. Xiao, F. Lei, Q. Zhu, K. Qin, X.-W. turkey/Ontario 7732/66. Archiv fur die Gesamte Zhang, X.-L. Zhang, D. Zhao, G. Wang, Y. Feng, Virusforschung 26:166–182. J. Ma, W. Liu, J. Wang, and G.F. Gao. 2005. 83. Nettles, V.F., J.M. Wood, and R.G. Webster. Highly pathogenic H5N1 infl uenza virus infec- 1985. Wildlife surveillance associated with an tion in migratory birds. Science 309:1206. outbreak of lethal H5N2 avian infl uenza in 74. Liu, J.H., K. Okazaki, H. Ozaki, Y. Sakoda, Q.M. domestic poultry. Avian Diseases 29:733–741. Wu, F.Y. Chen, and H. Kida. 2003. H9N2 infl u- 84. Nili, H., and K. Asasi. 2002. Natural cases and an enza viruses prevalent in poultry in China are experimental study of H9N2 avian infl uenza in phylogenetically distinct from A/quail/Hong commercial broiler chickens of Iran. Avian Kong/G1/97 presumed to be the donor of the Pathology 31:247–252. internal protein genes of the H5N1 Hong Kong/97 85. Ninomiya, A., A. Takada, K. Okazaki, K.E. virus. Avian Pathology 32:551–560. Shortridge, and H. Kida. 2002. Seroepidemio- 75. Loeffen, W.L.A., and G. Koch. 2003. Avian logical evidence of avian H4, H5, and H9 infl u- infl uenza (H7N7) in Dutch pigs. PROMED. enza A virus transmission to pigs in southeastern 20030520.1240. Available at http://www.promed- China. Veterinary Microbiology 88:107–114. mail.org/pls/promed/f?p = 2400:1202:444581495 86. OIE. 2004. Canada. New activities of the veteri- 7589253518::NO::F2400_P1202_CHECK_ nary services. In: World Animal Health. OIE: DISPLAY,F2400_P1202_PUB_MAIL_ID: Paris, pp. 81–89. X,21644. CIDC. Accessed on April 23, 2007. 87. Olsen, B., V.J. Munster, A. Wallensten, et al. 76. Lvov, D.K., V.M. Zdanov, A.A. Sazonov, N.A. 2006. Global patterns of infl uenza A virus in wild Braude, E.A. Vladimirtceva, L.V. Agafonova, birds. Science 312(21 April 2006):384–388. E.I. Skljanskaja, N.V. Kaverin, V.I. Reznik, T.V. 88. Peiris, M., K.Y. Yuen, C.W. Leung, K.H. Chan, Pysina, A.M. Oserovic, A.A. Berzin, I.A. Mjas- P.L. Ip, R.W. Lai, W.K. Orr, and K.F. Shortridge. 82 Avian Influenza

1999. Human infection with infl uenza H9N2 infl uenza H5N1 in viverrids: implications for [letter]. Lancet 354:916–917. wildlife health and conservation. Proceedings 89. Perdue, M.L., and D.L. Suarez. 2000. Structural Biological Sciences 273:1729–1732. features of the avian infl uenza virus hemaggluti- 101. Rohm, C., T. Horimoto, Y. Kawaoka, J. Suss, and nin that infl uence virulence. Veterinary Microbi- R.G. Webster. 1995. Do hemagglutinin genes of ology 74:77–86. highly pathogenic avian infl uenza viruses consti- 90. Perdue, M.L., D.L. Suarez, and D.E. Swayne. tute unique phylogenetic lineages? Virology 1999. Avian infl uenza in the 1990’s. Poultry and 209:664–670. Avian Biology Reviews 11:1–20. 102. Sandhu, T. 1981. Infl eunza A virus infection of 91. Perez, D.R., W. Lim, J.P. Seiler, G. Yi, M. Peiris, domestic ducks. In: R.W. Bankowski (ed.). Pro- K.F. Shortridge, and R.G. Webster. 2003. Role of ceedings of the First International Symposium on quail in the interspecies transmission of H9 infl u- Avian Infl uenza, April 1981, Beltsville, Mary- enza A viruses: molecular changes on HA that land. Symposium on Avian Infl uenza, US Animal correspond to adaptation from ducks to chickens. Health Association: Richmond, VA, pp. 93–99. Journal of Virology 77:3148–3156. 103. Sawabe, K., K. Hoshino, H. Isawa, T. Sasaki, T. 92. Perk, S., A. Panshin, E. Shihmanter, I. Gissin, S. Hayashi, Y. Tsuda, H. Kurahashi, K. Tanabayashi, Pokamunski, M. Pirak, and M. Lipkind. Ecology A. Hotta, T. Saito, A. Yamada, and M. Kobayashi. and molecular epidemiology of H9N2 avian 2006. Detection and isolation of highly patho- infl uenza viruses isolated in Israel during 2000– genic H5N1 avian infl uenza A viruses from blow 2004 epizootic. Developments in Biologicals fl ies collected in the vicinity of an infected poultry 124:201–209. farm in Kyoto, Japan, 2004. American Journal of 93. Perkins, L.E.L., and D.E. Swayne. 2002. Patho- Tropical Medicine and Hygiene 75:327–332. genicity of a Hong Kong-origin H5N1 highly 104. Schafer, W. 1955. Vergleichende sero-immuno- pathogenic avian infl uenza virus for emus, geese, logische untersuchungen uber die viren der infl u- ducks, and pigeons. Avian Diseases 46:53–63. enza unf klassichen gefl ugelpest. Zeitschrift fur 94. Petek, M. 1981. Current situation in Italy. In: Naturforschung 10B:81–91. R.W. Bankowski (ed.). Proceedings of the First 105. Scholtissek, C., I. Koennecke, and R. Rott. 1978. International Symposium on Avian Infl uenza, Host range recombinants of fowl plague (infl u- April 1981, Beltsville, Maryland. Symposium on enza A) virus. Virology 91:79–85. Avian Infl uenza, US Animal Health Association: 106. Senne, D.A., J.C. Pedersen, and B. Panigrahy. Richmond, VA, pp. 31–34. 2005. Live-bird markets in the Northeastern 95. Pingel, H. 2004. Duck and geese production United States: a source of avian infl uenza in com- around the world. World Poultry 20:26–28. mercial poultry. In: R.S. Schrijver and G. Koch 96. Power, C.A. 2005. An investigation into the (eds.). Avian Infl uenza. Prevention and Control. potential role of aerosol dispersion of dust from Springer: Dordrecht, pp. 19–24. poultry barns as a mode of disease transmission 107. Senne, D.A. 2003. Exotic Newcastle disease in during an outbreak of avian infl uenza (H7:N3) in California. Epidemiology and virus characteriza- Abbotsford, BC in 2004. Bulletin of the Aquacul- tion. Proceedings of the National Meeting on ture Association of Canada 105:7–14. Poultry Health and Processing 38:9–16. 97. Reid, A.H., and J.K. Taubenberger. 1999. The 108. Senne, D.A. 2004. Avian infl uenza. In: Proceed- 1918 fl u and other infl uenza pandemics: “over ings of the 108th Annual Meeting of the U.S. there” and back again. Laboratory Investigation Animal Health Association. US Animal Health 79:95–101. Association: Richmond, VA, pp. 545–547. 98. Reid, A.H., and J.K. Taubenberger. 2003. The 109. Senne, D.A., T.J. Holt, and B.L. Akey. 2005. An origin of the 1918 pandemic infl uenza virus: a overview of the 2002 outbreak of low-pathogenic continuing enigma. Journal of General Virology H7N2 avian infl uenza in Virginia, West Virginia 84:2285–2292. and North Carolina. In: R.S. Schrijver and G. 99. Reid, A.H., J.K. Taubenberger, and T.G. Fanning. Koch (eds.). Avian Infl uenza. Prevention and 2004. Evidence of an absence: the genetic origins Control. Springer: Dordrecht, pp. 41–47. of the 1918 pandemic infl uenza virus. Nature 110. Senne, D.A., B. Panigrahy, and R.L. Morgan. Reviews Microbiology 2:909–914. 1994. Effect of composting poultry carcasses on 100. Roberton, S., I, D.J. Bell, G.J.D. Smith, J.M. survival of exotic avian viruses: highly patho- Nicholls, K.H. Chan, D.T. Nguyen, P.Q. Tran, U. genic avian infl uenza (HPAI) virus and adenovi- Streicher, L.L.M. Poon, H. Chen, P. Horby, M. rus of egg drop syndrome-76. Avian Diseases Guardo, Y. Guan, and J.S.M. Peiris. 2006. Avian 38:733–737. 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 83

111. Senne, D.A., J.E. Pearson, and B. Panigrahy. A H5N1 in a dog. Emerging Infectious Diseases 1992. Live poultry markets: a missing link in the 12:1744–1747. epidemiology of avian infl uenza. In: B.C. Easter- 122. Songserm, T., R. Jam-on, N. Sae-Heng, and N. day (ed.). Proceedings of the Third International Meemak. 2006. Survival and stability of HPAI Symposium on Avian Infl uenza, May 27–29, H5N1 in different environments and susceptibil- 1992, Madison, Wisconsin. Symposium on Avian ity to disinfectants. Developments in Biologicals Infl uenza, US Animal Health Association: Rich- 124:254. mond, VA, pp. 50–58. 123. Spackman, E., D.A. Senne, S. Davison, and D.L. 112. Shope, R. 1931. Swine infl uenza. III. Filtration Suarez. 2003. Sequence analysis of recent H7 experiments and etiology. Journal of Experimen- avian infl uenza viruses associated with three dif- tal Medicine 54:373–385. ferent outbreaks in commercial poultry in the 113. Shortridge, K.F. 1981. Epidemiology of avian United States. Journal of Virology 77:13399– infl uenza and sources of infection in domestic 13402. species. In: R.W. Bankowski (ed.). Proceedings 124. Stallknecht, D.E. 1998. Ecology and epide- of the First International Symposium on Avian miology of avian infl uenza viruses in wild bird Infl uenza, April 1981, Beltsville, Maryland. Sym- populations: waterfowl, shorebirds, pelicans, cor- posium on Avian Infl uenza, US Animal Health morants, etc. In: D.E. Swayne and R.D. Slemons Association: Richmond, VA, pp. 54–68. (eds.). Proceedings of the Fourth International 114. Shortridge, K.F. 1981. Avian infl uenza in Hong Symposium on Avian Infl uenza, May 29–31, Kong. In: R.W. Bankowski (ed.). Proceedings of 1997, Athens, Georgia. Symposium on Avian the First International Symposium on Avian Infl uenza, US Animal Health Association: Rich- Infl uenza, April 1981, Beltsville, Maryland. Sym- mond, VA, pp. 61–69. posium on Avian Infl uenza, US Animal Health 125. Stallknecht, D.E., M.T. Kearney, S.M. Shane, Association: Richmond, VA, p. 29. and P.J. Zwank. 1990. Effects of pH, temperature, 115. Shortridge, K.F. 1982. Avian infl uenza A viruses and salinity on persistence of avian infl uenza of southern China and Hong Kong: ecological viruses in water. Avian Diseases 34:412–418. aspects and implications for man. Bulletin of the 126. Stallknecht, D.E., E. Nagy, D.B. Hunter, and World Health Organization 60:129–135. R.D. Slemons. 2007. Avian infl uenza. In: N.J. 116. Shortridge, K.F. 1999. Poultry and the infl uenza Thomas, D.B. Hunter, and C.T. Atkinson (eds.). H5N1 outbreak in Hong Kong, 1997: abridged Infectious Diseases of Wild Birds. Blackwell chronology and virus isolation. Vaccine 17:S26– Publishing: Ames, IA, pp. 108–130. S29. 127. Stallknecht, D.E., and S.M. Shane. 1988. Host 117. Shortridge, K.F., N.N. Zhou, Y. Guan, P. Gao, T. range of avian infl uenza virus in free-living birds. Ito, Y. Kawaoka, S. Kodihalli, S. Krauss, D. Veterinary Research Communications 12:125– Markhill, G. Murti, M. Norwood, D. Senne, L. 141. Sims, A. Takada, and R.G. Webster. 1998. Char- 128. Stallknecht, D.E., S.M. Shane, M.T. Kearney, acterization of avian H5N1 infl uenza viruses and P.J. Zwank. 1990. Persistence of avian infl u- from poultry in Hong Kong. Virology 252:331– enza viruses in water. Avian Diseases 34:406– 342. 411. 118. Sims, L.D., J. Domenech, C. Benigno, S. Kahn, 129. Stubbs, E.L. 1948. Fowl pest. In: H.E. Biester and A. Kamata, J. Lubroth, V. Martin, and P. Roeder. L.H. Schwarte (eds.). Diseases of Poultry, 2nd ed. 2005. Origin and evolution of highly pathogenic Iowa State University Press: Ames, IA, pp. 603– H5N1 avian infl uenza in Asia. Veterinary Record 614. 157:159–164. 130. Sturm-Ramirez, K.M., D.J. Hulse-Post, E.A. 119. Sims, L.D., T.M. Ellis, K.K. Liu, K. Dyrting, H. Govorkova, J. Humberd, P. Seiler, P. Puthava- Wong, M. Peiris, Y. Guan, and K.E. Shortridge. thana, C. Buranathai, T.D. Nguyen, A. Chaisingh, 2003. Avian infl uenza in Hong Kong 1997–2002. H.T. Long, T.S.P. Naipospos, H. Chen, T.M. Avian Diseases 47(Suppl):832–838. Ellis, Y. Guan, J.S.M. Peiris, and R.G. Webster. 120. Smith, P., and C. Daniel. 2000. The Chicken 2005. Are ducks contributing to the endemicity Book. University of Georgia Press: Athens, GA, of highly pathogenic H5N1 infl uenza virus in pp. 1–380. Asia? Journal of Virology 79:11269–11279. 121. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- 131. Suarez, D.L. 2000. Evolution of avian infl uenza Heng, N. Pariyothorn, S. Payungpom, A. Theam- viruses. Veterinary Microbiology 74:15–27. booniers, S. Chutinimitkul, R. Thanawongnuwech, 132. Suarez, D.L., M. Garcia, J. Latimer, D. Senne, and Y. Poovorawan. 2006. Fatal avian infl uenza and M. Perdue. 1999. Phylogenetic analysis of 84 Avian Influenza

H7 avian infl uenza viruses isolated from the live Y.M. Saif. 2005. Isolation and characterization of bird markets of the Northeast United States. H3N2 infl uenza A virus from turkeys. Avian Dis- Journal of Virology 73:3567–3573. eases 49:207–213. 133. Suarez, D.L., and C.S. Schultz. 2000. Immunology 144. Taubenberger, J.K. 2003. Fixed and frozen fl u: of avian infl uenza virus: a review. Developmental the 1918 infl uenza and lessons for the future. and Comparative Immunology 24:269–283. Avian Diseases 47:789–791. 134. Suarez, D.L., E. Spackman, and D.A. Senne. 145. Taubenberger, J.K., J.V. Hultin, and D.M. 2003. Update on molecular epidemiology of H1, Morens. 2007. Discovery and characterization H5, and H7 infl uenza virus infections in poultry of the 1918 pandemic infl uenza virus in his- in North America. Avian Diseases 47:888–897. torical context. Antiviral Therapy 12:581– 135. Swayne, D.E. 2000. Understanding the ecology 591. and epidemiology of avian infl uenza viruses: 146. Thomas, C., and D.E. Swayne. 2007. Thermal implications for zoonotic potential. In: C.C. inactivation of H5N1 high pathogenicity avian Brown and C.A. Bolin (eds.). Emerging Diseases infl uenza virus in naturally infected chicken meat. of Animals. ASM Press: Washington, D.C., pp. Journal of Food Protection 70:674–680. 101–130. 147. Timoney, P.J. 1996. Equine infl uenza. Compara- 136. Swayne, D.E. 2007. Understanding the complex tive Immunology, Microbiology and Infectious pathobiology of high pathogenicity avian infl u- Diseases 19:205–211. enza viruses in birds. Avian Diseases 51:242– 148. Trock, S.C. 1998. Epidemiology of infl uenza 249. in live bird markets and ratite farms. In: D.E. 137. Swayne, D.E., and B. Akey. 2005. Avian infl u- Swayne and R.D. Slemons (eds.). Proceedings of enza control strategies in the United States of the Fourth International Symposium on Avian America. In: R.S. Schrijver and G. Koch (eds.). Infl uenza, May 29–31, 1997, Athens, Georgia. Avian Infl uenza. Prevention and Control. Symposium on Avian Infl uenza, US Animal Springer: Dordrecht, pp. 113–130. Health Association: Richmond, VA, pp. 76– 138. Swayne, D.E., and J.R. Beck. 2005. Experimental 78. study to determine if low pathogenicity and high 149. Tsai, C.P., and M.J. Pan. 2003. New H1N2 and pathogenicity avian infl uenza viruses can be H3N1 infl uenza viruses in Taiwanese pig herds. present in chicken breast and thigh meat follow- Veterinary Record 153:408. ing intranasal virus inoculation. Avian Diseases 150. U.S. Department of Agriculture. 1999. Agricul- 49:81–85. tural Statistics 1999. U.S. Department of Agricul- 139. Swayne, D.E., M. Garcia, J.R. Beck, N. Kinney, ture: Washington, D.C., pp. 1–485. and D.L. Suarez. 2000. Protection against diverse 151. U.S. Department of Agriculture. 2007. Dairy and highly pathogenic H5 avian infl uenza viruses in poultry statistics. In: Agricultural Statistics. U.S. chickens immunized with a recombinant fowlpox Department of Agriculture: Washington, D.C., vaccine containing an H5 avian infl uenza hemag- pp. VIII-1–VIII-42. Available at http://www. glutinin gene insert. Vaccine 18:1088–1095. usda.gov/nass/pubs/agr01/acro01.htm. 140. Swayne, D.E., and D.A. Halvorson. 2008. Infl u- 152. United States Geological Survey. 2007. List of enza. In: Y.M. Saif, J.R. Glisson, A.M. Fadly, species affected by H5N1 (avian infl uenza). Ref- L.R. McDougald, and L. Nolan (eds.). Diseases erenced reports of highly pathogenic avian infl u- of Poultry, 12th ed. Blackwell: Ames, IA, in enza H5N1 in wildlife and domestic animals. press. United States Geological Survey: Madison, WI. 141. Swayne, D.E., M.J. Radin, T.M. Hoepf, and R.D. Available at http://www.nwhc.usgs.gov/disease_ Slemons. 1994. Acute renal failure as the cause information/avian_influenza/affected_species_ of death in chickens following intravenous inocu- chart.jsp. Accessed on July 13, 2007. lation with avian infl uenza virus A/chicken/ 153. van der Goot, J.A., M.C.M. de Jong, G. Koch, Alabama/7395/75 (H4N8). Avian Diseases and M. van Boven. 2003. Comparison of the 38:151–157. transmission characteristics of low and high 142. Swayne, D.E., and D.L. Suarez. 2000. Highly pathogenicity avian infl uenza A virus (H5N2). pathogenic avian infl uenza. Revue Scientifi que et Epidemiology and Infection 131:1003–1013. Technique Offi ce International des Epizooties 154. van der Goot, J.A., G. Koch, M.C.M. de Jong, 19:463–482. and M. van Boven. 2005. Quantifi cation of the 143. Tang, Y., C.W. Lee, Y. Zhang, D.A. Senne, R. effect of vaccination on transmission of avian Dearth, B. Byrum, D.R. Perez, D.L. Suarez, and infl uenza (H7N7) in chickens. Proceedings of the 4 / Epidemiology of Avian Influenza in Agricultural and Other Man-Made Systems 85

National Academy of Sciences U S A 102:18141– replication and characterization of infl uenza 18146. viruses in ducks. Virology 84:268–278. 155. van der Goot, J.A., M. van Boven, M.C. de Jong, 160. Xu, C.T., W.X. Fan, R. Wei, and H.K. Zhao. 2004. and G. Koch. 2007. Effect of vaccination on Isolation and identifi cation of swine infl uenza transmission of HPAI H5N1: the effect of a single recombinant A/swine/Shandong/1/2003(H9N2) vaccination dose on transmission of highly patho- virus. Microbes and Infection 6:919–925. genic avian infl uenza H5N1 in Peking ducks. 161. Xu, X., K. Subbarao, N.J. Cox, and Y. Guo. 1999. Avian Diseases 51:323–324. Genetic characterization of the pathogenic infl u- 156. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. enza A/goose/Guangdong/1/96 (H5N1) virus: Chambers, and Y. Kawaoka. 1992. Evolution and similarity of its hemagglutinin gene to those of ecology of infl uenza A viruses. Microbiology H5N1 viruses from the 1997 outbreaks in Hong Reviews 56:152–179. Kong. Virology 261:15–19. 157. Webster, R.G., W.J. Bean, Y. Kawaoka, and D. 162. Yingst, S.L., M.D. Saad, and S.A. Felt. 2006. Senne. 1986. Characterization of H5N2 infl uenza Qinghai-like H5N1 from domestic cats, northern viruses from birds in live poultry markets in Iraq. Emerging Infectious Diseases 12:1295–1297. United States. Proceedings of the United States 163. Zhang, G., D. Shoham, D. Gilichinsky, S. Animal Health Association 90:278–286. Davydov, J.D. Castello, and S.O. Rogers. 2006. 158. Webster, R.G., V.S. Hinshaw, W.J. Bean, K.L. Evidence of infl uenza a virus RNA in Siberian van Wyke, J.R. Geraci, D.J. St Aubin, and lake ice. Journal of Virology 80:12229–12235. G. Petursson. 1981. Characterization of an in - 164. Ziegler, A.F., S. Davison, H. Acland, and R.J. fl uenza A virus from seals. Virology 113:712– Eckroade. 1999. Characteristics of H7N2 (non- 724. pathogenic) avian infl uenza virus infections in 159. Webster, R.G., M. Yakhno, V.S. Hinshaw, W.J. commercial layers, in Pennsylvania, 1997–98. Bean, and K.G. Murti. 1978. Intestinal infl uenza: Avian Diseases 43:142–149. 5 Pathobiology of Avian Influenza Virus Infections in Birds and Mammals

David E. Swayne and Mary Pantin-Jackwood

INTRODUCTION genicity (LP) and high pathogenicity (HP), based Infl uenza A viruses have infected a wide variety of on the ability to produce disease and death in the domestic, captive, and wild animal species. In major domestic poultry species, the chicken (Gallus horses, pigs, and humans, these viruses are clinically domesticus) (130). Various pathophysiological and and/or economically important agents of disease and pathological changes are important to our under- endemic throughout the world (1, 27, 38, 84, 96, standing of the complex biology of low pathogenic- 140, 141, 145). By contrast, wild aquatic birds, espe- ity avian infl uenza (LPAI) and high pathogenicity cially of the orders Anseriformes (ducks, geese, and avian infl uenza (HPAI) viruses in various host swans) and Charadriiformes (shorebirds, gulls, terns, species and are discussed throughout this chapter. and auks) (55, 107, 113, 131, 141) are natural res- Several reviews on pathology of AI in poultry ervoirs of avian infl uenza (AI) viruses (i.e., infl u- have been published (2, 13, 27, 45, 114, 121, 130, enza A viruses that infect birds). These AI viruses 131). In the following sections, experimental studies are highly host adapted, typically replicating in epi- and information obtained from fi eld outbreaks on AI thelial cells of the gastrointestinal tract and produc- are summarized (1, 4, 5, 8, 10, 13–15, 23, 27, 28, ing subclinical infections in their native species. 50, 51, 61, 62, 66, 67, 74, 82, 85, 94, 96, 97, 105, Periodically, these AI viruses have been transmitted 108, 109, 117, 123, 124, 127–129, 149). to other hosts, including mammals and domestic poultry. Usually, these infections are sporadic and GENERAL CONCEPTS IN PATHOBIOLOGY transitory, producing subclinical infection with AI infection in domestic poultry produces syn- seroconversion, but infrequent illness may occur. dromes ranging from subclinical infection, to respi- However, AI viruses occasionally adapt to a new ratory disease and drops in egg production, to severe, species, such as gallinaceous poultry, establishing systemic disease with near 100% mortality. Infl u- infections and causing disease and even death. Such enza A virus infections of poultry can be divided viruses may become endemic within the population, into two distinct groups based on their ability to causing recurring disease problems. cause disease in chickens: HPAI and LPAI. The AI viruses can be categorized into subtypes based LPAI viruses produce respiratory disease and drops on serological typing of the two surface glycopro- in egg production in all types of poultry species. In teins, the hemagglutinin (H) and neuraminidase (N) chickens and other gallinaceous poultry, HPAI (131). There are 16 different hemagglutinin (H1– viruses cause severe systemic disease with very high H16) and 9 different neuraminidase (N1–N9) sub- mortality, but such viruses typically do not cause types (35, 145). In addition, AI viruses can be further illness or death in ducks and geese. However, since classifi ed into two different pathotypes, low patho- 1996, H5N1 HPAI viruses have emerged in Asia

Avian Influenza Edited by David E. Swayne 87 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 88 Avian Influenza that have evolved from producing disease and mor- especially if accompanied by secondary bacterial, tality only in gallinaceous poultry and domestic fungal, or viral infections. On rare occasions, the geese to causing clinical disease, lesions, and fre- LPAI viruses have spread systemically, replicating quent deaths in domestic ducks and a variety of and causing damage in the kidney tubules, pancre- captive and wild bird species (29, 86, 95). Clinical atic acinar epithelium, and other organs which signs of disease are extremely variable and depend contain epithelial cells with trypsin-like enzymes. on many factors, including host species, virus strain, By contrast, HPAI viruses in poultry have initial age, sex, concurrent infections, acquired immunity, replication in nasal epithelium, with visualization of and environmental factors. AI viral antigens in respiratory epithelium by 16 hours after exposure (121) (Figure 5.1). By 24 hours, Virus Replication Sites the nasal epithelium is ulcerated and inﬂ amed with To achieve multiple replication cycles and produc- virus in submucosal macrophages, heterophils, and tive infections, an AI virus must have its hemag- capillary endothelial cells (121, 122) (Figures 5.2 glutinin (HA0) cleaved into the HA1 and HA2 and 5.3). The macrophage and heterophil play key segments (122). For LPAI viruses, such cleavage is roles in this initial replication and dissemination of accomplished by trypsin-like enzymes located HPAI viruses. The virus replicates within endothe- within speciﬁ c epithelial cells, present in respiratory lial cells and spreads via the vascular or lymphatic secretions or produced by some types of bacteria. systems to infect and replicate in a variety of cell By contrast, the hemagglutinin of HPAI viruses can types within visceral organs, brain, and skin. This be cleaved by the same trypsin-like enzymes as for initial visceral replication may be seen as early as LPAI viruses or the hemagglutinin can be cleaved 24 hours after intranasal inoculation, and by 48 by ubiquitous proteases of the furin enzyme family hours, the virus titers are high and the lesions are that are contained in most cells within the body. This severe. However, with some HPAI viruses, the differential cleavage of hemagglutinin by different viremia may occur without extensive replication in classes of enzymes is responsible for variations in vascular endothelial cells and with more extensive the tissue sites for AI virus replication and lesion replication in parenchymal cells of visceral organs. production (Table 5.1). Clinical signs and death are due to multiorgan In poultry, the nasal cavity is the predominant failure. Damage caused by AI viruses is the result initial site of LPAI virus replication with release of of one of three processes: (1) direct virus replication virions and infection of other cells in the respiratory in cells, tissues, and organs; (2) indirect effects from tract and the intestinal tract (121, 122, 131). Illness production of cellular mediators, such as cytokines; or death is most often from respiratory damage, and (3) ischemia from vascular thrombosis (131).

Table 5.1. Histologic lesions associated with avian inﬂ uenza in naturally or intranasally infected chickens. References Lesion HPAI LPAI

Nasal cavity 49 13, 49, 74, 120 Nasal epithelial necrosis Rhinitis, sinusitis Trachea 74, 105, 126 13, 74, 105, 120 Tracheitis Lung 13, 74, 105, 120, 126 49, 74, 105, 120 Edema Interstitial pneumonia Heart 13, 17, 49, 62, 74, 96, 117, 120 Myocyte necrosis Myocarditis Table 5.1. Continued References Lesion HPAI LPAI

Brain 1, 61, 74, 96 Edema of neuropil Neuronal necrosis Gliosis Perivascular cuffi ng Meningoencephalitis Enteric tract 13, 49, 74, 96, 120 Hemorrhage Lymphoid depletion Epithelial necrosis Enteritis Pancreas 1, 13, 49, 74, 96, 120 Vacuolation Pancreatic necrosis Pancreatitis Spleen 17, 49, 74, 96, 117, 120 Lymphoid depletion Splenic necrosis Splenitis Hyperplasia of macrophage- phagocyte system Bursa, thymus, cecal tonsils 96, 120 Lymphoid depletion Phagocytic hyperplasia Necrosis Hemorrhage Adrenal 49, 96, 99 Adrenal necrosis Adrenal adenitis Integument 96 Subcutaneous edema Necrosis Bone marrow 74, 96, 99 Cellular depletion Necrosis Skeletal muscle 1, 49, 74, 96, 120 Myofi ber degeneration Kidney 74, 120 120 (IV inoculation) Tubule necrosis Liver 29, 96, 99 Hepatocellular necrosis Reproductive organs 13 Necrosis Infl ammation

89 90 Avian Influenza

Figure 5.1. Infl uenza A viral antigen in nasal Figure 5.3. Infl uenza A viral antigen in nasal epithelium of a chicken 16 hours post epithelium and underlying capillary intranasal inoculation with A/whooper swan/ endothelial cells and infl ammatory cells of a Mongolia/244/2005 H5N1 HPAI virus. chicken 24 hours post intranasal inoculation Immunohistochemical stain. Bar = 25 μm. with A/whooper swan/Mongolia/244/2005 Figure reprinted with permission from Avian H5N1 HPAI virus. Immunohistochemical stain. = μ Diseases. Copyright held by the American Bar 25 m. Figure reprinted with permission Association of Avian Pathologists, Athens, from Avian Diseases. Copyright held by the Georgia, USA. Source: D. Swayne, U.S. American Association of Avian Pathologists, Department of Agriculture, Agricultural Athens, Georgia, USA. Source: D. Swayne, Research Service, Athens, GA (121). For color U.S. Department of Agriculture, Agricultural detail, please see color plate section. Research Service, Athens, GA (121). For color detail, please see color plate section.

1975 H7 HPAI viruses that were HP for chickens produced either no disease or only mild clinical signs in domestic ducks (Anas platyrhynchos) (3). Differences in lethality and lesion production between bird species have also been observed in gallinaceous birds in experimental studies with various LPAI and HPAI viruses (68, 96, 143). In many situations, the differences in type and severity of disease were not the result of a virus’s ability to Figure 5.2. Ulcerative rhinitis in a chicken 24 infect or not infect a particular species since some hours post intranasal inoculation with A/ birds had infections without disease production. whooper swan/Mongolia/244/2005 H5N1 HPAI However, there has been an association between virus. Hematoxylin and eosin stain. Bar = high virus titers and the presence of necrotic and 75 μm. Figure reprinted with permission from inﬂ ammatory lesions. Avian Diseases. Copyright held by the American Association of Avian Pathologists, Pathobiological Mechanisms Athens, Georgia, USA. Source: D. Swayne, At the subcellular and cellular levels, HPAI viruses U.S. Department of Agriculture, Agricultural produce necrosis and apoptosis in AI virus–infected Research Service, Athens, GA (121). For color avian cells (43, 96). Necrosis has been associated detail, please see color plate section. with high level of virus replication as demonstrated by abundant AI viral nucleoprotein in the nucleus and cytoplasm of infected cells and by high AI virus Species Variation tissue titers (129). Necrosis has been most frequently The ability of individual AI viruses to cause disease reported in neurons of the brain, kidney tubule cells, and the ability of the host to respond to AI virus pancreatic acinar epithelium, cardiac myocytes, vary greatly by bird species. For example, four pre- adrenal cortical cells, and pulmonary epithelial cells 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 91

Figure 5.4. Lung of chicken 24 hours post intranasal inoculation with A/whooper swan/ Mongolia/244/2005 H5N1 HPAI virus, showing hypertrophy, degeneration, necrosis, and apoptosis of endothelial cells. Hematoxylin and eosin stain. Bar = 25 μm. Figure reprinted with permission from Avian Diseases. Copyright held by the American Association of Avian Pathologists, Athens, Georgia, USA. Source: D. Swayne, U.S. Department of Agriculture, Agricultural Research Service, Athens, GA (121). For color detail, please see color plate section.

of infected chickens (117). Apoptotic cell death has been demonstrated in various cell culture systems and involved cytokines including interferon-β and transforming growth factor β (43, 104, 132). In chicken embryos, apoptosis and necrosis may have similar biochemical features and indicate that differentiation morphologically and biochemically between them is neither easy nor clear (34). For the whole animal, HPAI viruses vary in the pathophysiological mechanisms responsible for the severe illness and death. In some gallinaceous Figure 5.5. Microthrombosis (arrowheads) in poultry, infection of vascular endothelium alters per- lung of a chicken 24 hours post intranasal inoculation with A/whooper swan/Mongolia/ meability, initiating a cascade of edema, hemor- 244/2005 H5N1 HPAI virus. Hematoxylin and rhage, and multiorgan failure. Histologically, eosin stain. Bar = 25 μm. Figure reprinted with changes in vascular endothelium include hypertro- permission from Avian Diseases. Copyright phy, degeneration, and necrosis or apoptosis and can held by the American Association of Avian be associated with microthrombosis (96, 117) Pathologists, Athens, Georgia, USA. Source: (Figures 5.4 and 5.5). The changes can progress to D. Swayne, U.S. Department of Agriculture, thrombocytopenia, consumptive coagulopathy, Agricultural Research Service, Athens, GA (121). thrombi, and emboli in vessels (Figure 5.5) and acti- For color detail, please see color plate section. vation of the coagulation cascade as evident by prolonged prothrombin time and high levels of tissue factor (76, 96). Some individual birds or certain INFLUENZA A IN GALLINACEOUS species may survive the peracute phase, and in these POULTRY birds, virus disseminates throughout the body. Simultaneous virus replication in multiple critical Low Pathogenicity Avian Infl uenza organs, such as in brain and autonomic nervous system, cardiac myocytes, endocrine tissue (e.g., Clinical Signs and Signalment adrenal gland), or pancreas, can result in single- For the LPAI viruses, high morbidity (>50%) and organ or multiorgan failure (95). low mortality rates (<5%) are typical, but mortality 92 Avian Influenza rates can be quite high if accompanied by secondary and have been accompanied by renal failure evident pathogens or if the disease occurs in young birds. as visceral urate deposition (“visceral gout”). In gallinaceous poultry, clinical signs refl ect Catarrhal to fi brinous enteritis, typhlitis, and procti- pathophysiological changes in the respiratory, diges- tis may be observed, especially in turkeys. Sporadi- tive, urinary, and reproductive systems (122). Most cally in turkeys, the pancreas may be pale and frequently, LPAI virus infections present with mild- mottled and contain random hemorrhages. to-severe respiratory signs such as coughing, sneezing, rales, rattles, and excessive lacrimation. In Microscopic Lesions reproductively active layers and breeders, increased LPAI viruses commonly produce heterophilic-to- broodiness and decreased egg production may be lymphocytic tracheitis and bronchitis (Table 5.2). present. General clinical signs of huddling, ruffl ed Ventromedial fi brinocellular to peribronchiolar lym- feathers, listlessness, decreased activity, decreased phocytic pneumonia may be present, which in severe feed and water consumption, and occasionally diar- cases can be accompanied by diffuse air capillary rhea may be present. edema. Rarely, nephrosis and nephritis have been reported. However, this renal tropism is virus-strain Gross Lesions specifi c and most consistently produced by experi- Gross lesions are variable depending on the virus mental intravenous inoculation. Pancreatic acinar strain, length of the time from infection to death, the necrosis has been reported in experimental studies and host species, and the presence of secondary patho- natural cases in turkeys during the 1999 Italian H7N1 gens (122). The infraorbital sinuses may be swollen, LPAI outbreak. Pancreatitis has been less common in especially in turkeys (Meleagris gallopavo), and chickens than in turkeys. Birds that die from LPAI have accompanying mucoid-to-mucopurulent nasal have lymphocyte depletion and necrosis or apoptosis discharge. The most frequent lesions are rhinitis and of lymphocytes in the cloacal bursa, thymus, spleen, sinusitis, whose character varies between catarrhal, and other areas with lymphocyte accumulations. fi brinous, serofi brinous, mucopurulent, and fi brinopurulent. The fi brinopurulent infl ammation usually Tissue and Cellular Sites of Viral is accompanied by secondary bacterial infections. Replication and Damage The tracheal mucosa may be edematous with con- Intranasal inoculation with LPAI viruses in chickens gestion, occasionally hemorrhages, and serous to and turkeys has demonstrated AI viral antigen com- caseous luminal exudates. The tracheal exudates monly in upper respiratory epithelial cells (Table occasional occlude airways with resulting asphyxi- 5.2). On intravenous inoculation, viral antigen was ation. Fibrinopurulent bronchopneumonia may be commonly demonstrated in necrotic renal tubule present and is usually accompanied by secondary epithelium and pancreatic acinar epithelium, but bacterial pathogens such as Pasteurella multocida or rarely in necrotic or apoptotic lymphocytes. Escherichia coli. Catarrhal to fi brinous to fi brinopurulent air sac- High Pathogenicity Avian Infl uenza culitis and coelomitis (“peritonitis”) may be present. Ovaries exhibit varying degrees of regression, begin- Clinical Signs and Signalment ning with hemorrhage adjacent to the stigmata of By defi nition, HPAI viruses express high lethality in large follicles and progressing to colliquation, or the chickens both in fi eld cases and experimentally in ova may rupture resulting in free yolk within the the intravenous pathogenicity test and in intranasal coelomic cavity (“egg yolk peritonitis”) with or infectivity and pathogenesis studies. Typically, without infl ammation. The oviduct may be edema- HPAI viruses also express high lethality in other tous and contain catarrhal to fi brinous luminal exu- gallinaceous birds, but the mean death times (MDTs) dates (salpingitis) before undergoing involution. are usually lengthened compared with those in The last few eggs laid before death may be fragile chickens and vary with individual HPAI virus due to reduced calcium in the eggshells. Such eggs strains. For example, in one intranasal experimental may be misshapened and have loss of shell pigment. study, H5 HPAI viruses isolated between 1959 and In a few natural cases in laying hens and intravenous 1984 most consistently produced high mortality inoculated chickens, swollen kidneys have occurred rates in turkeys and chickens, and fewer were 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 93

Table 5.2. Summary of histological lesions in experimental infections in chickens inoculated intranasally (IN) or intravenously (IV) with A/chicken/Jalisco/14589/94 (J12/94) and A/chicken/ Hidalgo/26654–1368/1994 (H5/94) Mexican H5N2 LPAI viruses and A/chicken/Pueblo/8623–607/ 1994 (P11/94) and A/chicken/Queretero/14588–19/1995 (Q1/95) Mexican H5N2 HPAI viruses (125). Organ Lesion H5/94 LPa J12/94 LPa P11/94 HPa Q1/95 HPa

Heart Myocyte necrosis − − ++ +++ Myocarditis − − +++ ++ Brain Neuron necrosis − − +++ +++ Meningoencephalitis − − +++ + Pancreas Vacuolation (+)(++) +++ +++ Acinar necrosis − − ++ +++ Kidney Tubule necrosis (+++)(++) ++ Interstitial nephritis (+++)(+++) +++ − Upper respiratory tract Rhinitis and tracheitis [++][++] ++ ++ Lung Pneumonia (+++)(++) + +++ a − = no lesion, + = mild lesion, ++ = moderate lesion, +++ = severe lesion; ( ) = intravenous inoculation only, [ ] = intranasal inoculation only; no ( ) or [ ] = both IN and IV inoculation. highly lethal for Japanese quail (Coturnix coturnix mus, paresis, paralysis, excitation, convulsions, japonicus). rolling or circling movements, tremors and incoor- In gallinaceous poultry, clinical signs refl ect dination, shaking of head, abnormal gait, paralysis HPAI virus replication and damage to major organ of wings, loss of balance and recumbency with ped- systems, including multiple visceral organs, cardio- aling movements, fl apping movements of the wings, vascular and nervous systems, and the integument. and unusual positions of head and appendages. Specifi c clinical manifestations depend on the level However, the appearance of neurological signs will of damage and which organs or organ systems are vary with the virus strains and the species of bird. affected. In the peracute stage, birds may be found For example, the prevalence of neurological dys- dead prior to the appearance of any clinical signs or function in experimental studies with a 1997 Hong with few clinical signs other than listlessness, Kong H5N1 HPAI virus varied between turkeys recumbency, and a comatose state. Closer observa- (41%), chukar partridge (Alectoris chukar) (28%), tion of remaining birds has revealed reduced activ- Bobwhite quail (Colinus virginianus) (14%), ring- ity, decreased sensitivity to stimuli, reduction in necked pheasant (Phasianus colchicus) (13%), and “in-house” noise, dehydration, and decreased feed Japanese quail (Coturnix coturnix japonicus) (18%) and water intake that rapidly progressed to severe (96). Neurological signs are not specifi c for HPAI listlessness and death. In some instances, 24 hours and may be identical but less frequent than neuro- before detection of mortality, birds exhibited logical signs of velogenic Newcastle disease and decreased activity, a reduction in normal vocaliza- other noninfectious and infectious diseases (96). tions, and decreased feed and water consumption. In Respiratory signs are less prominent than with breeders and layers, egg production will drop pre- LPAI virus infections but, if present, have included cipitously with typical declines leading to total ces- rales, sneezing, and coughing. Other galliforme sation of egg production within 6 days. Diarrhea birds may have less peracute disease than chickens may be evident as bile- or urate-stained loose drop- and turkeys, although clinical signs and the duration pings with variable amounts of intermixed mucus. of morbidity may be similar. If the clinical course was less peracute and birds survive for 3 to 7 days, individual birds may exhibit Gross Lesions nervous disorders such as tremors of head and neck, In gallinaceous poultry, HPAI virus infections inability to stand, torticollis, opisthotonus, nystag- produce a variety of edematous, hemorrhagic, and 94 Avian Influenza necrotic lesions in multiple visceral organs, cardio- from subcutaneous edema and may have accompa- vascular and nervous systems, and the integument nying petechial-to-ecchymotic subcutaneous hemor- (Figures 5.6 through 5.13). With the peracute phase rhages, especially of the nonfeathered skin (Figures (1 to 2 days post intranasal inoculation), no gross 5.6 through 5.8). Periorbital and intermandibular lesions are typically seen. In the acute phase (days edema may be seen in some birds. Some viruses 2 to 5), chickens have ruffl ed feathers and swelling produce hyperemia and edema of the eyelids, con- of the head, face, upper neck, leg shanks, and feet junctiva, and trachea. Necrotic foci, petechial-to- echymotic hemorrhage, and cyanosis of the wattles, combs, and snood may be common, and such lesions are used in syndromic clinical surveillance to identify suspect HPAI cases. The cyanosis results from ischemic necrosis following vascular infarction. The gross lesions in internal organs vary with virus strain but most consistently include hemorrhages on serosal or mucosal surfaces and foci of necrosis within parenchyma of multiple visceral organs (Figures 5.9 through 5.13). Especially prominent are hemorrhages in the coronary fat and on the epicardium (Figure 5.9), on the serosa and the mucosa of the proventriculus and ventriculus, and Figure 5.6. Multifocal necrosis and within the pectoral muscles (Figure 5.13). Occa- hemorrhage of comb and wattles in adult sional hemorrhages are present on the inner surface White Leghorn hen 7 days post intranasal of the sternum and in the cecal tonsils and Meckel’s inoculation with A/chicken/12508/86 derivative diverticulum (Figure 5.10). The pancreas may have H5N2 HPAI virus. 2 cm = bar. Reprinted with red to light orange to brown mottling from necrosis. permission (122). Source: M. Brugh, U.S. Ruptured ova with free yolk in the coelomic cavity Department of Agriculture, Agricultural have been reported in layer, broiler and turkey Research Service, Athens, GA. For color breeders. With the recent H5N1 HPAI viruses and detail, please see color plate section. classic fowl plague viruses, necrosis and hemor-

Figure 5.8. Severe subcutaneous Figure 5.7. Edema of comb, wattles, and hemorrhage and edema of feet and leg periorbital tissues in adult White Leghorn shanks post intranasal inoculation with A/ hen, 5 days post intranasal inoculation with Hong Kong/156/97 (H5N1). Bar = 2 cm. A/chicken/Puebla/8623-607/1994 H5N2 HPAI Reprinted with permission (117). Source: D. virus. 2 cm = bar. Source: D. Swayne, U.S. Swayne, U.S. Department of Agriculture, Department of Agriculture, Agricultural Agricultural Research Service, Athens, GA. Research Service, Athens, GA. For color For color detail, please see color plate detail, please see color plate section. section. Figure 5.9. Petechial hemorrhages in Figure 5.11. Generalized congestion with epicardial fat 4 days post intranasal hemorrhage and edema in the lung from a 4- inoculation with A/chicken/NJ/12508/86 week-old chicken 1.5 days post intranasal (H5N2). Bar = 3 cm. Reprinted with inoculation with A/chicken/Hong permission (122). Source: M. Brugh, U.S. Kong/220/1997 (H5N1) virus. Bar = 0.75 cm. Department of Agriculture, Agricultural Figure reprinted with permission from Avian Research Service, Athens, GA. For color Diseases. Copyright held by the American detail, please see color plate section. Association of Avian Pathologists, Athens, Georgia, USA. Source: D. Swayne, U.S. Department of Agriculture, Agricultural Research Service, Athens, GA (95). For color detail, please see color plate section.

Figure 5.12. Focal mucosal hemorrhage visible from the serosal surface of an ileal Peyer’s patch from a 4-week-old chicken 1.5 days post intranasal inoculation with A/ chicken/Hong Kong/220/1997 (H5N1) virus. Figure 5.10. Hemorrhage in cecal tonsils and Bar = 0.5 cm. Figure reprinted with rectum, 1 day post intravenous inoculation permission from Avian Diseases. Copyright with A/Hong Kong/483/97. 1 cm = bar. Source: held by the American Association of Avian D. Swayne, U.S. Department of Agriculture, Pathologists, Athens, Georgia, USA. Source: Agricultural Research Service, Athens, GA. D. Swayne, U.S. Department of Agriculture, For color detail, please see color plate Agricultural Research Service, Athens, GA section. (95). For color detail, please see color plate section.

95 96 Avian Influenza

Figure 5.13. Multifocal hemorrhage in the fascial plane of the gastrocnemius muscle (pars intermedia) from a 4-week-old guineafowl 2 days post intranasal inoculation with A/chicken/Hong Kong/220/1997 (H5N1) Figure 5.14. Severe congestion with virus. Bar = 0.5 cm. Figure reprinted with microthrombosis (arrow), interstitial edema, permission from Avian Diseases. Copyright and interstitial heterophilic infi ltration in lung held by the American Association of Avian of chicken 1.5 days post intranasal Pathologists, Athens, Georgia, USA. Source: inoculation with A/chicken/Hong D. Swayne, U.S. Department of Agriculture, Kong/220/1997 (H5N1). Hematoxylin and Agricultural Research Service, Athens, GA eosin stain. Bar = 50 μm. Figure reprinted (95). For color detail, please see color plate with permission from Avian Diseases. section. Copyright held by the American Association of Avian Pathologists, Athens, Georgia, USA. Source: D. Swayne, U.S. Department of Agriculture, Agricultural Research Service, rhage in Peyer’s patches of the small intestine Athens, GA (95). For color detail, please see (Figure 5.12) have been common, as has been severe color plate section. edema and hemorrhage in the lungs (Figure 5.11), and with some viruses, gross edema of the brain. With most HPAI viruses, necrotic foci have been and/or wattles (12%), hemorrhages in the proven- reported in the heart and, occasionally, in the liver triculus (4%), and/or no gross lesions (17%) (28). and kidneys. The kidney lesions may be accompa- The fi rst two lesions are nonspecifi c and commonly nied by urate deposits. Lungs have focal ventral to seen with multiple different viral and bacterial diffuse interstitial pneumonia with edema. The lungs agents (28). can be congested or hemorrhagic. In immature birds, the cloacal bursa and thymus may be atrophic with Microscopic Lesions or without hemorrhage. The spleen may be normal Microscopic lesions in chickens and turkeys are in size or enlarged. When enlarged, the parenchyma more consistent than are gross lesions in HPAI can contain pale necrotic foci. cases. For experimental studies, histopathological The early hemorrhagic lesions point to severe descriptions vary with individual virus strains, inoc- alternations in the cardiovascular system, princi- ulum dose, strain or breed of chicken, route of inoc- pally the vascular endothelium, and the subsequent ulation, and passage history. Typically, histological visceral necrotic changes resulting from the viremia lesions consist of necrosis and/or infl ammatory and virus replication in the viscera. changes in multiple organs, most consistently and The frequency of gross lesions varies with the severely in the skin, brain, heart, pancreas, lungs, virus strain and species of bird and is not consis- adrenal glands, and primary and secondary lym- tently apparent in all birds. For example, in the phoid organs (Table 5.3 and Figures 5.14 through H7N7 HPAI outbreak in the Netherlands, laying 5.17). In the peracute phase (1 to 2 days post inocu- chickens from outbreak farms at necropsy had peri- lation [DPI]), most parenchymal cells of organs lack tonitis (62%), tracheitis (43%), edema of the neck microscopic lesions. If present, necrosis and infl am- Table 5.3. Immunohistochemical demonstration of AI viral nucleoprotein in experimental infections of chickens inoculated intranasally (IN) or intravenously (IV) with A/chicken/ Jalisco/14589/94 (J12/94) and A/chicken/Hidalgo/26654–1368/1994 (H5/94) Mexican H5N2 LPAI viruses and A/chicken/Pueblo/8623–607/1994 (P11/94) and A/chicken/Queratero/14588– 19/1995 (Q1/95) Mexican H5N2 HPAI viruses (125). Organ Cell Type H5/94 LPa J12/94 LPa P11/94 HPa Q1/95 HPa

Heart Myocytes − − ++ +++ Autonomic neurons −− −+ Brain Neurons − − +++ +++ Ependyma/choroid plexus −− ++ Pancreas Acinar epithelium − − + +++ Kidney Tubule epithelium (+++)(++) ++ Circulatory system Vascular endothelium −− ++ Upper respiratory Epithelium [++][++] ++ tract a AI antigen staining score: − = none, + = infrequent or rare, ++ = frequent, +++ = common; () = IV inoculation only, [] = IN inoculation only, no () or [] = both IN and IV inoculation.

Figure 5.15. AI viral antigen in vascular Figure 5.16. Mild perivascular edema and endothelium and phagocytic leukocytes within minimal cardiac myocyte hyalinization in 4- the pulmonary parenchyma and small caliber week-old Japanese quail 2 days post interstitial vessels 1.5 days post intranasal intranasal inoculation with A/chicken/Hong inoculation with A/chicken/Hong Kong/220/ Kong/220/1997 (H5N1). Hematoxylin and 1997 (H5N1). Immunohistochemical stain. Bar eosin stain. Bar = 50 μm. Figure reprinted = 50 μm. Figure reprinted with permission with permission from Avian Diseases. from Avian Diseases. Copyright held by the Copyright held by the American Association American Association of Avian Pathologists, of Avian Pathologists, Athens, Georgia, USA. Athens, Georgia, USA. Source: D. Swayne, U. Source: D. Swayne, U.S. Department of S. Department of Agriculture, Agricultural Agriculture, Agricultural Research Service, Research Service, Athens, GA (95). For color Athens, GA (95). For color detail, please see detail, please see color plate section. color plate section.

97 98 Avian Influenza

nonfeathered skin within dermal and hypodermal capillaries and small blood vessels that is accompanied by vasculitis, perivascular to generalized edema, subcutaneous edema, and necrosis of capillary endothelium and accompanied by epidermal vesicle formation progressing to full-thickness necrosis; and (4) necrosis in skeletal myofi bers, renal tubules, vascular endothelial cells, corticotrophic cells of adrenal glands, and pancreatic acinar epithelium. Areas of necrosis and infl ammation have associated abundant infl uenza viral protein. In the acute phase (survival to 2 to 5 days), the necrosis will be less severe, but the intensity of lymphohistiocytic infl ammation will increase. Depletion of lymphocytes and necrosis Figure 5.17. Extensive intranuclear and and/or apoptosis will be common in cloacal bursa, intracytoplasmic localization of AI viral thymus, and spleen, but AI viral antigen is rarely antigen in cardiac myocytes and in vascular seen in lymphocytes, more often in vascular endo- endothelial cells (arrow) 2 days post thelial cells or phagocytic cells. The lungs most con- intranasal inoculation with A/chicken/Hong sistently have moderate-to-severe, diffuse edema Kong/220/1997 (H5N1). Immunohistochemical with necrosis of blood and air capillary endothelium, stain. Bar = 50 μm. Figure reprinted with congestion, hemorrhage, and interstitial pneumoni- permission from Avian Diseases. Copyright tis. The lesions in respiratory tract vary widely from held by the American Association of Avian Pathologists, Athens, Georgia, USA. Source: minimal to severe. In other gallinaceous species, D. Swayne, U.S. Department of Agriculture, lesions are similar as described earlier, but in general, Agricultural Research Service, Athens, GA because the birds survive longer than chickens or (95). For color detail, please see color plate turkeys, the necrotic and infl ammatory characters of section. lesions in parenchymal organs are more common and prominent. matory changes are usually mild and multifocal in Tissue and Cellular Sites of Viral distribution with most immunohistochemical dem- Replication and Damage onstration of HPAI virus replication in the vascular HPAI viruses grow to high titers in the respiratory endothelial cells and cardiac myocytes (Figures 5.14 and intestinal tracts of chickens and turkeys and are and 5.15) (117). In the acute phase (2 to 5 days), shed in the respiratory secretions and feces. In lesions appear in multiple organs with the predomi- immunohistochemical studies, HPAI viral antigen nant lesion being necrosis and, to a lesser extent, has been demonstrated in both vascular and paren- apoptotic cell death with associated infl ammation, chymal cells (Table 5.3). In birds surviving beyond hemorrhage, and edema. The longer the birds 3 days, the dominant lesion is necrosis, which can survive, the less prominent are the necrosis and result from either infarction if accompanied by vas- apoptosis, and the more prominent is the infl amma- cular damage, such as thrombosis and embolism, or tion. Necrotic cells have associated AI viral antigen, from direct viral replication and damage to the except the apoptotic lymphocytes, which have parenchymal cells (45). Edema, congestion, and inconsistent to lack of demonstrable AI antigen. hemorrhage are consequences of the vascular Common lesions have included (1) lymphocytic damage, especially visible as reddening and swell- meningoencephalitis with neuronal necrosis, neuro- ing in the heads, legs, and feet (45). The vascular nophagia, and focal gliosis, occasionally with edema damage and viremia precede the parenchymal and hemorrhage; (2) focal degeneration to multifo- disease (45). The necrotic and infl ammatory changes cal-diffuse coagulative necrosis of cardiac myocytes in parenchyma of organs are due to the direct effect usually with accompanying lymphohistiocytic of the virus on target cells. The degree of virus dis- infl ammation (Figure 5.16); (3) microthrombosis of semination and the resulting variety of lesions are 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 99 dependent on the length of survival, which is depen- reported in rheas have included heterophilic-to- dent on resistance of the host (i.e., age, species, pyogranulomatous sinusitis, bronchitis, and pneu- strain, etc.) and virulence of the virus (46). For monia with necrosis of respiratory epithelium. In example, some HPAI virus isolates may be neuro- ostriches, histological lesions of splenic and hepatic tropic without extensive vascular endothelial necrosis, enteritis, and sinusitis were seen. replication (78). Thus, two forms of neural pathogenesis may occur: (1) early necrotizing form within High Pathogenicity Avian Infl uenza 3 days after inoculation, producing disseminated vascular endothelial disease, which resulted in Wild Birds microgliosis and necrosis of brain parenchyma; and Prior to 2002, there were few reports of HPAI virus (2) a later form between 4 and 7 days after inocula- infections and lesions in wild birds. In 1961, high tion, also involving infection of ependymal cells, mortality was reported in common terns (Sterna and resulting in associated ventriculitis and periven- hirundo) in South Africa from infections with an tricular necrosis and infl ammation (61). H5N3 HPAI virus (10). Since 2002, a variety of wild aquatic and terrestrial wild birds have died from INFLUENZA A IN WILD BIRDS, DOMESTIC infection with the Asian lineage of H5N1 HPAI WATERFOWL AND RATITES viruses.

Low Pathogenicity Avian Infl uenza Domestic Ducks and Geese Mortality in domestic ducks has been infrequent Wild Birds before the H5N1 HPAI outbreaks in Asia. However, LPAI viruses have been isolated from a variety of death in domestic ducks (Anas platyrhynchos) due to wild aquatic bird species, which are primarily the HPAI was reported in an intranasal experimental genetic primordial reservoirs (see Chapter 3, Ecology infection with an A/fowl/Germany/34 (H7N1) of Avian Infl uenza in Wild Birds). In such wild (Rostock virus) virus (3). In 1999–2000, deaths birds, LPAI viruses preferentially infect intestinal occurred in Muscovy ducks (Cairina moschata) fol- epithelial cells and LPAI virus is excreted in the lowing natural infections with H7N1 HPAI virus in feces. Typically, such LPAI virus infections have Italy. The lesions and viral antigen were primarily been asymptomatic in wild birds. demonstrated in the brain (16). Experimental infection of domestic Pekin ducks with HPAI viruses has Domestic Ducks and Geese shown either no virus replication, or limited replica- In domestic ducks and geese, LPAI infection has tion, and few clinical signs (3, 4, 97). However, the varied in clinical outcome from asymptomatic infec- pathobiology of HPAI infections in ducks has tions to respiratory disease, including sinusitis, con- changed with some Asian lineage H5N1 AI viruses junctivitis, and other respiratory lesions. Co-infections (since 2002) replicating systemically and producing with bacteria have been common and usually associ- neurological disease and death (29, 47, 58, 69, 79, ated with production of clinical disease. 115, 116, 148). Neurological disease and lesions were reported in domestic geese (Anser anser domes- Ratites ticus) inoculated with 1997 H5N1 HPAI virus and In ratites (ostriches [Struthio camelus], emus [Drom- lesions were predominantly seen in the brain (97). aius novaehollandiae], and rheas [Rhea americana]), LPAI viruses typically have produced respiratory Ratites signs and in some cases green diarrhea or “green In fi eld cases of HPAI, ostriches had reduced activ- urine.” The most frequent gross lesions in rheas and ity and decreased appetite and exhibited listlessness. emus have included ocular and/or nasal discharge; The birds had ruffl ed feathers, sneezing, open mouth fi brinous sinusitis, tracheitis, bronchitis, and air breathing, swollen throat and neck, and nervous sacculitis; interstitial pneumonitis with necrosis of signs such as incoordination, torticollis, paralysis respiratory epithelium; congested visceral organs; of the wings, and tremors of the head and neck. hemorrhage in trachea; and occasional fi brinous Gross and microscopic lesions reported included perihepatitis and pericarditis. Histological lesions edema of head and neck, severe hemorrhagic 100 Avian Influenza enteritis, enlarged and fi rm pancreas from necrosis, although the heart and pancreas were also frequently mild to severe air sacculitis, hepatitis, peritonitis, affected organs (Figures 5.20 through 5.24). Group renomegaly, and splenomegaly. 3 contained Pekin ducks, house sparrows (Passer domesticus), and laughing gulls (Larus atricilla). Unique Pathobiology of Asian Lineage of H5N1 The virus replicated to low titers, typically only in HPAI the respiratory tract and occasionally in the heart and The Asian lineage H5N1 HPAI viruses have changed gonad. Clinical signs were minimal and mortality over the past 10 years with differences in pathobiol- was lacking (Figures 5.25 through 5.26). Group 4 ogy for individual virus strains and for certain bird contained rock pigeons (Columba livia) and Euro- species (17, 79, 86, 95, 96, 98, 99, 115, 117, 125, pean starlings (Sturnus vulgaris). The virus failed to 138). In the initial chicken outbreaks in Hong Kong produce evidence of infection or infection was infre- during March 1997, the H5N1 HPAI virus was quent based on serological and virological assays, studied as the prototype Asian lineage H5N1 HPAI and the viral titers were minimal and without patho- virus in various domestic and wild bird species (95, logical consequence. 96, 98, 99). Using the standard 106 mean embryo The virulence of the Asian H5N1 HPAI viruses infectious doses (EID50) given intranasally in 20 dif- for chickens isolated over the next 9 years was 100% ferent animal species, the animals were placed into lethal, but the pathobiology varied slightly based on four different pathobiological groups (groups 1 the MDT, which ranged from 1 to 4.1 days on intra- through 4) based on virus replication, pathology, venous inoculation (69, 79, 97, 117, 138). Similarly, morbidity, and mortality (95) (Table 5.4). The quan- inoculation of chickens with H5N1 HPAI viruses by tity of virus replication was directly associated with a simulated natural route of exposure (i.e., intranasal severity of lesions produced and ultimately with pathogenicity tests) and a controlled virus dose (106 death of the individual. Animals in pathobiology group 1 were composed of gallinaceous birds (chickens, turkeys, Japanese quail, Bobwhite quail, pearl guineafowl [Numida meleagris], ring-neck pheasant, and chukar partridges) and zebra fi nches (Tae- niopygia guttata). Group 1 had systemic AI virus infection and experienced 100% morbidity and greater than 75% mortality. Typically, the birds exhibited severe listlessness before death, some with neurological dysfunction. The birds that died per- acutely may not have exhibited any clinical signs. Chickens and turkeys had the shortest MDTs and had prominent virus replication in vascular endothelium and phagocytic leukocytes (Figures 5.14 through 5.17). The other gallinaceous birds and zebra fi nches had slightly longer MDTs and had Figure 5.18. Focal vacuolar degeneration to virus replication predominantly in parenchymal necrosis of adrenal corticotrophic and cells such as in the heart, adrenal gland, pancreas, chromaffi n cells in a Zebra fi nch 5 days post and brain, with accompanying necrotic and infl am- intranasal inoculation with A/chicken/Hong matory lesions (Figures 5.18 and 5.19). Group 2 was Kong/220/1997 (H5N1). Hematoxylin and composed of domestic geese (Anser anser domesti- eosin stain. Bar = 50 μm. Figure reprinted cus), emus, house fi nches (Carpodacus mexicanus), with permission from Avian Diseases. and budgerigars (Melopsittacus undulatus). These Copyright held by the American Association birds had severe lesions in two or three critical of Avian Pathologists, Athens, Georgia, USA. organs with delayed morbidity compared with group Source: D. Swayne, U.S. Department of 1 and the lower mortality (<75%). The virus had a Agriculture, Agricultural Research Service, Athens, GA (95). For color detail, please see strong tropism for the central nervous system result- color plate section. ing in common observation of neurological signs, 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 101

Table 5.4. Summary data obtained from the intranasal inoculation of multiple avian and mammalian species with the A/chicken/Hong Kong/220/97 (H5N1) avian inﬂ uenza virus, including the average morbidity and mortality, average distribution and severity of gross and histological lesions, average distribution of viral antigen, and viral titers reisolated from brain, lung, and kidney (95). Gross Histological Viral Virus Species Morbiditya Mortalityb Lesionsc Lesionsd Antigene Reisolationf

WL chickens +++ +++ +++ +++ +++ +++ WR chickens +++ +++ +++ +++ +++ +++ J. quail +++ +++ +++ +++ +++ +++ B. quail +++ +++ +++ +++ +++ +++ Turkeys +++ +++ +++ +++ +++ +++ Guineafowl +++ +++ +++ +++ +++ +++ Pheasants +++ +++ +++ +++ +++ +++ Partridges +++ +++ ++ ++ ++ +++ Z. ﬁ nches +++ +++ + ++ +++ +++ Geese ++ − + ++ ++ ++ Emus ++ − + ++ ++ ++ H. ﬁ nches +++ ++ + ++ +++ ++ Budgerigars +++ ++ − ++ ++ ++ H. sparrows +−−++± Ducks −−−+±+ Gulls −−−±−± Starlings −−−−−± Pigeons −−−−−− Rats −−−−−− Rabbits −−−−−− a Morbidity: +++ = ≥ 75%; ++ = 50 to 74%; + = less than 50%; − = none. b Mortality: +++ = ≥ 75%; ++ = 50 to 74%; + = less than 50%; − = none. c Gross lesions: +++ = lesions common and in multiple organs; ++ = lesions sporadic and in few organs; + = lesions infrequent; − = lesions not observed. d Histological lesions: +++ = lesions common and in multiple organs; ++ = lesions sporadic and in few organs; + = lesions infrequent; ± = lesions rare and mild; − = lesions not observed. e Viral antigen: +++ = widespread; ++ = multifocal; + = infrequent; ± = rare; − = no viral antigen. f 5.0 Virus reisolation: +++ = high viral titers (≥10 ELD50/g tissue) obtained consistently from all brain, lung, 5.0 and kidney; ++ = high viral titers (≥10 ELD50/g tissue) obtained primarily from brain; + = low to moderate 4.1 viral titers obtained (≤10 ELD50/g tissue) from lung and/or kidney, negative reisolation from brain; ± = 1.9 virus reisolated at low titers from only lung or kidney (≤10 ELD50/g tissue); − = virus not reisolated from brain, lung, or kidney.

EID) produced 100% mortality, but the MDTs were the longest MDTs were seen for viruses isolated longer compared with the intravenous test, and sim- from domestic ducks and geese, suggesting some ilarly varied by virus strain with ranges of 1.5 to 2.1 selection or host adaptation for speciﬁ c species. days for isolates from chickens and humans and 2.4 These waterfowl isolates replicated and were shed to 5.5 days for isolates from ducks and geese (121) at lower oropharyngeal titers than other viruses. The (Table 5.5). Typically, the shortest MDTs were seen heart had higher tissue titers of virus than in the with chicken-, crow-, and human-origin viruses, and other tissues, suggesting that lethality was associ- 102 Avian Influenza

Figure 5.19. Diffuse demonstration of avian inﬂ uenza viral antigen in the adrenal gland in a Zebra ﬁ nch 5 days post intranasal inoculation with A/chicken/Hong Kong/220/1997 (H5N1). Immunohistochemical stain. Bar = 50 μm. Figure reprinted with permission from Avian Diseases. Copyright held by the American Association of Avian Figure 5.20. Two-week-old Embden goose Pathologists, Athens, Georgia, USA. Source: with torticollis 8 days post intranasal D. Swayne, U.S. Department of Agriculture, inoculation with A/chicken/Hong Kong/220/ Agricultural Research Service, Athens, GA 1997 (H5N1). Figure reprinted with permission (95). For color detail, please see color plate from Avian Diseases. Copyright held by the section. American Association of Avian Pathologists, Athens, Georgia, USA. Source: D. Swayne, U.S. Department of Agriculture, Agricultural ated with high titer replication and damage to vital Research Service, Athens, GA (95). For color organs. detail, please see color plate section.

Domestic Ducks During the past 11 years, the Asian lineage H5N1 HPAI viruses have evolved into multiple distinctly different strains that vary in pathogenicity for various bird species. For example, experimental inoculation of domestic ducks with some H5N1 HPAI strains isolated from ducks in China between 1999 and 2002 produced respiratory and alimentary tract infection with shedding but did not cause illness or Figure 5.21. Mottling of the pancreas in a 2- death (18). Using a 2-week-old domestic duck model week-old Embden goose 7 days post 6 and intranasal inoculation with 10 EID50 of virus, intranasal inoculation with A/chicken/Hong changes were noted in pathogenicity such that spe- Kong/220/1997 (H5N1). Bar = 1 cm. Figure ciﬁ c strains had evolved with the ability to produce reprinted with permission from Avian illness and death and could replicate in internal Diseases. Copyright held by the American organs such as the brain, causing neurological signs Association of Avian Pathologists, Athens, (86, 125) (Table 5.6 and Figures 5.27 and 5.28). The Georgia, USA. Source: D. Swayne, U.S. isolates from 1997–2000 only replicated in the respi- Department of Agriculture, Agricultural Research Service, Athens, GA (95). For color ratory tract and produced associated mild respiratory detail, please see color plate section. lesions (97). However, a strain isolated from frozen Figure 5.22. Focally extensive neuronal Figure 5.23. Severe necrosis of the necrosis and vacuolation of neuropil in the pancreatic acinar epithelium with scattered cerebellum of a House ﬁ nch 13 days post heterophils in a pancreas from a 2-week-old intranasal inoculation with A/chicken/Hong goose 4 days post intranasal inoculation Kong/220/1997 (H5N1). Hematoxylin and with A/chicken/Hong Kong/220/1997 (H5N1). eosin stain. Bar = 50 μm. Inset: Demonstration Bar = 50 μm. Inset: Demonstration of chicken/ of viral antigen in neurons and glial cells. HK viral antigen in pancreatic acinar epithelial Immunohistochemical stain. Figure reprinted cells. Immunohistochemical stain. Bar = with permission from Avian Diseases. 75 μm. Figure reprinted with permission from Copyright held by the American Association Avian Diseases. Copyright held by the of Avian Pathologists, Athens, Georgia, USA. American Association of Avian Pathologists, Source: D. Swayne, U.S. Department of Athens, Georgia, USA. Source: D. Swayne, U. Agriculture, Agricultural Research Service, S. Department of Agriculture, Agricultural Athens, GA (95). For color detail, please see Research Service, Athens, GA (95). For color color plate section. detail, please see color plate section.

Table 5.5. Differences in virus replication titers and mean death times (MDTs) of H5N1 HPAI 6 viruses in intranasally inoculated 3- to 6-week-old chickens (10 EID50 per bird).

Virus Titers (EID50/ml or g) Virus Age (weeks) MDT Oral Cloacal Brain Heart

A/chicken/Hong Kong/220/97 4 1.5 4.7 4.2 6 A/goose environment/Hong 4 5.5 1.7 − Kong/437–6/99 A/duck/Anyang/AVL-1/01 4 2.9 3.1 6.7 A/goose/Vietnam/113/01 4 2.6 A/goose/Vietnam/324/01 4 2.4 A/chicken/Korea/ES/03 3–6 2 6.2 6 7.2 9.3 A/chicken/Indonesia/7/03 4–6 2.1 6.4 5.6 7.6 A/crow/Thailand/1C/04 4 1.8 6.8 4 6.9 9 A/Vietnam/1203/04 4 1.5 6.3 5.9 7.7 10 A/whooper swan/Mongolia/05 4 2.3 7.6 5.3 6.9 10.6 Blank cells = not examined, − = negative for virus.

103 104 Avian Influenza

Figure 5.24. Focal cardiac myocyte Figure 5.25. Interstitial infi ltration with fragmentation and necrosis with mixed mixed mononuclear cells and fewer mononuclear cell infl ammation in the heart heterophils adjacent to a tertiary bronchus in of a 2-week-old emu 5 days post intranasal a 4-week-old duck 4 days post intranasal inoculation with A/chicken/Hong inoculation with A/chicken/Hong Kong/220/1997 (H5N1). Hematoxylin and Kong/220/1997 (H5N1). Hematoxylin and = μ eosin stain. Bar 50 m. Inset: Demonstration eosin stain. Bar = 50 μm. Figure reprinted of chicken/HK viral antigen in cardiac with permission from Avian Diseases. myofi bers and infi ltrating macrophages. Copyright held by the American Association Immunohistochemical stain. Figure reprinted of Avian Pathologists, Athens, Georgia, USA. with permission from Avian Diseases. Source: D. Swayne, U.S. Department of Copyright held by the American Association Agriculture, Agricultural Research Service, of Avian Pathologists, Athens, Georgia, USA. Athens, GA (95). For color detail, please see Source: D. Swayne, U.S. Department of color plate section. Agriculture, Agricultural Research Service, Athens, GA (95). For color detail, please see color plate section. dissemination (group 3). Many isolated from 2003 caused severe lesions in cardiovascular system duck meat imported from China into South Korea (group 2), while some newer strains produced severe during 2001 was able to replicate and spread sys- lesions in multiple organs and tissues, including the temically in intranasally inoculated domestic ducks brain with respiratory signs and diarrhea (group 1) but did not produce clinical signs or death, although (Table 5.7 and Figures 5.27 through 5.29). For the virus was isolated from, and visualized by immuno- group 1 viruses, the lesions and pathogenesis of the histochemistry in, skeletal and cardiac muscle and infection and disease were very similar between brain (138). New strains were isolated in 2002 and chickens and 2-week-old ducklings. 2003 that caused variable mortality in experimen- Lesions indicative of vascular damage such as tally inoculated domestic ducks (18, 29, 47, 69, 79, severe pulmonary edema, congestion, hemorrhage, 115, 116). Typically, this mortality resulted from and microthrombosis in capillaries, as well as virus systemic infections and had an associated increase demonstration in vascular endothelium that have in virus titers within the respiratory tract and brain been consistently observed in chickens and other (Table 5.6) compared with previous isolates that gallinaceous species infected with H5N1 HPAI (96, resulted in subclinical infections. Pathobiologically, 130), have not been observed in ducks. In ducks, the initial H5N1 strains were in pathobiological these viruses caused lesions, and viral antigens were group 4, while beginning in 2001, some strains demonstrated in multiple organs including respira- caused severe respiratory infection with occasional tory tract (Figures 5.30 and 5.31), the pancreas, 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 105

tion of 5-week-old ducks with an H5N1 duck isolate from an outbreak in Japan did not produce mortality, but virus was recovered from multiple organs, including the brain, and neurological signs were observed in association with high virus titers in brain (58). Such age-dependent lethality may explain the lack of or low mortality reported for naturally infected ducks. Interestingly, viruses isolated in Vietnam in 2005 have shown an additional increase in pathogenicity in ducks compared with the previous studied viruses as indicated by the shorter MDT of 2.7 days when inoculated into 2-week-old ducks and causing 100% mortality in 5-week-old ducks (87) (Table 5.6). This increase in pathogenicity is the consequence of an increase in viral replication titers in tissues and an expanded tissue tropism. Field obser- Figure 5.26. Focal mononuclear infi ltration vations concur with these results, with increased with few heterophils in the myocardium of a House sparrow 7 days post intranasal number of cases of H5N1 HPAI among domestic inoculation with A/chicken/Hong ducks, with accompanying high mortality, reported Kong/220/1997 (H5N1). Hematoxylin and in Vietnam during the last year (81). eosin stain. Bar = 50 μm. Inset: Demonstration of minimal viral antigen in myocytes and Wild Birds macrophages. Immunohistochemical stain. Beginning in 2002, Asian lineage H5N1 HPAI Figure reprinted with permission from Avian viruses have emerged with the ability to cause severe Diseases. Copyright held by the American illness and death in Anseriformes. Such virus strains Association of Avian Pathologists, Athens, have also killed birds in at least 10 other avian Georgia, USA. Source: D. Swayne, U.S. orders, including Charadriiformes (gulls and shore- Department of Agriculture, Agricultural birds), Ciconiiformes (storks, herons, and egrets), Research Service, Athens, GA (95). For color detail, please see color plate section. Columbiformes (pigeons and doves), Falconiformes (eagles, goshawks, kestrel, buzzard, and kites), Gru- iformes (coots, moorhen, and swamphen), Passeri- formes (crow, sparrow, starling, fi nch, mesia, munia, central nervous system (Figure 5.33), adrenal glands and mynah), Pelecaniformes (pelicans and cormo- (Figure 5.34), and myocardium (Figure 5.32) as has rants), Phoenicopteriformes (fl amingoes), Podici- been reported for gallinaceous poultry (14, 15, 46, pediformes (grebes), and Strigiformes (owls) (19, 61, 62, 69, 74, 86, 96, 97) (Figures 5.32 through 29, 33, 80). In late November and December 2002, 5.35). However, the pathogenicity of the H5N1 H5N1 outbreaks in two Hong Kong parks, Kowloon HPAI viruses differed in domestic ducks where Park and Penfold Park, caused the deaths of many lethality was age dependent, with some strains captive, feral, and free-living avian species, includ- causing high mortality only in 2-week-old, not 5- ing a variety of ducks, geese, and swans; greater week-old, domestic ducks (86, 88). This difference fl amingos; a grey heron; a little egret, and a black- in age susceptibility was also observed during a headed gull (29). The range of pathological changes natural outbreak of H5N1 HPAI in commercial ducks present in the various water birds examined in this in South Korea during 2003–2004. In 14-day-old outbreak resembled those reported generally for meat ducks, the virus produced increased morbidity HPAI viruses in chickens (29). However, the lesions and up to 12% mortality with microscopic lesions were in generally more severe in the respiratory tract observed in the pancreas, liver, brain, and heart. with more extensive neurological lesions in the However, adult ducks at eight breeder duck farms captive birds than seen in chickens. Since 2002, showed decreased egg production and feed con- sporadic deaths within wild birds associated with sumption but no mortality (64). Experimental infec- H5N1 HPAI have continued (83). Beginning in Table 5.6. Mortality, mean death times (MDTs), and viral replication titers from oropharyngeal and cloacal swabs, and brain of 2-week-old ducks intranasally infected with Asian-origin H5N1 infl uenza viruses. Virus Isolationd Oral Titers Cloacal Brain Virusa Mortalityb MDTc 3 dpi Titers 3 dpi Titers 3 dpi A/duck/Vietnam/218/2005 8/8 2.7 6.5 3.3 NA A/duck/Vietnam/203/2005 8/8 3.4 4.8 1.5 NA A/whooper swan/Mongolia/244/2005 7/8 4.3 NA NA NA A/Vietnam/1203/2004 7/8 4.2 4.9 2.0 4.6 A/Thailand PB/6231/2004 3/8 6.3 3.7 1.3 4.3 A/crow/Thailand/2004 8/8 4.4 4.4 1.9 6.4 A/egret/HK/757.2/2002 7/8 4.1 5.8 2.3 5.0 A/chicken/Indonesia/7/2003 4/8 7 2.5 − NA A/chicken/Korea/ES/2003 2/8 4 1.6 1.6 NA A/goose/Hong Kong/739.2/2002 7/8 4 3.8 NA NA A/Hong Kong/213/2003 0/8 − 2.7 NA NA A/goose/Vietnam/113/2001 0/8 − 1.8 <1.6 1.5 A/chicken/Hong Kong/317.5/2001 0/8 − 1.9 2.5 NA A/duck/Anyang/ALV-1/2001 0/8 − 2.5 1.3 2.1 A/environment/HK/437–6/1999 0/8 − 2.1 2.6 − A/chicken/Hong Kong/220/1997 0/8 − 2.0 1.2 − Data from previously published and unpublished experiments (69, 79, 86, 88, 97, 138). dpi = days postinoculation. a 5 Ducks were inoculated intranasally with 10 EID50 of the viruses or exposed by contact. b Number of dead ducks/number of inoculated or exposed ducks. c Mean death time in days. d Mean log10 titers expressed as EID50/ml from oropharyngeal and cloacal swabs were sampled from three 0.97 individual ducks on the days indicated. The limit of detection was 10 EID50/ml. Brain titers were reported as EID50/g for a 10% tissue homogenate.

Table 5.7. Pathobiology group and lesion severity and distribution for selected H5N1 HPAI 6 viruses following intranasal inoculation (10 EID50) into 2-week-old domestic ducks (121). Pathobiology Group Viruses Affected Systems 1 A/duck/Vietnam/218/2005 Multiple systems—most organs A/duck/Vietnam/203/2005 and tissues A/whooper swan/Mongolia/244/2005 A/Vietnam/1203/2004 A/crow/Thailand/2004 A/egret/Hong Kong/757.2/2002 2 A/Thailand PB/6231/2004 Cardiovascular = respiratory = A/chicken/Indonesia/7/2003 nervous 3 A/duck meat/Anyang/2001 Respiratory >> cardiovascular > A/goose/Vietnam/113/2001 skeletal muscle > nervous A/Hong Kong/213/2003 A/chicken/Korea/ES/2003 4 1997–2001 H5N1 HPAI viruses No lesions > mild respiratory

106 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 107

Figure 5.29. Bile-stained loose droppings from a 2-week-old Pekin duck at 3 days after intranasal inoculation with A/egret/ HK/757.2/02 H5N1 HPAI virus. Figure reprinted with permission from Avian Diseases. Copyright held by the American Association of Avian Pathologists, Athens, Georgia, USA. Source: M. Pantin-Jackwood, U.S. Department of Agriculture, Agricultural Research Service, Athens, GA (86). For color detail, please see color plate section. Figures 5.27 and 5.28. Two-week-old Pekin ducks showing severe neurological signs at 3 days after intranasal inoculation with the A/ (2003) and inoculated in high doses (106 and 108 egret/HK/757.2/02 H5N1 HPAI virus. Figures EID50/bird) into the upper respiratory tract in pigeons reprinted with permission from Avian have produced infrequent illness and death (60, Diseases. Copyright held by the American 121). Most of the pigeons did not become ill, but Association of Avian Pathologists, Athens, some of these asymptomatic birds did become Georgia, USA. Source: M. Pantin-Jackwood, infected, with virus being shed from oropharynx and U.S. Department of Agriculture, Agricultural Research Service, Athens, GA (86). For color cloaca. In the pigeons that died, neurological signs detail, please see color plate section. were present and the most severe lesions and highest virus titers were in the brain. Additional lesions were necrosis in heart, skeletal muscle, adrenal glands, and pancreas. The AI virus was commonly spring 2005, H5N1 HPAI outbreaks involving large demonstrated in neurons and ependymal cells of numbers of wild birds were reported. brain, autonomic neurons, and cardiac myocytes. In the past 3 years, wild birds in the orders Colum- Some AI viral antigen was visualized in skeletal biformes, Passeriformes, and Anseriformes have myofi bers, adrenal corticotrophic cells, granulo- been identifi ed with fatal H5N1 HPAI infections. cytes, capillary endothelium, a few pancreatic acinar Historically, rock pigeons (order Columbiformes, cells, and smooth muscle in large arteries. Pigeons family Columbidae) have been resistant to natural in contact with inoculated birds did not become and experimental infections by most LPAI and infected, suggesting pigeon infections are possible HPAI viruses (52), but some cases of dead pigeons with some H5N1 HPAI strains, but such infections with isolation of H5N1 HPAI have been reported require high exposure doses and transmission (FAO, 2006). Intranasal inoculation of pigeons with between birds in these populations is ineffi cient. A/chicken/Hong Kong/220/1997 H5N1 HPAI virus This raises uncertainties that H5N1 HPAI virus will failed to produce infection, disease, or death (97). become established in feral pigeon populations, However, recent studies with three H5N1 HPAI which is important because of the close proximity viruses isolated from Thailand (2004) and Indonesia to poultry and humans. 108 Avian Influenza

Figure 5.30. Moderate necrotizing rhinitis, Figure 5.31. Degeneration and necrosis of with submucosal congestion and edema, and the tracheal epithelium with mucocellular glandular hyperplasia of the nasal epithelium exudate containing sloughed epithelial cells of a 2-week-old duck that died 3 days after of the trachea of a 2-week-old duck intranasal inoculation with the A/crow/ intranasally inoculated with A/crow/ Thailand/04 H5N1 HPAI virus. Hematoxylin Thailand/04 and found dead at 4 days after and eosin stain. Inset: Demonstration of viral inoculation. Hematoxylin and eosin stain. antigen in the epithelial cells. Figure reprinted Inset: Viral antigen staining present in the with permission from Avian Diseases. epithelial cells. Figure reprinted with Copyright held by the American Association permission from Avian Diseases. Copyright of Avian Pathologists, Athens, Georgia, USA. held by the American Association of Avian Source: M. Pantin-Jackwood, U.S. Pathologists, Athens, Georgia, USA. Source: Department of Agriculture, Agricultural M. Pantin-Jackwood, U.S. Department of Research Service, Athens, GA (86). For color Agriculture, Agricultural Research Service, detail, please see color plate section. Athens, GA (86). For color detail, please see color plate section.

Natural infections and death associated with H5N1 HPAI virus have also been reported for northern pintail (Anas acuta), blue-winged teal corvids (crows, jays, etc.) (65). An experimental (Anas crecca), redhead (Aythya americana), and study in American crows (Corvus brachyrhychos) wood ducks (Aix sponsa) with A/whooper swan/ revealed listlessness, hunched posture, rufﬂ ed feath- Mongolia/244/2005 or A/duck meat/Anyang/2001 ers, and loss of appetite (121). Lesions seen in H5N1 HPAI viruses produced disease and death severely ill birds included necrotizing pancreatitis, only in wood ducks (12). Infected wood ducks had nonsuppurative meningoencephalitis with neuron cloudy eyes, rufﬂ ed feathers, rhythmic dilation and necrosis, splenic and hepatic hemachromatosis, constriction of the pupils, severe weakness, incoor- myocarditis with necrosis, multifocal adrenalitis dination, and tremors and seizures. Histologically, with associated necrosis of corticotrophic cells, severe diffuse neuronal necrosis in the brain, necro- transmural lymphocytic typhlitis and serositis, and tizing pancreatitis and adrenalitis, and multifocal mild myenteric ganglioneuritis. Virus was demon- myocardial necrosis were common lesions. The mal- strated in neurons, pancreatic acinar cells, and gran- lards, northern pintail, blue-winged teal, and redhead ulocytes and precursors in bone marrow. These ducks became infected as evidenced by serology and lesions were similar to those reported for natural virus isolation results but shed low levels of virus H5N1 HPAI virus infections in another corvid for 1 to 3 days and did not develop clinical disease. species, the magpie (Pica pica sericea) (65). Infection of swans with H5N1 HPAI virus has The numbers of studies that have examined infec- only been described infrequently (29). However, tion in non-domestic ducks have been few. Intrana- during the outbreaks in Europe, swans appeared to sal inoculation of mallard (Anas platyrhynchos), be primarily affected and were designated as an indi- 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 109

Figure 5.32. Extensive intranuclear and Figure 5.33. Strongly positive viral staining intracytoplasmic viral antigen in degenerated present in neurons of the cerebrum of a 2- and necrotic myocytes of the heart of a 2- week-old duck intranasally inoculated with the week-old duck intranasally inoculated with the A/Vietnam/1203/04 H5N1 HPAI virus and found A/Thailand PB/6231/04 H5N1 HPAI virus and dead at 4 days after inoculation. Bar = 50 μm. found dead at 5 days after inoculation. Bar = Figure reprinted with permission from Avian 50 μm. Figure reprinted with permission from Diseases. Copyright held by the American Avian Diseases. Copyright held by the Association of Avian Pathologists, Athens, American Association of Avian Pathologists, Georgia, USA. Source: M. Pantin-Jackwood, Athens, Georgia, USA. Source: M. Pantin- U.S. Department of Agriculture, Agricultural Jackwood, U.S. Department of Agriculture, Research Service, Athens, GA (86). For color Agricultural Research Service, Athens, GA (86). detail, please see color plate section. For color detail, please see color plate section.

cator species of the presence of H5N1 HPAI virus in wild birds (133, 134). Mute (Cygnus olor) and whooper (Cygnus cygnus) swans that died of natural infection were examined (133). The most common lesions observed were multifocal hemorrhage and necrosis in the pancreas, pulmonary congestion and edema, and subpericardial hemorrhages. Histologi- cal lesions reported were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasma- cytic encephalitis with neuronal necrosis. Mild lymphocyte necrosis was present in Peyer’s patches and spleen; cortical and medullar adrenal necrosis was also observed. AI viral antigen was demonstrated Figure 5.34. Viral staining of the corticotrophic in pancreas, adrenal gland, liver, and brain. The cells of the adrenal gland of a 2-week-old lethal outcome was attributed to the systemic viral duck intranasally inoculated with A/Vietnam/ infection. 218/05, 2 days after inoculation. Bar = 50 μm. Figure reprinted with permission from Avian AVIAN INFLUENZA IN MAMMALS Diseases. Copyright held by the American Interspecies transmission of inﬂ uenza A viruses has Association of Avian Pathologists, Athens, occurred occasionally, mainly from aquatic birds to Georgia, USA. Source: M. Pantin-Jackwood, mammalian species. The outbreaks have tended to be U.S. Department of Agriculture, Agricultural Research Service, Athens, GA (86). For color self-limiting, and the introduced viruses were usually detail, please see color plate section. not maintained in the new species. Infection of 110 Avian Influenza

Netherlands in 2003, 89 people became infected and there was one fatal case (36). In 2004, two poultry workers developed conjunctivitis and mild respiratory symptoms after an outbreak of an H7N3 HPAI in Canada (139). However, and of most concern, have been the cases of H5N1 HPAI infection in humans occurring in Asia, Eurasia, the Middle East, and Africa since 2003. More complete reviews of AI in humans are found in the literature (24, 89, 118) and in Chapter 20 (Public Health Implications of Avian Infl uenza Viruses). The clinical manifestations of AI infections in humans varied depending on the virus subtype and strain causing the infection. In the H7N7 2003 out- Figure 5.35. Viral staining of the myocardial break in the Netherlands, 82 of the 89 cases (92.1%) cells of skeletal muscle of a 2-week-old duck manifested as conjunctivitis, and the remaining intranasally inoculated with A/crow/ patients presented with infl uenza-like illness. One Thailand/04 and euthanized at 4 days after veterinarian had an infl uenza-like illness, which pro- inoculation. Bar = 50 μm. Figure reprinted with permission from Avian Diseases. gressed to pneumonia 7 days later. The pneumonia Copyright held by the American Association persisted despite treatment, and the patient died of of Avian Pathologists, Athens, Georgia, USA. acute respiratory distress syndrome (ARDS) 15 days Source: M. Pantin-Jackwood, U.S. after exposure (36). Department of Agriculture, Agricultural Most cases of human H5N1 HPAI infections have Research Service, Athens, GA (86). For color been characterized by a severe infl uenza-like detail, please see color plate section. syndrome, clinically indistinguishable from severe human infl uenza, with symptoms of fever, cough, and shortness of breath and radiological evidence of mammals, including humans, with infl uenza viruses pneumonia (24, 147). Besides respiratory symp- of avian origin has been reported sporadically. toms, a large proportion of patients also complain of However, Asian-origin H5N1 HPAI viruses have gastrointestinal symptoms such as diarrhea, vomit- reportedly caused infection and disease in several ing, and abdominal pain. Bleeding from nose and mammalian species, some of which were fatal. gums and encephalopatic illness has also been reported. The clinical course of the illness in severe Avian Infl uenza in Humans cases was characterized by rapid development of Prior to 1997, the transmission of AI viruses to severe bilateral pneumonia. Complications include humans was not considered to be a serious human ARDS, renal failure, and multi-organ failure. Clini- threat, based on the fi ndings that avian viruses do cal laboratory fi ndings included an early onset of not replicate effi ciently in experimentally infected lymphopenia, thrombocytopenia, and increased humans (9) and that no fatal cases of human infec- levels of serum transaminases. High levels of cyto- tions had been reported during any outbreaks of AI. kines and chemokines have been observed, suggest- Until 1997, only three cases of direct avian-to- ing a role of immune-mediated pathology in the human transmission of infl uenza viruses had been pathogenesis of H5N1 infections (25, 91, 137). described (145). In 1997, avian H5N1 HPAI viruses Other fi ndings include diffuse alveolar damage with were transmitted to humans causing 33% mortality interstitial fi brosis, hepatic central lobular necrosis, in hospitalized cases (22, 119). In 1998 and 1999, acute renal tubular necrosis, and lymphoid deple- H9N2 viruses transmitted from birds to pigs and tion. Although the gastrointestinal, hepatic, renal, humans in China and Hong Kong (21, 90). Human and hematological manifestations could suggest infections with symptoms typical of infl uenza were wider tissue tropism, there was little evidence of AI reported in China and Hong Kong (92). During an virus replication in organs outside the respiratory outbreak of H7N7 HPAI viruses in poultry in the tract (137). 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 111

Table 5.8. Lesions associated with natural fatal H5N1 HPAI virus infections in mammals (25, 56, 103, 111, 112, 135, 136).a Organ Lesion Humans Felids Dogs Civets

Heart Myocyte necrosis/myocarditis −+−− Brain Neuron necrosis/nonsuppurative + +++ − +++ encephalitis Meningitis Liver Necrosis ++++− Kidney Tubule necrosis/interstitial ++++++ nephritis Upper respiratory tract Rhinitis and tracheitis +++−++ Lung Interstitial pneumonia +++ +++ +++ +++ Bronchitis Edema Immune organs Lymphoid depletion ++−− a − = no lesion, + = mild lesion, ++ = moderate lesion, +++ = severe lesion.

AI viruses in humans induced pathological pulmonary necrotizing changes have been accompa- changes throughout the respiratory tract, but the nied by rupture of alveoli and bronchioles. most signifi cant lesions were present in the lower respiratory tract (Table 5.8) (reviewed in Wright and Avian Infl uenza in Pigs, Horses, Seals, Whales, Webster [144]). Acute diffuse infl ammation of the and Mink larynx, trachea, and bronchi were observed with Pigs are susceptible to experimental infection with a mucosal infl ammation and edema. Microscopically, range of avian and human infl uenza viruses (57). columnar ciliated cells were vacuolated and edema- However, interspecies transmission of AI viruses to tous and lost cilia before becoming desquamated. pigs in nature is not often documented (89). Avian Within 1 day after the onset of symptoms, desqua- H1N1 viruses have established themselves in pigs mation of the ciliated epithelium and mucus-produc- in Europe (93), and other viruses have been trans- ing epithelial cells was observed; in other areas, a iently detected in pig populations, including avian thickened, hyalinized basement membrane was virus subtypes H1N1 in Asia, H4N6 in Canada, H9N2 exposed. Submucosal edema and hyperemia occurred in China, and H5N1 in Asia (20, 40, 53, 84, 90). In with infi ltration by neutrophils and mononuclear horses, AI viruses can replicate in the trachea without cells. Viral antigen was present, predominantly in producing disease signs (75), but to date only the epithelial cells and mononuclear cells but also infre- H3N8 and H7N7 subtypes have been established quently within epidermal cells in the basal cell layer. as adapted endemic infl uenza A virus lineages in In more severe cases of primary viral pneumonia, horses. there was interstitial pneumonitis with marked In 1979 to 1980, approximately 20% of the harbor hyperemia and broadening of the alveolar walls, seal (Phoca vitulina) population of the northeast with predominantly mononuclear leukocyte infi ltra- coast of the United States died of a severe respiration and capillary dilation and thrombosis. The tory infection with consolidation of the lungs typical alveolar walls were denuded of epithelium; intral- of primary viral pneumonia (38). Infl uenza virus veolar edema and exudates were present; hyaline particles were found in high concentrations in the membranes covered alveolar walls; and intralveolar lungs and brains of the dead seals. Antigenic and hemorrhage occurred. Infl uenza virus specifi c genetic analyses (142) established that this virus antigen was present in type 1 and 2 alveolar epithe- derived all its genes from H7N7 LPAI viruses and lial cells, as well as in intralveolar macrophages. The was associated with severe disease in mammals. 112 Avian Influenza

During 1983, an H4N5 LPAI virus also caused A domestic cat naturally infected with H5N1 pneumonia and death among harbor seals off the HPAI virus presented with fever, panting, and coast of New England (42). H13N2 and H13N9 depression (112). The cat had convulsions and died LPAI viruses have been isolated from the lungs and 2 days after onset of illness. Necropsy of the cat hilar lymph nodes of a stranded pilot whale (Globi- showed cerebral congestion, conjunctivitis, pulmo- cephala melas) (41). nary edema, severe pneumonia, renal congestion, Infl uenza A viruses have been isolated from mink and hemorrhage in the intestinal serosa. Histopatho- (Mustela vison), which, together with ferrets, belong logical examination demonstrated nonsuppurative to the genus Mustela and are naturally susceptible encephalitis with gliosis, mononuclear cell infi ltra- to infl uenza A viruses. Outbreaks of H10N4 LPAI tion, vasculitis, and congestion in both cerebrum and viruses in mink have caused respiratory disease, cerebellum. In the lung, severe pulmonary edema, characterized by interstitial pneumonia, which was interstitial pneumonia, and congestion were noted, also reproduced experimentally and shown to spread as well as multifocal necrosis in the liver, nephritis, to contacts (11, 30–32, 59). and lymphoid depletion in the spleen. In October 2004, a dog (Canis familiaris) infected Avian Infl uenza (H5N1) in Felids, Dogs, and with H5N1 HPAI virus was reported in Thailand Civets (111). The dog had consumed duck carcasses from Natural infection with H5N1 HPAI has been an area with reported H5N1 HPAI virus infections observed in tigers (Panthera tigris) and leopards in ducks. The dog developed high fever, panting, (Panthera pardus and Neofelis nebulosa) in zoos in and lethargy and died within 24 hours. Lesions seen Thailand (56, 135) and in domestic cats (Felis catus) included bloody nasal discharge; severe pulmonary under natural and experimental conditions (63, 102, congestion and edema; and congestion of the spleen, 112, 136, 146). Transmission between felids has kidney, and liver (Table 5.8). Infl uenza virus was also been reported. During the H5N1 virus outbreak isolated from lung, liver, kidney, and urine speci- in Thailand in 2003, two tigers and two leopards at mens (111). Histopathological examination of the a zoo developed clinical signs, including high fever lung showed severe pulmonary edema and intersti- and respiratory distress, followed by death (56, 135). tial pneumonia with infl ammatory cell infi ltration. The animals had been fed fresh chicken carcasses Hemolysis with brownish black particles was found from a local slaughterhouse. At that time, many in the pulmonary parenchyma, and the liver showed chickens were dying of H5N1 virus infection. The focal necrosis. The kidneys showed mild nephritis four animals had severe pulmonary consolidation with tubular degeneration. Viral antigen staining and multifocal hemorrhage in several organs, includ- was found in pulmonary alveolar cells, hepato- ing lung, heart, thymus, stomach, intestine, liver, cytes, renal tubular epithelium, and glomerular and lymph nodes (135) (Table 5.8). All animals that epithelium. died had serosanguinous nasal discharge, and some Cases of H5N1 HPAI have also been reported in had neurological signs. The lungs were severely Owston’s civets (Chrotogale owstoni) in Vietnam congested and had hemorrhage. Serosanguinous that showed appetite loss and neurological symp- exudate was seen throughout the tracheal and bron- toms, including convulsions, and high mortality chiolar lumen, and pleural effusion was also reported. (103). Necropsy revealed pulmonary edema with Microscopic lesions were characterized by loss of mild parenchymal infl ammation (Table 5.8). Mod- bronchiolar and alveolar epithelium; thickening of erately severe meningitis was present, together with alveolar walls; and fl ooding of alveolar lumens with cerebral edema and hypoxic change of the neurons. edema fl uid mixed with fi brin, erythrocytes, neutro- Foci of necrosis were seen in the liver. Within the phils, and macrophages. Microscopic fi ndings lung there was AI viral antigen staining of bronchial showed moderate congestion of the brain with epithelium and pneumocytes. Infl uenza virus antigen mild nonsuppurative meningoencephalitis, severe was also detected in the infl ammatory exudates in diffuse lung hemorrhage and edema, and moderate the meninges. Prominent staining of the neurons of multifocal necrotizing hepatitis. Virus was demon- the cerebral cortex and cerebellum was seen but was strated in bronchiolar epithelium, neurons and not accompanied by a signifi cant infl ammatory cell hepatocytes. infi ltrate. In contrast to previous instances of infec- 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 113 tion of tigers and leopards, the civets were not fed most frequently observed in the lungs of H5N1- dead poultry and the source for the infection of the infected animals consisting of consolidation of the civets remains unknown. lungs. A spectrum of histopathological features was observed including acute bronchiolitis, broncho- AVIAN INFLUENZA EXPERIMENTAL pneumonia, and interstitial pneumonia. Immunohis- INFECTIONS IN MAMMALS tochemical staining for the presence of viral antigen The pathogenicity of AI viruses has been studied in yielded rare positive cells in the lungs of H5N1- a number of mammalian models, including mice and infected animals. Histopathological features detected rats (26, 37, 48, 71, 73, 79, 100, 106), hamsters (44), in the brains included the presence of glial nodules, ferrets (39, 44, 72, 73, 79, 150), monkeys (6, 7, 77, 101), perivascular infi ltration of lymphocytes and poly- cats (44, 63, 102), and pigs (20, 44, 48, 57, 106). morphonuclear cells in the parenchyma, neurono- The murine model has been commonly used to phagia, and increased lymphocytic infi ltrate in the study H5N1 HPAI virus pathogenicity in mammals. choroid plexus (150). Many H5N1 HPAI viruses However, unlike human H1N1 and H3N2 infl uenza isolated since 2004 have been highly lethal for mice A viruses, the human- and avian-origin H5N1 iso- and ferrets and exhibited a substantially greater level lates from 1997 did not require adaptation to be of virulence in ferrets than other H5N1 viruses from pathogenic in mice (Mus musculus) and were catego- 1997 through 2003 (73). These viruses induced a rized as viruses that either were of high pathogenicity rapid disease progression with high lethality rates and replicated systemically (including in the brain) and replicated to high titers in the respiratory tract or are of low pathogenicity and replicated only in the and spread to multiple organs, including brain. lungs and upper respiratory tract of mice (37, 70, 71). Different species of monkeys have also been used In general, the pathogenicity of H5N1/97 isolates as models for studying infl uenza infections. Cyno- in mice corresponded to the severity of disease in molgus macaques (Macaca fascicularis) infected humans (54). Some H5N1 HPAI viruses caused with infl uenza virus A/Hong Kong/156/97 (H5N1) severe disease in mice, characterized by hypother- developed acute respiratory distress syndrome and mia, rapid weight loss, and 75% to 100% mortality fever associated with a necrotizing interstitial pneu- (26). The viruses caused necrosis in respiratory epi- monia (101). The monkeys had extensive pulmonary thelium of the nasal cavity, trachea, bronchi, and consolidation in both lungs. Histologically, the bronchioles with accompanying infl ammation. lesions consisted of necrotizing bronchointerstitial Ferrets (Mustela putorius furo) are naturally sus- pneumonia, with extensive loss of alveolar and ceptible to infection with human H1N1 and H3N2 bronchiolar epithelium, and fl ooding of the alveoli infl uenza A viruses, and infections produce disease with edema fl uid, fi brin, erythrocytes, cell debris, that resembles infl uenza in humans—that is, infec- neutrophils, and macrophages. Lesions in tissues tion and lesions largely confi ned to the nasal mucosa outside the respiratory tract consisted of a mild sup- but occasionally the lower respiratory tract (110). purative tonsillitis, extensive lymphocytic necrosis Infection of ferrets with H5N1 HPAI viruses has a in spleen and lymph nodes, and multifocal renal resulted in upper and lower respiratory tract infec- tubular necrosis. Positive AI viral antigen was tion, severe lethargy, fever, weight loss, transient detected only in alveolar macrophages, pneumo- lymphopenia, and, in some animals, gastrointestinal cytes, bronchiolar and bronchial epithelial cells, abnormalities such as yellow-colored diarrhea (39, neutrophils, and unidentifi ed mononuclear cells in 150). Some H5N1 HPAI viruses resulted in systemic the respiratory tract. No infected cells were demon- infection with isolation of virus from multiple sys- strated in the spleen, heart, or brains by immunohis- temic organs, although at substantially lower titers tochemistry in any of these animals, revealing that, than in respiratory tissues. The ferrets exhibited in contrast to the situation with mice, the virus did respiratory signs including nasal discharge, sneez- not replicate outside the respiratory tract in the ing, visual signs of dyspnea, and extreme lethargy. monkeys. Severely affected ferrets exhibited substantial weight Pigtail macaque (Macaca namestrina) experi- loss over the course of infection. Neurological signs mentally infected with a genetically reconstructed occasionally were observed and included ataxia, strain of human infl uenza H1N1 presented with hindlimb paresis, and torticollis. Gross lesions were weight loss, clear nasal discharge, moderate fever, 114 Avian Influenza throat infl ammation, and loss of appetite (7). The antigen was demonstrated in intestinal autonomic monkeys had mild suppurative tracheitis, mild neurons, suggesting such lesions are not specifi c for tracheobronchial lymphadenopathy, and multifocal direct intestinal infection and could result from sys- to coalescing vascular congestion, edema, and mild temic virus infection (12). to moderate consolidation of the lung parenchyma. Experimental studies with Asian H5N1 viruses in The pulmonary histopathology was consistent with pigs have demonstrated viral replication in the upper progressive primary viral pneumonia as seen in respiratory tract of infected pigs but no transmission humans. Ten different LPAI viruses administered to contact pigs (20, 57, 106). Clinical signs consisted to squirrel monkeys produced a spectrum of replica- of mild cough, hyperthermia, and anorexia. Micro- tion and virulence, and induction of pneumonia scopically, some of these viruses produced moderate and systemic illness occurring with some of the interstitial pneumonia (20). viruses (77). Domestic cats experimentally inoculated with CONCLUSIONS H5N1 virus, intratracheally or by feeding them A variety of AI viruses have caused infections in virus-infected chickens, developed severe diffuse birds and mammals resulting in varying clinical and alveolar damage, and transmitted the virus to in- pathobiological features depending on multiple contact sentinel cats (63, 102). Two days after inoc- factors, including host species and virus strain. LPAI ulation, all cats showed clinical signs, including virus typically produced clinical signs and lesions in hyperthermia, lethargy, protrusion of the third the respiratory and intestinal tracts of domestic eyelid, conjunctivitis, labored breathing, and multi- poultry. By contrast, HPAI viruses typically produce focal to coalescing pulmonary lesions. Cats fed a similar severe, systemic disease with high mor- virus-infected chicks also had enlargement of and tality in chickens and other gallinaceous birds. multifocal petechial hemorrhages in the tonsils, However, these same HPAI viruses usually produce mandibular lymph nodes and retropharyngeal lymph no infections or limited infection with no clinical nodes, and multiple petechial hemorrhages in the signs or only mild disease in domestic ducks and liver. Intratracheally inoculated cats had had multi- wild birds. Over the past decade, the emergent H5N1 ple to coalescing foci of infl ammation and necrosis HPAI viruses have shifted to increased virulence for that centered on the bronchioles. Some alveoli were chickens as evident by shorter MDTs and a greater covered by hyaline membranes. The epithelium of propensity for massive disseminated replication in bronchiolar and alveolar walls had necrosis and vascular endothelial cells. These viruses have hyperplasia. The brains had multiple randomly dis- changed from producing inconsistent respiratory tributed foci of necrosis and infl ammation, charac- infections in ducks to some strains being highly terized by aggregates of neutrophils and glial cells, lethal in ducks with virus found in multiple visceral interstitial edema, neuronal necrosis, and perivascu- organs and brain. The most recent Asian H5N1 lar mononuclear infi ltrates. The leptomeninges had HPAI viruses have also infected some wild birds multifocal to diffuse mononuclear infi ltrates. The producing systemic infections and death. Across all lesions were generally more severe in the cerebrum bird species, the ability to produce severe disease than in the brain stem, and were mild or absent in and death is associated with high virus replication the cerebellum. The heart had multiple foci of necro- titers in the host, especially in specifi c tissues such sis and mononuclear cell infl ammation in the myo- as brain and heart. The AI viruses on occasion have cardium. Necrotic lesions were also observed in the caused infection in a number of mammalian species, kidney, liver and adrenal gland. Staining for AI virus including humans, domestic cats, dogs, and large antigen was closely associated with the presence of felids. Experimental infection with AI viruses has histological lesions. In cats experimentally fed on produced pathological changes that varied depend- infected chicks, virus-associated ganglioneuritis ing on the animal species and the virus strain. also occurred in the submucosal and myenteric plexi of the small intestine, which the authors suggested REFERENCES resulted from direct infection from intestinal lumen 1. Acland, H.M., L.A. Silverman Bachin, and R.J. (102). However, intranasally inoculated ducks had Eckroade. 1984. Lesions in broiler and layer similar ganglioneuritis in the intestines and AI viral chickens in an outbreak of highly pathogenic 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 115

avian infl uenza virus infection. Veterinary Pathol- 13. Capua, I., and F. Mutinelli. 2001. A Color Atlas ogy 21:564–569. and Text on Avian Infl uenza. Papi Editore: 2. Alexander, D.J. 1993. Orthomyxovirus infec- Bologna, Italy, pp. 1–127. tions. In: J.B. McFerran and M.S. McNulty (eds.). 14. Capua, I., F. Mutinelli, C. Terregino, G. Cattoli, Virus Infections in Birds. Elsevier Science: R.J. Manvell, and F. Burlini. 2000. Highly patho- London, pp. 287–316. genic avian infl uenza (H7N1) in ostriches farmed 3. Alexander, D.J., W.H. Allan, D.G. Parsons, and in Italy. Veterinary Records 146:356. G. Parsons. 1978. The pathogenicity of four avian 15. Capua, I., S. Marangon, L. Selli, D.J. Alexander, infl uenza viruses for fowls, turkeys and ducks. D.E Swayne, M.D. Pozza, E. Parenti, and F.M. Research in Veterinary Science 24:242–247. Cancellotti. 1999. Outbreaks of highly pathogenic 4. Alexander, D.J., G. Parsons, and R.J. Manvell. avian infl uenza (H5N2) in Italy during October 1986. Experimental assessment of the pathoge- 1997-January 1998. Avian Pathology 28:455–460. nicity of eight avian infl uenza A viruses of H5 16. Capua, I., and F. Mutinelli. 2001. Mortality in subtype for chickens, turkeys, ducks and quail. Muscovy ducks (Cairina moschata) and domestic Avian Pathology 15:647–662. geese (Anser anser var. domestica) associated 5. Allwright, D.M., W.P. Burger, A. Geyer, and with natural infection with a highly pathogenic A.W. Terblanche. 1993. Isolation of an infl uenza avian infl uenza virus of H7N1 subtype. Avian A virus from ostriches. Avian Pathology 22:59– Pathology 30:179–183. 65. 17. Cauthen, A.N., D.E. Swayne, S. Schultz-Cherry, 6. Baas, T., C.R. Baskin, D.L. Diamond, A. Garcia- M.L. Perdue, and D.L. Suarez. 2000. Continued Sastre, H. Bielefeldt-Ohmann, T.M. Tumpey, circulation in China of highly pathogenic avian M.J. Thomas, V.S. Carter, T.H. Teal, N. Van infl uenza viruses encoding the hemagglutinin Hoeven, S. Proll, J.M. Jacobs, Z.R. Caldwell, gene associated with the 1997 H5N1 outbreak in M.A. Gritsenko, R.R. Hukkanen, D.G. Camp, poultry and humans. Journal of Virology 74: 2nd, R.D. Smith, and M.G. Katze. 2006. Inte- 6592–6599. grated molecular signature of disease: analysis 18. Chen, H., G. Deng, Z. Li, G. Tian, Y. Li, P. Jiao, of infl uenza virus-infected macaques through L. Zhang, Z. Liu, R.G. Webster, and K. Yu. 2004. functional genomics and proteomics. Journal of The evolution of H5N1 infl uenza viruses in ducks Virology 80:10813–10828. in southern China. Proceedings of the National 7. Baskin, C.R., A. Garcia-Sastre, T.M. Tumpey, H. Academy of Science U S A 101:10452–1047. Bielefeldt-Ohmann, V.S. Carter, E. Nistal-Villan, 19. Chen, H., G.D.J. Smith, S.Y. Zhang, K. Qin, J. and M.G. Katze. 2004. Integration of clinical Wang, K.S. Li, R.G. Webster, J.S.M. Peiris, and data, pathology, and cDNA microarrays in infl u- Y. Guan. 2005. H5N1 virus outbreak in migratory enza virus-infected pigtailed macaques (Macaca waterfowl: a worrying development could help to nemestrina). Journal of Virology 78:10420–10432. spread this dangerous virus beyond its stronghold 8. Beard, C.W., and D.H. Helfer. 1972. Isolation of in Southeast Asia. Nature (London) 436:191– two turkey infl uenza viruses in Oregon. Avian 192. Diseases 16:1133–1136. 20. Choi, Y.K., T.D. Nguyen, H. Ozaki, R.J. Webby, 9. Beare, A.S., and R.G. Webster. 1991. Replication P. Puthavathana, C. Buranathal, A. Chaisingh, P. of avian infl uenza viruses in humans. Archives of Auewarakul, N.T. Hanh, S.K. Ma, P.Y. Hui, Y. Virology 119:37–42. Guan, J.S. Peiris, and R.G. Webster. 2005. Studies 10. Becker, W.B. 1966. The isolation and classifi ca- of H5N1 infl uenza virus infection of pigs by tion of Tern virus: infl uenza A-Tern South Africa- using viruses isolated in Vietnam and Thailand in 1961. Journal of Hygiene 64:309–320. 2004. Journal of Virology 79:10821–10825. 11. Berg, M., L. Englund, I.A. Abusugra, B. Klinge- 21. Choi, Y.K., H. Ozaki, R.J. Webby, R.G. Webster, born, and T. Linne. 1990. Close relationship J.S. Peiris, L. Poon, C. Butt, Y.H. Leung, and Y. between mink infl uenza (H10N4) and concomi- Guan. 2004. Continuing evolution of H9N2 infl u- tantly circulating avian infl uenza viruses. Archives enza viruses in Southeastern China. Journal of of Virology 113:61–71. Virology 78:8609–8614. 12. Brown, J.D., D.E. Stallknecht, J.R. Beck, D.L. 22. Claas, E.C., A.D. Osterhaus, R. van Beek, J.C. De Suarez, and D.E. Swayne. 2006. Susceptibility of Jong, G.F. Rimmelzwaan, D.A. Senne, S. Krauss, North American ducks and gulls to H5N1 highly K.F. Shortridge, and R.G. Webster. 1998. Human pathogenic avian infl uenza viruses. Emerging infl uenza A H5N1 virus related to a highly patho- Infectious Diseases 12:1663–1670. genic avian infl uenza virus. Lancet 351:472–477. 116 Avian Influenza

23. Cooley, A.J., H. Van Campen, M.S. Philpott, 34. Fernanadez, P.A., R.J. Rotello, Z. Rangini, A. B.C. Easterday, V.S. Hinshaw. 1989. Pathologi- Doupe, H.C.A. Drexler, and J.Y. Yuan. 1994. cal lesions in the lungs of ducks with infl uenza A Expression of a specifi c marker of avian pro- viruses. Veterinary Pathology 26:1–5. grammed cell death in both apoptosis and necro- 24. de Jong, M.D., and T.T. Hien. 2006. Avian infl u- sis. Proceedings of the National Academy of enza A (H5N1). Journal of Clinical Virology Science USA 91:8641–8645. 35:2–13. 35. Fouchier, R.A., V. Munster, A. Wallensten, 25. de Jong, M.D., C.P. Simmons, T.T. Thanh, V.M. T.M. Bestebroer, S. Herfst, D. Smith, G.F. Rim- Hien, G.J. Smith, T.N. Chau, D.M. Hoang, N.V. melzwaan, B. Olsen, and A.D. Osterhaus. 2005. Chau, T.H. Khanh, V.C. Dong, P.T. Qui, B.V. Characterization of a novel infl uenza A virus Cam, Q. Ha do, Y. Guan, J.S. Peiris, N.T. Chinh, hemagglutinin subtype (H16) obtained from black- T.T. Hien, and J. Farrar. 2006. Fatal outcome of headed gulls. Journal of Virology 79:2814–2822. human infl uenza A (H5N1) is associated with 36. Fouchier, R.A., P.M. Schneeberger, F.W. high viral load and hypercytokinemia. Nature Rozendaal, J.M. Broekman, S.A. Kemink, V. Medicine 12:1203–1207. Munster, T. Kuiken, G.F. Rimmelzwaan, M. 26. Dybing, J.K., S. Schultz-Cherry, D.E. Swayne, Schutten, G.J. Van Doornum, G. Koch, A. D.L. Suarez, and M.L. Perdue. 2000. Distinct Bosman, M. Koopmans, and A.D. Osterhaus. pathogenesis of Hong Kong-origin H5N1 viruses 2004. Avian infl uenza A virus (H7N7) associated in mice compared with that of other highly patho- with human conjunctivitis and a fatal case of genic H5 avian infl uenza viruses. Journal of acute respiratory distress syndrome. Proceedings Virology 74:1443–1450. of the National Academy of Science USA 27. Easterday, B.C., V.S Hinshaw, and D.A. Halvor- 101:1356–1361. son. 1997. Infl uenza. In: B.W. Calnek, H.J. 37. Gao, P., S. Watanabe, T. Ito, H. Goto, K. Wells, Barnes. C.W. Beard, L.R. McDougald, and Y.M. M. McGregor, A.J. Cooley, and Y. Kawaoka. Saif (eds.). Diseases of Poultry, 10th ed. Iowa 1999. Biological heterogeneity, including sys- State University Press: Ames, IA, pp. 583–605. temic replication in mice, of H5N1 infl uenza A 28. Elbers, A.R., T.H. Fabri, T.S. de Vries, J.J. de virus isolates from humans in Hong Kong. Journal Wit, A. Pijpers, and G. Koch. 2004. The highly of Virology 73:3184–3189. pathogenic avian infl uenza A (H7N7) virus epi- 38. Geraci, J.R., D.J. St Aubin, I.K. Barker, R.G. demic in The Netherlands in 2003—lessons Webster, V.S. Hinshaw, W.J. Bean, H.L. Ruhnke, learned from the fi rst fi ve outbreaks. Avian Dis- J.H. Prescott, G. Early, A.S. Baker, S. Madoff, eases 48:691–705. and R.T. Schooley. 1982. Mass mortality of 29. Ellis, T.M., R.B. Bousfi eld, L.A. Bissett, K.C. harbor seals: pneumonia associated with infl u- Dyrting, G.S. Luk, S.T. Tsim, K. Sturm-Ramirez, enza A virus. Science 215:1129–1131. R.G. Webster, Y. Guan, and J.S. Malik Peiris. 39. Govorkova, E.A., J.E. Rehg, S. Krauss, H.L. Yen, 2004. Investigation of outbreaks of highly patho- Y. Guan, M. Peiris, T.D. Nguyen, T.H. Hanh, P. genic H5N1 avian infl uenza in waterfowl and Puthavathana, H.T. Long, C. Buranathai, W. Lim, wild birds in Hong Kong in late 2002. Avian R.G. Webster, and E. Hoffmann. 2005. Lethality Pathology 33:492–505. to ferrets of H5N1 infl uenza viruses isolated from 30. Englund, L. 2000. Studies on infl uenza viruses humans and poultry in 2004. Journal of Virology H10N4 and H10N7 of avian origin in mink. Vet- 79:2191–2198. erinary Microbiology 74:101–107. 40. Guan, Y., K.F. Shortridge, S. Krauss, P.H. Li, Y. 31. Englund, L., and C. Hard af Segerstad. 1998. Two Kawaoka, and R.G. Webster. 1996. Emergence avian H10 infl uenza A virus strains with different of avian H1N1 infl uenza viruses in pigs in China. pathogenicity for mink (Mustela vison). Archives Journal of Virology 70:8041–8046. of Virology 143:653–666. 41. Hinshaw, V.S., W.J. Bean, J. Geraci, P. Fiorelli, 32. Englund, L., B. Klingeborn, and T. Mejerland. G. Early, and R.G. Webster. 1986. Characteriza- 1986. Avian infl uenza A virus causing an out- tion of two infl uenza A viruses from a pilot whale. break of contagious interstitial pneumonia in Journal of Virology 58:655–656. mink. Acta Veterinaria Scandinavica 27:497– 42. Hinshaw, V.S., W.J. Bean, R.G. Webster, J.E. 504. Rehg, P. Fiorelli, G. Early, J.R. Geraci, and D.J. 33. FAO. 2004. Update on the avian infl uenza situa- St Aubin. 1984. Are seals frequently infected tion (as of 15/06/2004). FAO AIDE News AI with avian infl uenza viruses? Journal of Virology Bulletin 16:1–10. 51:863–865. 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 117

43. Hinshaw, V.S., C.W. Olsen, N. Dybdahlsissoko, of the haemagglutinin subtype H7. Deutsche and D. Evans. 1994. Apoptosis: a mechanism of Tierarztliche Wochenschrift 111:467–472. cell killing by infl uenza A and B viruses. Journal 53. Karasin, A.I., C.W. Olsen, I.H. Brown, S. Carman, of Virology 68:3667–3673. M. Stalker, and G. Josephson. 2000. H4N6 infl u- 44. Hinshaw, V.S., R.G. Webster, B.C. Easterday, enza virus isolated from pigs in Ontario. Cana- and W.J. Bean, Jr. 1981. Replication of avian dian Veterinary Journal 41:938–939. infl uenza A viruses in mammals. Infection and 54. Katz, J.M., X. Lu, T.M. Tumpey, C.B. Smith, Immunity 34:354–361. M.W. Shaw, and K. Subbarao. 2000. Molecular 45. Hooper, P., and P. Selleck. 1998. Pathology correlates of infl uenza A H5N1 virus pathogen- of low and high virulent infl uenza virus infec- esis in mice. Journal of Virology 74:10807– tions. In: Proceedings of the Fourth Interna- 10810. tional Symposium on Avian Infl uenza, D.E. 55. Kawaoka, Y., T.M. Chambers, W.L. Sladen, and Swayne and R.D. Slemons, eds. U.S Animal R.G. Webster. 1988. Is the gene pool of infl uenza Health Association: Richmond, Virginia, pp. viruses in shorebirds and gulls different from that 134–141. in wild ducks? Virology 163:247–250. 46. Hooper, P.T., G.W. Russell, P.W. Selleck, and 56. Keawcharoen, J., K. Oraveerakul, T. Kuiken, W.L. Stanislawek. 1995. Observations on the R.A. Fouchier, A. Amonsin, S. Payungporn, S. relationship in chickens between the virulence of Noppornpanth, S. Wattanodorn, A. Theamboo- some avian infl uenza viruses and their pathoge- niers, R. Tantilertcharoen, R. Pattanarangsan, N. nicity for various organs. Avian Diseases 39:458– Arya, P. Ratanakorn, D.M. Osterhaus, and Y. 464. Poovorawan. 2004. Avian infl uenza H5N1 in 47. Hulse-Post, D.J., K.M. Sturm-Ramirez, J. tigers and leopards. Emerging Infectious Diseases Humberd, P. Seiler, E.A. Govorkova, S. Krauss, 10:2189–2191. C. Scholtissek, P. Puthavathana, C. Buranathai, 57. Kida, H., T. Ito, J. Yasuda, Y. Shimizu, C. Itakura, T.D. Nguyen, H.T. Long, T.S. Naipospos, H. K.F. Shortridge, Y. Kawaoka, and R.G. Webster. Chen, T.M. Ellis, Y. Guan, J.S. Peiris, and R.G. 1994. Potential for transmission of avian infl u- Webster. 2005. Role of domestic ducks in the enza viruses to pigs. Journal of General Virology propagation and biological evolution of highly 75:2183–2188. pathogenic H5N1 infl uenza viruses in Asia. Pro- 58. Kishida, N., Y. Sakoda, N. Isoda, K. Matsuda, M. ceedings of the National Academy of Science Eto, Y. Sunaga, T. Umemura, and H. Kida. 2005. U S A 102:10682–10687. Pathogenicity of H5 infl uenza viruses for ducks. 48. Isoda, N., Y. Sakoda, N. Kishida, G.R. Bai, K. Archives of Virology 150:1383–1392. Matsuda, T. Umemura, and H. Kida. 2006. Patho- 59. Klingeborn, B., L. Englund, R. Rott, N. Juntti, genicity of a highly pathogenic avian infl uenza and G. Rockborn. 1985. An avian infl uenza A virus, A/chicken/Yamaguchi/7/04 (H5N1) in dif- virus killing a mammalian species—the mink. ferent species of birds and mammals. Archives of Brief report. Archives of Virology 86:347–351. Virology 151:1267–1279. 60. Klopfl eisch, R., O. Werner, E. Mundt, T. Harder, 49. Jones, Y.L., and D.E. Swayne. 2004. Compara- and J.P. Teifke. 2006. Neurotropism of highly tive pathobiology of low and high pathogenicity pathogenic avian infl uenza virus A/chicken/ H7N3 Chilean avian infl uenza viruses in chick- Indonesia/2003 (H5N1) in experimentally in- ens. Avian Diseases 48:119–128. fected pigeons (Columbia livia f. domestica). 50. Jorgensen P.H., O.L. Nielsen, H.C. Hansen, R.J. Veterinary Pathology 43:463–470. Manvell, J. Banks, D.J. Alexander. 1998. Isola- 61. Kobayashi, Y., T. Horimoto, Y. Kawaoka, D.J. tion of infl uenza virus subtype H5N2 and avian Alexander, and C. Itakura. 1996. Neuropatho- paramyxovirus type 1 from a fl ock of ostriches in logical studies of chickens infected with highly Europe. Avian Pathology 27:15–20. pathogenic avian infl uenza viruses. Journal of 51. Jungherr, E.L., E.E. Tyzzer. C.A. Brandly, and Comparative Pathology 114:131–147. H.E. Moses. 1946. The comparative pathology of 62. Kobayashi, Y., T.H., Y. Kawaoka, D.J. Alexan- fowl plague and Newcastle disease. American der, and C. Itaruka. 1996. Pathological studies of Journal of Veterinary Research 7:1946. chickens experimentally infected with two highly 52. Kaleta, E.F., and A. Honicke. 2004. Review of pathogenic avian infl uenza viruses. Avian Pathol- the literature on avian infl uenza A viruses in ogy 25:285–304. pigeons and experimental studies on the suscep- 63. Kuiken, T., G. Rimmelzwaan, D. van Riel, G. van tibility of domestic pigeons to infl uenza A viruses Amerongen, M. Baars, R. Fouchier, and A. Oster- 118 Avian Influenza

haus. 2004. Avian H5N1 infl uenza in cats. Science L.M. Chen, P. Thawatsupha, M. Chittaganpitch, 306:241. S. Waicharoen, D.T. Nguyen, T. Nguyen, H.H. 64. Kwon, Y.-K., M.-C. Kim, H.-W. Sung, Y.-J. Lee, Nguyen, J.H. Kim, L.T. Hoang, C. Kang, L.S. J.-G. Choi, E.-K. Lee and J.-H. Kim. 2005. Highly Phuong, W. Lim, S. Zaki, R.O. Donis, N.J. Cox, pathogenic avian infl uenza (H5N1) in the com- J.M. Katz, and T.M. Tumpey. 2005. Avian infl u- mercial domestic ducks of South Korea. Avian enza (H5N1) viruses isolated from humans in Pathology 34:367–370. Asia in 2004 exhibit increased virulence in 65. Kwon, Y.K., S.J. Joh, M.C. Kim, Y.J. Lee, J.G. mammals. Journal of Virology 79:11788–11800. Choi, E.K. Lee, S.H. Wee, H.W. Sung, J.H. 74. Mo, I.P., M. Brugh, O.J. Fletcher, G.N. Rowland, Kwon, M.I. Kang, and J.H. Kim. 2005. Highly and D.E. Swayne. 1997. Comparative pathology pathogenic avian infl uenza in magpies (Pica pica of chickens experimentally inoculated with avian sericea) in South Korea. Journal of Wildlife Dis- infl uenza viruses of low and high pathogenicity. eases 41:618–623. Avian Diseases 41:125–136. 66. Laudert, E., D. Halvorson, V. Sivanandan, and 75. Mumford, J.A., and T.M. Chambers. 1998. D. Shaw. 1993. Comparative evaluation of tissue Equine infl uenza. In: Nicholson K.G., R.G. trophism characteristics in turkeys and mallard Webster, and A.J. Hay (eds.). Textbook of Infl u- ducks after intravenous inoculation of type enza. Blackwell Sciences: Oxford, pp. 146–162. A infl uenza viruses. Avian Diseases 37:773– 76. Muramoto, Y., H. Ozaki, A. Takada, C.H. Park, 780. Y. Sunden, T. Umemura, Y. Kawaoka, H. Matsuda, 67. Laudert, E., V. Sivanandan, and D. Halvorson. and H. Kida. 2006. Highly pathogenic H5N1 1993. Effect of an H5N1 avian infl uenza virus infl uenza virus causes coagulopathy in chickens. infection on the immune system of mallard ducks. Microbiology and Immunology 50:73–81. Avian Diseases 37:845–853. 77. Murphy, B.R., V.S. Hinshaw, D.L. Sly, W.T. 68. Laudert E., V.S., D. Halvorson, D. Shaw, and London, N.T. Hosier, F.T. Wood, R.G. Webster, R.G. Webster. 1993. Biological and molecular and R.M. Chanock. 1982. Virulence of avian characterization of H13N3 infl uenza type A infl uenza A viruses for squirrel monkeys. Infec- viruses isolated from turkeys and surface water. tion and Immunity 37:1119–1126. Avian Diseases 37:793–799. 78. Narayan, O. 1972. Pathogenesis of lethal infl u- 69. Lee, C.W., D.L. Suarez, T.M. Tumpey, H.W. enza virus infection in turkeys. II. Central nervous Sung, Y.K. Kwon, Y.J. Lee, J.G. Choi, S.J. Joh, system phase of infection. Journal of Compara- M.C. Kim, E.K. Lee, J.M. Park, X. Lu, J.M. Katz, tive Pathology 82:139–146. E. Spackman, D.E. Swayne, and J.H. Kim. 2005. 79. Nguyen, D.C., T.M. Uyeki, S. Jadhao, T. Maines, Characterization of highly pathogenic H5N1 M. Shaw, Y. Matsuoka, C. Smith, T. Rowe, X. avian infl uenza A viruses isolated from South Lu, H. Hall, X. Xu, A. Balish, A. Klimov, T.M. Korea. Journal of Virology 79:3692–3702. Tumpey, D.E. Swayne, L.P. Huynh, H.K. 70. Lipatov, A.S., E.A. Govorkova, R.J. Webby, H. Nghiem, H.H. Nguyen, L.T. Hoang, N.J. Cox, Ozaki, M. Peiris, Y. Guan, L. Poon, and R.G. and J.M. Katz. 2005. Isolation and characteriza- Webster. 2004. Infl uenza: emergence and control. tion of avian infl uenza viruses, including highly Journal of Virology 78:8951–8959. pathogenic H5N1, from poultry in live bird 71. Lu, X., T.M. Tumpey, T. Morken, S.R. Zaki, N.J. markets in Hanoi, Vietnam, in 2001. Journal of Cox, and J.M. Katz. 1999. A mouse model for the Virology 79:4201–4212. evaluation of pathogenesis and immunity to infl u- 80. OIE. 2003. Pathogenic H5N1 Avian Infl uenza in enza A (H5N1) viruses isolated from humans. Waterfowl and Wild Birds. Final report, Hong Journal of Virology 73:5903–5911. Kong, China, July 2003. 72. Maines, T.R., L.M. Chen, Y. Matsuoka, H. Chen, 81. OIE. 2007. Highly Pathogenic Avian Infl uenza in T. Rowe, J. Ortin, A. Falcon, T.H. Nguyen, Q. Vietnam. Follow-up report No.9. OIE Update on Mai le, E.R. Sedyaningsih, S. Harun, T.M. Avian Infl uenza in Animals. Available at http:// Tumpey, R.O. Donis, N.J. Cox, K. Subbarao, and www.oie.int/downld/AVIAN%20INFLUENZA/ J.M. Katz. 2006. Lack of transmission of H5N1 A_AI-Asia.htm. avian-human reassortant infl uenza viruses in a 82. Olivier, A.J. Ecology and epidemiology of avian ferret model. Proceedings of the National infl uenza in ostriches. Developments in Biologi- Academy of Science U S A 103:12121–12126. cals 124:51–57. 73. Maines, T.R., X.H. Lu, S.M. Erb, L. Edwards, J. 83. Olsen, B., V.J. Munster, A. Wallensten, J. Guarner, P.W. Greer, D.C. Nguyen, K.J. Szretter, Waldenstrom, A.D. Osterhaus, and R.A. Fouch- 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 119

ier. 2006. Global patterns of infl uenza A virus in 95. Perkins, L.E., and D.E. Swayne. 2003. Compara- wild birds. Science 312:384–388. tive susceptibility of selected avian and mam- 84. Olsen, C.W. 2002. The emergence of novel malian species to a Hong Kong-origin H5N1 swine infl uenza viruses in North America. Virus high-pathogenicity avian infl uenza virus. Avian Research 85:199–210. Diseases 47:956–967. 85. Panigrahy, B., D.A. Senne, and J.E. Pearson. 96. Perkins, L.E., and D.E. Swayne. 2001. Pathobiol- 1995. Presence of avian infl uenza virus (AIV) ogy of A/chicken/Hong Kong/220/97 (H5N1) subtypes H5N2 and H7N1 in emus (Dromaius avian infl uenza virus in seven gallinaceous novaehollandiae) and rheas (Rhea americana): species. Veterinary Pathology 38:149–164. virus isolation and serologic fi ndings. Avian 97. Perkins, L.E., and D.E. Swayne. 2002. Patho- Diseases 39:64–67. genicity of a Hong Kong-origin H5N1 highly 86. Pantin-Jackwood, M.J., and D.E. Swayne. 2007. pathogenic avian infl uenza virus for emus, geese, Pathobiology of Asian highly pathogenic avian ducks, and pigeons. Avian Diseases 46:53–63. infl uenza H5N1 virus infections in ducks. Avian 98. Perkins, L.E., and D.E. Swayne. 2002. Suscepti- Diseases 51:250–259. bility of laughing gulls (Larus atricilla) to H5N1 87. Pantin-Jackwood, M.J., D.L. Suarez, and D.E. and H5N3 highly pathogenic avian infl uenza Swayne. 2006. Increased pathogenicity of Vietnam viruses. Avian Diseases 46:877–885. H5N1 avian infl uenza viruses in ducks. In: Pro- 99. Perkins, L.E., and D.E. Swayne. 2003. Varied ceedings of 110th United States Animal Health pathogenicity of a Hong Kong-origin H5N1 avian Association Meeting, Minneapolis, MN, p. 93. infl uenza virus in four passerine species and bud- 88. Pantin-Jackwood, M.J., D.L. Suarez, E. Spack- gerigars. Veterinary Pathology 40:14–24. man, and D.E. Swayne. 2007. Age at infec- 100. Rigoni, M., K. Shinya, A. Toffan, A. Milani, F. tion affects the pathogenicity of Asian highly Bettini, Y. Kawaoka, G. Cattoli, and I. Capua. pathogenic avian influenza H5N1 viruses 2007. Pneumo- and neurotropism of avian origin in ducks. Virus Research, doi: 10_1016/j. Italian highly pathogenic avian infl uenza H7N1 virusres.2007.06.006. isolates in experimentally infected mice. Virol- 89. Peiris, J.S., M.D. de Jong, and Y. Guan. 2007. ogy 364(1):28–35. Avian infl uenza virus (H5N1): a threat to human 101. Rimmelzwaan, G.F., T. Kuiken, G. van Ameron- health. Clinical Microbiology Reviews 20:243– gen, T.M. Bestebroer, R.A. Fouchier, and A.D. 267. Osterhaus. 2001. Pathogenesis of infl uenza A 90. Peiris, J.S., Y. Guan, D. Markwell, P. Ghose, (H5N1) virus infection in a primate model. R.G. Webster, and K.F. Shortridge. 2001. Journal of Virology 75:6687–6691. Cocirculation of avian H9N2 and contemporary 102. Rimmelzwaan, G.F., D. van Riel, M. Baars, T.M. “human” H3N2 infl uenza A viruses in pigs in Bestebroer, G. van Amerongen, R.A. Fouchier, southeastern China: potential for genetic reas- A.D. Osterhaus, and T. Kuiken. 2006. Infl uenza sortment? Journal of Virology 75:9679–9686. A virus (H5N1) infection in cats causes systemic 91. Peiris, J.S., W.C. Yu, C.W. Leung, C.Y. Cheung, disease with potential novel routes of virus spread W.F. Ng, J.M. Nicholls, T.K. Ng, K.H. Chan, S. within and between hosts. American Journal of T. Lai, W.L. Lim, K.Y. Yuen, and Y. Guan. 2004. Pathology 168:176–183. Re-emergence of fatal human infl uenza A subtype 103. Roberton, S.I., D.J. Bell, G.J. Smith, J.M. Nich- H5N1 disease. Lancet 363:617–619. olls, K.H. Chan, D.T. Nguyen, P.Q. Tran, U. 92. Peiris, M., K.Y Yuen, C.W. Leung, K.H. Chan, Streicher, L.L. Poon, H. Chen, P. Horby, M. P.L. Ip, R.W. Lai, W.K. Orr, and K.F. Shortridge. Guardo, Y. Guan, and J.S. Peiris. 2006. Avian 1999. Human infection with infl uenza H9N2. infl uenza H5N1 in viverrids: implications for Lancet 354:916–917. wildlife health and conservation. Proceedings of 93. Pensaert, M., K. Otis, J. Vandeputte, M.M. the Royal Biological Society 273:1729–1732. Kaplan, and P.A. Bachmann. 1981. Evidence for 104. Schultz-Cherry, S., R.M. Krug, and V.S. Hinshaw. the natural transmission of infl uenza A viruses 1996. Infl uenza virus neuraminidase activates from wild ducks to swine and its potential impor- latent transforming growth factor beta. Journal of tance for man. Bulletin of the World Health Orga- Virology 70:8624–8629. nization 59:75–78. 105. Shalaby, A.A., R.D. Slemons, and D.E. Swayne. 94. Perdue, M.L., D.L. Suarez, and D.E. Swayne. 1994. Pathological studies of A/chicken/Alabama/ 1999. Avian infl uenza in the 1990’s. Poultry and 7395/75 (H4N8) infl uenza virus in specifi c pathogen- Avian Biology Reviews 11:1–20. free laying hens. Avian Diseases 38:22–32. 120 Avian Influenza

106. Shortridge, K.F., N.N. Zhou, Y. Guan, P. Gao, T. Ellis, Y. Guan, J.S. Peiris, and R.G. Webster. Ito, Y. Kawaoka, S. Kodihalli, S. Krauss, D. 2005. Are ducks contributing to the endemicity Markwell, K.G. Murti, M. Norwood, D. Senne, of highly pathogenic H5N1 infl uenza virus in L. Sims, A. Takada, and R.G. Webster. 1998. Asia? Journal of Virology 79:11269–11279. Characterization of avian H5N1 infl uenza viruses 117. Suarez, D.L., M.L. Perdue, N. Cox, T. Rowe, from poultry in Hong Kong. Virology 252:331– C. Bender, J. Huang, and D.E. Swayne. 1998. 342. Comparisons of highly virulent H5N1 infl uenza 107. Slemons, R.D., D.C. Johnson, J.S. Osborn, and F. A viruses isolated from humans and chickens Hayes. 1974. Type-A infl uenza viruses isolated from Hong Kong. Journal of Virology 72:6678– from wild free-fl ying ducks in California. Avian 6688. Diseases 18:119–124. 118. Subbarao, K., and J. Katz. 2000. Avian infl uenza 108. Slemons, R.D., and D.E. Swayne. 1992. Nephro- viruses infecting humans. Cellular and Molecular tropic properties demonstrated by A/chicken/ Life Science 57:1770–1784. Alabama/75 (H4N8) following intravenous chal- 119. Subbarao, K., A. Klimov, J. Katz, H. Regnery, lenge of chickens. Avian Diseases 36:926–931. W. Lim, H. Hall, M. Perdue, D. Swayne, C. 109. Slemons, R.D., and D.E. Swayne. 1990. Replica- Bender, J. Huang, M. Hemphill, T. Rowe, M. tion of a waterfowl-origin infl uenza virus in the Shaw, X. Xu, K. Fukuda, and N. Cox. 1998. kidney and intestine of chickens. Avian Diseases Characterization of an avian infl uenza A (H5N1) 34:277–284. virus isolated from a child with a fatal respiratory 110. Smith, H., and C. Sweet. 1988. Lessons for human illness. Science 279:393–396. infl uenza from pathogenicity studies with ferrets. 120. Swayne, D.E. 1997. Pathobiology of H5N2 Review of Infectious Diseases 10:56–75. Mexican avian infl uenza virus infections of 111. Songserm T, A.A., R. Jam-on, N. Sae-Heng, N. chickens. Veterinary Pathology 34:557–567. Pariyothorn, and S. Payungporn. 2006. Fatal 121. Swayne, D.E. 2007. Understanding the complex avian infl uenza A H5N1 in a dog. Emerging pathobiology of high pathogenicity avian infl u- Infectious Diseases 12:1744–1747. enza viruses in birds. Avian Diseases 51:242– 112. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- 249. Heng, N. Meemak, N. Pariyothorn, S. Payung- 122. Swayne, D.E., and D.A. Halvorson. 2008. Infl u- porn, A. Theamboonlers, and Y. Poovorawan. enza. In: Y.M. Saif, J.R. Glisson, A.M. Fadly, 2006. Avian infl uenza H5N1 in naturally infected L.R. McDougald, and L. Nolan (eds.), Diseases domestic cat. Emerging Infectious Diseases of Poultry, 12th ed. Blackwell Publishing: Ames, 12:681–683. IA, in press. 113. Stallknecht, D.E. 1998. Ecology and epidemi- 123. Swayne, D.E., J.R. Beck, M.L. Perdue, M. Brugh, ology of avian infl uenza viruses in wild bird and R.D. Slemons. 1996. Assessment of the populations: waterfowl, shorebirds, pelicans, cor- ability of ratite-origin infl uenza viruses to infect morants, etc. In: D.E. Swayne and R.D. Slemons and produce disease in rheas and chickens. Avian (eds.). Proceedings of the Fourth International Diseases 40:438–447. Symposium on Avian Infl uenza, US Animal 124. Swayne, D.E., M.D. Ficken, and J.S. Guy. 1992. Health Association: Richmond, VA, pp. 61– Immunohistochemical demonstration of infl uenza 69. A nucleoprotein in lungs of turkeys with natural 114. Stubbs, E.L. 1948. Fowl pest. In: H.E. Biester and and experimental infl uenza respiratory disease. L.H. Schwarte (eds.). Diseases of Poultry, 2nd ed. Avian Pathology 21:547–557. Iowa State University Press: Ames, IA, pp. 603– 125. Swayne, D.E., and M.J. Pantin-Jackwood. 2006. 614. Pathogenicity of avian infl uenza viruses in 115. Sturm-Ramirez, K.M., T. Ellis, B. Bousfi eld, L. poultry. Developments in Biologicals 124:61– Bissett, K. Dyrting, J.E. Rehg, L. Poon, Y. Guan, 67. M. Peiris, and R.G. Webster. 2004. Reemerging 126. Swayne, D.E., and R.D. Slemons. 1994. Com- H5N1 infl uenza viruses in Hong Kong in 2002 parative pathology of a chicken-origin and two are highly pathogenic to ducks. Journal of Virol- duck-origin infl uenza virus isolates in chickens: ogy 78:4892–4901. the effect of route of inoculation. Veterinary 116. Sturm-Ramirez, K.M., D.J. Hulse-Post, E.A. Pathology 31:237–245. Govorkova, J. Humberd, P. Seiler, P. Putha- 127. Swayne, D.E., and R.D. Slemons. 1995. Com- vathana, C. Buranathai, T.D. Nguyen, A. Chais- parative pathology of intravenously inoculated ingh, H.T. Long, T.S. Naipospos, H. Chen, T.M. wild duck- and turkey-origin type A infl uenza 5 / Pathobiology of Avian Influenza Virus Infections in Birds and Mammals 121

viruses in chickens. Avian Diseases 39:74– ated with avian infl uenza A H5N1 virus. Journal 84. of Medical Virology 63:242–246. 128. Swayne, D.E., and R.D. Slemons. 1992. Evalua- 138. Tumpey, T.M., D.L. Suarez, L.E. Perkins, D.A. tion of the kidney as a potential site of avian Senne, J.G. Lee, Y.J. Lee, I.P. Mo, H.W. Sung, infl uenza virus persistence in chickens. Avian and D.E. Swayne. 2002. Characterization of a Diseases 36:937–944. highly pathogenic H5N1 avian infl uenza A virus 129. Swayne, D.E., and R.D. Slemons. 1990. Renal isolated from duck meat. Journal of Virology pathology in specifi c-pathogen-free chickens 76:6344–6355. inoculated with a waterfowl-origin type A infl u- 139. Tweed, S.A., D.M. Skowroski, S.T. David, A. enza virus. Avian Diseases 34:285–294. Larder, M. Petric, W. Lees, Y. Li, J. Katz, M. 130. Swayne, D.E., and D.L. Suarez. 2000. Highly Krajden, R. Tellier, C. Halpert, M. Hirst, C. pathogenic avian infl uenza. Revue Scientifi que et Astell, D. Lawrence, and A. Mak. 2004. Human Technique (International Offi ce of Epizootics) illness from avian infl uenza H7N3, British 19:463–482. Columbia. Emerging Infectious Diseases 10: 131. Swayne, D.E., and D.A. Halvorson. 2003. 2196–2199. Infl uenza. In: Y.M. Saif, H.J. Barnes, J.R. 140. Van Reeth, K. and B.C. Easterday. 1999. Swine Glisson, A.M. Fadly, L.R. McDougald, and D.E. infl uenza. In: B.E. Straw, S. D’Allaire, W.L. Swayne. (eds.). Diseases of Poultry, 11th ed. Mengeling and D.J. Taylor (eds.). Diseases of Iowa State University Press: Ames, IA, pp. 135– Swine. Iowa State University Press: Ames, IA, 160. pp. 277–290. 132. Takisawa, T.R.F., T. Miyawaki, K. Ohashi, and 141. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. Y. Nakanishi. 1995. Activation of the apoptotic Chambers, and Y. Kawaoka. 1992. Evolution and fasr antigen-encoding gene upon infl uenza virus ecology of infl uenza A viruses. Microbiology infection involving spontaneously produced beta- Reviews 56:152–179. interferon. Virology 209:288–296. 142. Webster, R.G., V.S. Hinshaw, W.J. Bean, K.L. 133. Teifke, J.P., R. Klopfl eisch, A. Globig, E. Starick, Van Wyke, J.R. Geraci, D.J. St. Aubin, and G. B. Hoffmann, P.U. Wolf, M. Beer, T.C. Metten- Petursson. 1981. Characterization of an infl uenza leiter, and T.C. Harder. 2007. Pathology of natural A virus from seals. Virology 113:712–724. infections by H5N1 highly pathogenic avian 143. Wood, J.M., R.G. Webster, and V.F. Nettles. infl uenza virus in mute (Cygnus olor) and 1985. Host range of A/chicken/Pennsylvanis/83 whooper (Cygnus cygnus) swans. Veterinary (H5N2). Avian Diseases 29:198–207. Pathology 44:137–143. 144. Wright, P.F. and R.G. Webster. 2001. Ortho- 134. Terregino, C., A. Milani, I. Capua, A.M. Marino, myxoviruses. In: D.M. Knipe and P.M. Howley and N. Cavaliere. 2006. Highly pathogenic avian (eds.). Fields Virology, 4th ed., vol. 1. Lippincott infl uenza H5N1 subtype in mute swans in Italy. Williams and Wilkins: Philadelphia, pp. 1533– Veterinary Records 158:491. 1579. 135. Thanawongnuwech, R., A. Amonsin, R. Tantil- 145. Wright, P.F., G. Neumann, and Y. Kawaoka. ertcharoen, S. Damrongwatanapokin, A. Theam- 2006. Orthomyxoviruses. In: D.M. Knipe and boonlers, S. Payungporn, K. Nanthapornphiphat, P.M. Howley (eds.). Fields Virology, 5th ed., vol. S. Ratanamungklanon, E. Tunak, T. Songserm, 2. Lippincott Williams and Wilkins: Philadelphia, V. Vivatthanavanich, T. Lekdumrongsak, S. Kes- pp. 1691–1740. dangsakonwut, S. Tunhikorn, and Y. Poovorawan. 146. Yingst, S.L., M.D. Saad, and S.A. Felt. 2006. 2005. Probable tiger-to-tiger transmission of Qinghai-like H5N1 from domestic cats, northern avian infl uenza H5N1. Emerging Infectious Iraq. Emerging Infectious Diseases 12:1295– Diseases 11:699–701. 1297. 136. Thiry, E., A. Zicola, D. Addie, H. Egberink, K. 147. Yuen, K.Y., P.K. Chan, M. Peiris, D.N. Tsang, Hartmann, H. Lutz, H. Poulet, and M.C. Horzinek. T.L. Que, K.F. Shortridge, P.T. Cheung, W.K. 2007. Highly pathogenic avian infl uenza H5N1 To, E.T. Ho, R. Sung, and A.F. Cheng. 1998. virus in cats and other carnivores. Veterinary Clinical features and rapid viral diagnosis of Microbiology 122(1–2):25–31. human disease associated with avian infl uenza A 137. To, K.F., P.K. Chan, K.F. Chan, W.K. Lee, W.Y. H5N1 virus. Lancet 351:467–471. Lam, K.F. Wong, N.L. Tang, D.N. Tsang, R.Y. 148. Zhou, J.Y., H.G. Shen, H.X. Chen, G.Z. Tong, Sung, T.A. Buckley, J.S. Tam, and A.F. Cheng. M. Liao, H.C. Yang, and J.X. Liu. 2006. Charac- 2001. Pathology of fatal human infection associ- terization of a highly pathogenic H5N1 infl u- 122 Avian Influenza

enza virus derived from bar-headed geese in commercial layers, in Pennsylvania, 1997–98. China. Journal of General Virology 87:1823– Avian Diseases 43:142–149. 1833. 150. Zitzow, L.A., T. Rowe, T. Morken, W.J. Shieh, 149. Ziegler, A.F., S. Davison, H. Acland, and R.J. S. Zaki, and J.M. Katz. 2002. Pathogenesis of Eckroade. 1999. Characteristics of H7N2 (nonavian inﬂ uenza A (H5N1) viruses in ferrets. pathogenic) avian inﬂ uenza virus infections in Journal of Virology 76:4420–4429. 6 The Global Nature of Avian Influenza

David E. Swayne

INTRODUCTION of AI in the live poultry market (LPM) system in Avian infl uenza (AI) virus is a global virus that northeastern United States during 1986 and in fol- knows no geographic boundaries, has no political lowing years, reports in other nontraditional avian agenda, and can infect commercial and noncom- species such as ratites and game birds, and elimina- mercial poultry, indoor- and outdoor-reared poultry, tion of range rearing of turkeys, surveillance has pet birds, wild birds, and a variety of other avian and increased and the number of AI virus isolations has nonavian species. AI viruses have been isolated grown geometrically, with the LPM birds and from poultry, captive birds, and wild birds in Africa, nontraditional poultry species becoming the major Asia, Australia, Europe, and North and South source of LPAI isolates (134). The LPM system is America, and anti-AI antibodies have even been typically supplied with poultry from small, special- identifi ed in Antarctic penguins (70, 122). However, ized mixed bird farms and not from the integrated reports of AI infection and the diseases they cause commercial poultry production systems except for in domestic poultry and other birds vary with spent laying chickens. Spent layers have been the individual countries, regions, and continents. The primary introduction point of H7N2 LPAI viruses reported frequency of AI is greatly skewed by the from LPM into commercial poultry (146). availability of diagnostics, quantity and quality of Active serological surveillance is done annually on surveillance conducted, the type of birds and pro- over 95% of broiler, layer, and turkey breeder fl ocks duction sector tested, the time of year, geographic in the United States through the National Poultry location, climatic conditions, and other undefi ned Improvement Plan (NPIP) (A. Rhorer, personal com- factors. However, failure to conduct adequate sur- munication, March 2, 2007). The NPIP AI monitor- veillance and diagnostics on poultry and other birds ing program began in 1998 with commercial broiler is not evidence to support the absence of AI virus or and layer breeder fl ocks, and expanded in 2000 to associated infections. include commercial turkey breeders. In 2002, the For example, diagnostics and surveillance in the program was changed from AI monitored to H5/H7 United States prior to the 1983–1984 H5N2 high AI monitored. In 2006, 181,000 AGID serological pathogenicity AI (HPAI) outbreak was done mostly tests were performed on 4551 breeder fl ocks in the on commercial poultry and the occasional backyard United States, encompassing a population of 47 fl ock using virus isolation in embryonating chicken million birds (135). In 2006, this NPIP program was eggs and serology with the agar gel immunodiffu- expanded to include monitoring for H5 and H7 AI in sion (AGID) test. From 1964 through 1985, the list meat chickens and turkeys at slaughter plants with of isolates (all low pathogenicity AI [LPAI] viruses over 90% of meat birds in the United States being except for 1984 HPAI viruses) or anti–infl uenza A in the NPIP monitoring program. In addition, the antibodies were primarily from range-reared turkeys National Chicken Council has a preslaughter testing and of limited number (132). With the description program for all commercial broiler fl ocks.

In 2006, 2,969,160 AGID serological tests were uniform international terminology of “highly patho- performed in the United States using reference genic avian infl uenza (HPAI)” was not adopted until reagents from the National Veterinary Services Lab- 1981, when HPAI became the offi cial designation oratories, which yielded 468 commercial turkey and for the virulent form of AI at the First International 4 commercial chicken fl ocks positive to antibodies Symposium on Avian Infl uenza (14). “High patho- to swine infl uenza viruses and only 1 commercial genicity” is an equivalent grammatical variant of turkey fl ock positive to an AI virus (H6N2) (135). “highly pathogenic” and can be used interchange- Most of these turkey fl ocks were vaccinated against ably (122). H1 and H3 swine infl uenza viruses, which resulted From the 1870s into the early 1900s, fowl plague in the positive serological results. In the LPM spread from Northern Italy into Europe with reports system, 8437 samples examined for AI virus yielded in Austria, Belgium, France, Germany, Great Britain, 134 LPAI viruses (135). However, these results do Hungary, the Netherlands, Romania, Russia, and not indicate that the United States has a higher fre- Switzerland (39, 57, 67, 69, 111, 112). By the mid- quency of LPAI than other countries—only that twentieth century, fowl plague had been diagnosed testing of large numbers of samples from high-risk in North Africa (Egypt), the Middle East, Asia sectors of poultry yielded AI viruses and/or anti-AI (China and Japan), South America (Argentina and antibodies. As surveillance expands in other coun- Brazil), North America (United States), and much tries and continents and diagnostics improve, new of Europe. Fowl plague was endemic in parts of sources of AI viruses will be identifi ed. Europe and Africa into the 1930s (3). Interestingly, in many situations fowl plague disappeared at the GENERAL HISTORY same time Newcastle disease was recognized as a To understand the global nature of AI, we must disease of poultry. For example, fowl plague was recognize the changing defi nition of AI infections reported in Italy into the early 1930s but extin- and the diseases they cause, based on scientifi c guished itself by 1937 when the epidemic of New- improvements in diagnosis and new knowledge in castle disease began (91). With the discovery of ecology and epidemiology over the past 125 years. Newcastle disease, and its similarity to fowl plague Historically, three major events have changed our in lesions, clinical presentation, and high morbidity defi nition of avian infl uenza and thus impacted the and mortality rates, this resulted in confusion on reported frequency of AI in the world: (1) early diagnosis of fi eld cases and their respective viral diagnosis of fowl plague in domestic poultry was etiologies. In some cases, the HPAI term “fowl based primarily on clinical features, lesions, and pest” was used interchangeable with “Newcastle animal studies; (2) recognition of LPAI viruses and disease,” and later “pseudo-fowl plague” and their infections in domestic poultry based on serol- “pseudo-fowl pest.” ogy and virus isolation; and (3) discovery of AI in The viral etiology for fowl plague was unknown asymptomatic wild bird reservoirs (122). until 1955 when fowl plague was determined to be caused by infl uenza A virus (95). Those early out- Fowl Plague in Poultry breaks of fowl plague were caused by HPAI viruses Historically, AI is of relatively recent description in that are classifi ed today as H7N1 and H7N7 sub- the poultry health literature with historical records types (37). However, at the time it was generally reporting the fi rst cases of AI as a highly lethal, considered that the fowl plague viruses were all the systemic disease of chickens in Italy during 1878, same because the antigenic and genetic differences that is, HPAI (90). This severe systemic disease has between “individual strains” were not known (D. most frequently been termed fowl plague or fowl Alexander, personal communication, February 27, pest, but other names have been used, including 2007). As a result, viruses that were exchanged peste aviaire, Gefl ugelpest, typhus exudatious gal- between laboratories may not have kept the original linarium, Brunswick bird plague, Brunswick disease, names or were renamed for the shipping laboratory. fowl disease, fowl or bird grippe, and others (111, Today, any conclusions concerning the source and 112) [see Chapter 7, The Beginning and Spread of date of many historical fowl plague isolates must be Fowl Plague (H7 High Pathogenicity Avian Infl u- interpreted with caution. For example, Petek (1981) enza) Across Europe and Asia (1878–1955)]. The states that the virus FPV-(fowl plague virus)-Brescia 6 / The Global Nature of Avian Influenza 125 was isolated in 1935 and has been erroneously called viruses of low virulence; therefore, these AI viruses Brescia/02 but should be correctly labeled A/ did not meet biological or molecular criteria for chicken/Brescia/35 (H7N1). However, there are HPAI viruses (43). references to FPV Brescia that predate 1935 (26) The earliest known LPAI virus was the “Dinter” and some samples before 1935 may have been or “N” strain, isolated in 1949 from chickens correctly maintained as A/chicken/Brescia/02 (91) in Germany [A/chicken/Germany/49 (H10N7)]. (D. Alexander, personal communication, February However, this virus was not known to be an LPAI 27, 2007). virus until 1960 [reviewed in (39)]. Between 1953 The early fowl plague cases in chickens and other and 1963, LPAI viruses were isolated from a series gallinaceous poultry were diagnosed primarily based of respiratory disease cases in domestic ducks in on the sudden high mortality; presence of specifi c Canada, Czechoslovakia, England, and the Ukraine. lesions such as cyanotic combs, hemorrhage in ven- This was followed by descriptions in turkeys from triculus and proventriculus and petechia on the heart; 1963 to 1965 of LPAI virus infections being a cause and identifi cation of a fi lterable virus [see Chapters of respiratory disease and drops in egg production 7 and 8, The Beginning and Spread of Fowl Plague in Canada and the United States. However, addi- (H7 High Pathogenicity Avian Infl uenza) Across tional cases of LPAI virus were not described again Europe and Asia (1878–1955) and High Pathogenic- in chickens until 1966 in Italy, along with the fi rst ity Avian Infl uenza in the Americas respectively]. cases in pheasants and quail. Throughout the latter Such virus isolates cross-reacted in hemagglutina- half of the 1960s, reports of respiratory disease and tion inhibition test using antisera from recovered isolation of LPAI viruses were common in turkeys birds, which resulted in the conclusion that a posi- and domestic ducklings. These early LPAI viruses tive “H7” HI test indicated a fowl plague virus were a variety of hemagglutinin and neuraminidase (HPAI) or infection by such an agent. However, in subtypes. 1959, 1961, and 1966, a clinical disease, indistin- Initially, H5 and H7 subtypes of infl uenza A virus guishable from classic fowl plague (i.e., H7) was were only associated with fowl plague viruses, but identifi ed in chickens, common terns, and turkeys, in 1966 and 1968, LPAI viruses were isolated from respectively, but these viruses were not inhibited by turkeys with low mortality or ill-defi ned syndromes antisera from fowl plague–recovered birds (i.e., that were typed as the H5 subtype, that is, the fi rst these viruses were not H7). This gave rise to the H5 LPAI viruses (2, 39, 106). In 1971, a turkey fl ock briefl y used term “fowl plague-like” and was the in Oregon that experienced mild respiratory disease fi rst indication that another hemagglutinin subtype, with diarrhea had an H7N3 AI virus isolated (22). H5, could be an HPAI virus. Thus, the original AI Since 1971, numerous H5 and H7 LPAI viruses infections in poultry were detected by severe clinical have been isolated and characterized, thus dispeling disease and linked serologically to two subtypes: H5 the myth that subtypes H5 and H7 equate with HP. and H7 HPAI viruses. In fact, only a small portion of the diverse H5 and These early HPAI (fowl plague) outbreaks are H7 AI viruses are HP; most are LPAI viruses (2, 37, covered in detail in Chapters 7 and 8. 50). Furthermore, the development of the AGID serological test in 1970 and its subsequent adoption Recognition of Low Pathogenicity as the primary international test to identify AI in- Avian Infl uenza in Poultry and Other fected chicken and turkey fl ocks expanded the iden- Man-made Systems tifi cation of LPAI in the 1970s and 1980s (20, 21). Mild clinical forms of AI (those that produced respiratory disease and drops in egg production) were Low and High Pathogenicity Avian Infl uenza fi rst recognized in various domestic poultry species Viruses in Wild Birds beginning in 1949, with occasional reports through Although early reports of fowl plague suspected the mid-1960s (39). These forms of AI have been wild birds in the transmission, the fi rst proof of AI called low pathogenic, pathogenic, non-HP and LP virus infection in wild birds was in common terns AI. In 2002, at the Fifth International Symposium with high mortality in South Africa during 1961 on Avian Infl uenza, the term “low pathogenicity (23). In the late 1960s, a survey of migratory water- (LP)” was adopted as the offi cial designation for AI fowl showed serological evidence of infection by AI 126 Avian Influenza viruses (38). However, the fi rst isolates of LPAI (LPNAI), which includes all H5 and H7 LPAI; and viruses were not made until 1972 from migratory (3) all other LPAI that are not notifi able to OIE, ducks in a Newcastle disease virus surveillance which includes H1–H4, H6, and H8–H16 LPAI (5, program in California (105) and from a pelagic 80). The third category—non-H5 and non-H7 seabird (shearwater) in Australia (33). Since then, LPAI—is not reported to OIE but may be reportable numerous surveys have been conducted, mostly in to national and state/provincial authorities. How- North American birds, and have demonstrated ever, based on pathobiological characteristics (e.g., asymptomatic infection by AI viruses in healthy disease, lesions, and signalment), the LPNAI and wild aquatic birds, principally in the orders Anseri- LPAI are indistinguishable except that some H5 and formes and Charadriiformes (50, 51, 56, 108–110). H7 LPNAI viruses have shown the ability to change Since the initial reports, extensive surveys have been to HPAI viruses, which is the reason for their listing conducted in Russia, Israel, China, Europe, and as international viruses for control. The defi nitions other countries (61, 63, 83, 102). These surveys have for HPNAI and LPNAI are as follows (80): yielded thousands of LPAI viruses of all 16 hemagglutinin and 9 neuraminidase subtypes from asymp- 1. HPNAI viruses have an intravenous pathogenic- tomatic wild birds. However, some HPAI viruses ity index (IVPI) in 6-week-old chickens greater have been isolated from wild birds: (1) during an than 1.2 or, as an alternative, cause at least 75% epidemic with high mortality in common terns (A/ mortality in 4-to 8-week-old chickens infected tern/South Africa/61 [H5N3]); (2) single isolations intravenously. H5 and H7 viruses that do not of A/fi nch/Germany/72 (H7N1), A/gull/Germany/79 have an IVPI of greater than 1.2 or cause less (H7N7), and A/peregrine falcon/UAE/2384/98 than 75% mortality in an intravenous lethality (H7N3); and (3) during the recent H5N1 HPAI out- test should be sequenced to determine whether break in Asia, Europe, and Africa with multiple iso- multiple basic amino acids are present at the lations from wild birds (4, 23, 30, 62, 64). The cleavage site of the hemagglutinin molecule ecology of AI viruses in wild birds has been covered (HA0); if the amino acid motif is similar to that in Chapter 3 (Ecology of Avian Infl uenza in Wild observed for other HPNAI isolates, the isolate Birds) and will not be discussed further in this or being tested should be considered as HPNAI. later chapters. Gallinaceous species of birds, both 2. LPNAI are all infl uenza A viruses of H5 and H7 domestic and wild, are not natural reservoirs of AI subtype that are not HPNAI viruses. viruses (86, 114). Although the in vivo pathotyping test is based on REGULATORY ASPECTS testing only chickens, most AI viruses give similar Since the early descriptions of fowl plague, various in vivo test results when using related species of governments and other entities have practiced erad- gallinaceous birds (10, 88). By contrast, most AI ication as the primary means to deal with HPAI and viruses that are highly lethal or HP for chickens have protect the food supply (136). Initially, eradication produced no infections or asymptomatic infections programs focused on identifying HPAI viruses using in the domestic duck, except for some strains of the in vivo chicken pathogenicity tests (i.e., pathotype) Eurasian H5N1 HPAI virus that are also highly and differentiation of these viruses from LPAI lethal for young domestic ducks but not for older viruses. However, in 1994, specifi c molecular and ducks (8,54, 89). Pathogenicity test results are spe- in vitro criteria were added as alternatives to in vivo cifi c for the host used in the test (122). testing to defi ne HPAI viruses (133). Today, the World Organization for Animal Health (Offi ce Terminology International des Epizooties [OIE]), an intergovern- The terminology used to describe AI varies with ment organization, sets the international sanitary and individual reports, publications, lectures, and other health standards for animals, including AI, and, such media forms used to disseminate information. The codes are used to safeguard international trade in terms “HPAI,” “HPNAI,” “LPAI,” and “LPNAI” poultry and poultry products. Using the OIE code were defi ned earlier. However, throughout this book, and other standards, AI can be divided into three the term “LPAI” will be used to indicate any of the categories: (1) HP notifi able AI (HPNAI), which 16 hemagglutinin subtypes of LPAI. Other terms includes all H5 and H7 HPAI; (2) LP notifi able AI may vary in various publications and in the chapters 6 / The Global Nature of Avian Influenza 127 that follow. For example, the term “outbreak” can quent in chickens, turkeys, and ducks (2, 4). be used to mean a single farm, as in Italy, or can However, in the integrated commercial poultry refer to the complete epidemic involving a strain or systems in developed countries, AI has been a rare lineage of virus in a country or region. A “case” occurrence considering the 25 to 30 billion chickens typically means a single diagnostic submission from raised each year (131). Cases have been reported in a farm or an outbreak on a single farm. captive wild birds held as caged pets or in quarantine stations, private collections/reserves, and zoological LOW PATHOGENICITY AVIAN parks (2, 4). INFLUENZA IN POULTRY AND CAPTIVE For poultry, the reported frequency is highest in BIRDS birds raised on small mixed species farms with There is no international mandate or uniform stan- outdoor access (village and rural poultry) or those dards used around the world for LPAI surveillance, raised for the LPM systems, which typically use few and no requirements to report LPAI to the OIE other veterinary services, have poor control of bird move- than LPNAI (H5/H7) in poultry. Therefore, pub- ment, and lack biosecurity. However, incidence and lished reports of LPAI are sporadic and infrequent, distribution vary greatly with geographic region, with most reports being in peer-reviewed scientifi c country, species and age of bird, time of year, and literature concerning single cases or clusters of the environmental or agricultural system occupied cases. However, several national and international (122). A few examples of LPAI in different man- organizations have attempted to compile reports on made systems are discussed next. LPAI from individual countries, regions and continents, especially the fi rst through the sixth Interna- Examples of Low Pathogenicity Avian Infl uenza tional Symposia on Avian Infl uenza (1981–2006) in Man-made Systems (15, 35, 36, 118, 126, 128). Surveillance for AI and Historically, LPAI viruses have been reported in reporting have been most common from the United range-reared turkeys in Minnesota, United States, States, the European Union, Australia, and Canada, following exposure to LPAI virus–infected wild with sporadic reports from other countries. Some waterfowl during the fall staging and migration countries lack the veterinary diagnostic infrastruc- south for the winter (48). However, the number of ture or fi nancial resources to conduct adequate diag- infected turkey fl ocks has varied by year, with a low nostics and surveillance for LPAI or place a low of 2 fl ocks in 1983 and highs of 141, 178, and 258 priority on LPAI compared with other animal dis- fl ocks in 1978, 1995, and 1988, respectively (47). eases, while some countries chose a policy of “do To eliminate this problem, the Minnesota turkey not look and you do not have AI.” industry decided in 1998 to eliminate outdoor raising As reported in Chapter 4 (Epidemiology of Avian of turkeys, which resulted in reduced numbers of Infl uenza in Agricultural and Other Man-made cases, with only 33 infl uenza A–infected fl ocks from Systems), mankind has developed new avian anthro- 1996 to 2000; most of these were infections with pocentric systems through captivity, domestication, H1N1 swine infl uenza viruses and not AI virus rearing of birds at the agriculture–wild bird inter- infections (46). In addition to direct exposure face, nonindustrial and industrial agriculture, increasing infection rates, turkeys have a greater national and international commerce, and nontradi- susceptibility to wild bird infl uenza A viruses, which tional raising practices (117). Avian infl uenza contributes to more cases in turkeys than in chickens viruses can survive and be perpetuated in fi ve dif- (122). ferent categories of man-made ecosystems (117, Various subtypes of LPAI viruses have been iso- 122): (1) bird collection, trading, maintenance, and lated from poultry in the LPM system of the north- exhibition systems; (2) village, backyard, and hobby eastern United States (1986–present), but since the fl ocks, especially outdoor rearing and mixing of bird implementation of a control program in 2002, the species; (3) LPM systems; (4) range- or outdoor- rate of infection has declined from a high of 60% of reared commercial poultry; and (5) integrated indoor the markets to below 20% with less than 1.1% of the commercial poultry. The frequency of LPAI viruses samples from markets being positive for LPAI in domestic poultry, captive birds, and wild birds is viruses (71, 135). However, these LPMs and the largely unknown, but in most developed countries, small farms that supply the birds have become a infections are sporadic in poultry, being most fre- major reservoir of LPAI viruses in the United States 128 Avian Influenza and have served as the source of LPAI viruses that which can result in mortality as high as 80% in crossed over to infect small and large commercial turkeys and 75% quail (91, 122). In some cases, fl ocks (97, 101): (1) H5N2 LPAI virus in Pennsyl- LPAI viruses have caused severe and economically vania that infected 100 commercial fl ocks during important disease in the fi eld when accompanied by 1983 and mutated to H5N2 HPAI virus (45, 139); secondary infection and other stressors. Reproduc- (2) H5N2 LPAI virus that infected 21 fl ocks in New tion of the fi eld syndrome by experimental inocula- York, New Jersey, Massachusetts, and Ohio during tion of chickens with LPAI viruses alone usually 1986 (45); (3) H7N2 LPAI virus that infected 24 produces no morbidity or mortality, thus pointing to commercial poultry fl ocks in Pennsylvania between the need for concurrent bacterial or other infections 1996 and 1998 (32, 107, 119, 146); (4) H7N2 LPAI in fi eld disease. virus that infected seven commercial poultry fl ocks in Pennsylvania during 2001–2002 (34, 107, 119); HIGH PATHOGENICITY AVIAN (5) H7N2 LPAI virus that infected 210 commercial INFLUENZA (1959 TO 2007) fl ocks in Virginia/West Virginia/North Carolina Over the past 48 years, since the development of during 2002 (97, 99, 107, 119); (6) H7N2 LPAI consistent diagnostic and control strategies, 26 epi- virus that infected 3.9 million chickens in a large demics or limited outbreaks of HPAI have been layer company (four farms) in Connecticut during documented worldwide and are compiled in Table 2003 (97, 119); (7) H7N2 LPAI virus that infected 6.1. All of these HPAI viruses were of the H5 or H7 32,000 layers in a single fl ock in Rhode Island hemagglutinin subtype. There have been no HPAI during 2003 (97, 119); and (8) H7N2 LPAI virus outbreaks with AI viruses of the other 14 hemag- that infected three broiler fl ocks in Delaware and glutinin subtypes (H1–H4, H6, H8–H16). However, Maryland during 2004 (98). several non–H5/H7 AI viruses have expressed high Other examples of poultry infection (>100 fl ocks) lethality in chickens in the intravenous pathogenic- by LPAI viruses include (1) H5N2 LPAI virus, ity test, including A/mandarin duck/Singapore/805/ endemic in noncommercial and commercial chick- F-72/7/1993 (H10N5), A/turkey/England/384/79 ens in Mexico beginning in 1993 and continuing to (H10N4), and derivatives of A/chicken/Alabama/ the present (86, 94, 137, 138); (2) H9N2 LPAI virus, 1975 (H4N8), but these viruses were not highly endemic in noncommercial and commercial chick- lethal on intranasal inoculation and lacked the hem- ens in many developing countries of Asia and the agglutinin cleavage site sequence compatible with Middle East beginning in the late 1990s and continu- HPAI virus (25, 143). In addition, the mechanism ing today (7, 24, 65); (3) H7N1 epidemic of LPAI responsible for the high lethality was renal failure in turkeys in Italy during 1999 that mutated to HPAI from extensive replication in the kidney tubular epi- virus; (5) H7N3 LPAI epidemic in Northern Italy thelium, a mechanism previously reported to occur during 2002–2003 (65); and (4) H1N1, H1N2, and in intravenous inoculated chickens using a variety H3N2 swine infl uenza viruses, meat, and breeder of different LPAI viruses (120, 123–125, 143). turkeys in multiple countries (37, 116, 129). Addi- Thus, the “HP” results for these three AI isolates tional outbreaks in poultry (commercial and non- were a laboratory phenomenon and these three commercial), ratites, pet birds, fi ghting cocks, and viruses are not HPAI viruses. other birds have been described over the past 25 Clinically, these 26 HPAI epidemics or limited years and are reported in the proceedings of the fi rst outbreaks initially were recognized with three dis- through sixth International Symposia on Avian tinctly different presentations: (1) initial detection as Infl uenza (15, 35, 36, 118, 126, 128). Compiling high mortality disease in chickens and other gallina- these LPAI outbreaks is beyond the scope of this ceous poultry, that is, detection of HPAI virus in the brief chapter. index case with high mortality rates (epidemics 1–3, 5–7, 9–13, 15, 17, 18, 21, 24, 25); (2) detection of Features of Low Pathogenicity Avian Infl uenza HPAI virus in domestic waterfowl or gallinaceous Infections in the fi eld with LPAI virus infection poultry as the index case but without high mortality typically produces respiratory disease or drops in (epidemics 4 and 16); and (3) appearance of H5 or egg production, but mortality is usually low unless H7 LPAI in the index case with abrupt change to accompanied by secondary agents such as bacteria, HPAI in the index or additional cases after a period Table 6.1. Twenty-six documented epidemics or limited outbreaks of HPAI since discovery of AI virus as cause of fowl plague in 1955, modifi ed from (6,121,122,127). Number Affected With High Hemagglutinin Proteolytic Cleavage Mortality or Were Specifi c No. Date Prototype AI Virus Subtype Site Deduced Amino Acid Sequence Depopulateda References

1 1959 A/chicken/Scotland/59 H5N1 PQRKKR/GLF Chickens (Gallus gallus 60, 87 (D.J. domesticus): number of Alexander, farms and total number of personal birds affected not reported communication, 2000) 2 1961 A/tern/South Africa/61 H5N3 PQRETRRQKR/GLF 4 small areas, 1300 common 23, 60 terns (Sterna hirundo) 129 3 1963 A/turkey/England/63 H7N3 PETPKRRRR/GLF 2 farms; 29,000 breeder 140, 144 turkeys (Meleagridis gallopavo) 4 1966 A/turkey/Ontario/7732/66 H5N9 PQRRRKKR/GLF 2 farms; 8,100 breeder turkeys 58, 60 5 1975–76 A/chicken/Victoria/75 H7N7 PEIPKKREKR/GLF 1 farm; 25,000 laying 13, 19, 130 (Sometimes noted as chickens, 17,000 broilers, A/chicken/Victoria/76) and 16,000 ducks (Anas platyrhyncos) 6 1979 A/chicken/Leipzig H7N7 PEIPKKKKR/GLF 1 farm: 600,000 chickens, 80 6, 49, 92 (Germany)/79 PEIPKRKKR/GLF geese (formerly East PEIPKKRKKR/GLF Germany) PEIPKKKKKKR/GLF 7 1979 A/turkey/England/199/79 H7N7 PEIPKKRKR/GLF 3 commercial farms of 1, 9, 12, 144 PEIPKRRRR/GLF turkeys: 9262 turkeys PEIPKKREKR/GLF Table 6.1. Continued Number Affected With High Hemagglutinin Proteolytic Cleavage Mortality or Were Speciﬁ c No. Date Prototype AI Virus Subtype Site Deduced Amino Acid Sequence Depopulateda References

8 1983–84 A/chicken/Pennsylvania/ H5N2 PQKKKR/GLF (LPAIV) 452 ﬂ ocks, 17 million birds; 37, 40, 60, 132 1/83 (LPAIV) most were chickens or A/chicken/Pennsylvania/ PQKKKR/GLF (HPAIV)—HPAIV turkeys, a few chukar 1370/83 (HPAIV) lacked a glycosylation site at amino partridges (Alectoris acid residue 13 chukar) and guinea fowl (Numida meleagris) 9 1983 A/turkey/Ireland/1378/83 H5N8 PQRKRKKR/GLF 3 farms; 800 meat turkeys 4, 60, 68 died on original farm; 8,640 turkeys, 28,020 chickens and 270,000 ducks

130 were depopulated on original and 2 adjacent farms 10 1985 A/chicken/Victoria/1/85 H7N7 PEIPKKREKR/GLF 1 farm; 24,000 broiler 18, 31, 100 breeders, 27,000 laying chickens, 69,000 broilers and 118,518 unspecifi ed- type of chickens 11 1991 A/turkey/England/50– H5N1 PQRKRKTR/GLF 1 farm; 8000 turkeys 11, 100, 144 92/91 12 1992 A/chicken/Victoria/1/92 H7N3 PEIPKKKKR/GLF 2 farms, 1 backyard fl ock and 96, 104, 141 1 hatchery; 12,700 broiler breeders, 5,700 ducks, 105,000 day-old chicks 13 1994 A/chicken/Queensland/ H7N3 PEIPRKRKR/GLF 1 farm; 22,000 laying 86, 141, P. Selleck, 477/94 chickens personal communication, 2007 14 1994–95 A/chicken/ H5N2 PQRETR/GLF (LPAIV) Chickens: stamping-out policy 37, 44, 85, 138 Mexico/31381–7/1994 was not used for control. (LPAIV) Concurrent circulation of A/chicken/Puebla/8623– PQRKRKTR/GLF (HPAIV) LP and HPAI virus strains, 607/94 (HPAIV) but HPAI virus only from A/chicken/Queretaro/ PQRKRKRKTR/GLF (HPAIV) late 1994 to mid-1995. 14588–19/95 (HPAIV) Unknown number of birds infected with HPAI virus, but 360 commercial chicken fl ocks were “depopulated” for AI in 1995 through controlled marketing 15 1994–95, A/chicken/ H7N3 PETPKRKRKR/GLF “Stamping-out” policy was 16, 37, 72, 73, 75, 2001, Pakistan/447/95 not used for control. 76

131 2004 A/chicken/Pakistan/1369- PETPKRRKR/GLF Surveillance, quarantine, CR2/95 vaccination, and controlled A/chicken/Pakistan/16/95 PETPKRRNR*GLF marketing were used as the control strategy. Two incursions: (1) 3.2 million broilers and broiler breeder chickens (northern part of country: 1994–1995), and (2) 2.52 million layers (Karachi, 2004) 16 1996– A/goose/ H5N1 PQRERRRKKR/GLF Unknown number of 42, 103, 113, 145 Guangdong/1/1996 commercial and A/chicken/Hong noncommercial ﬂ ocks Kong/220/97 (village poultry); over 220 million birds dead or culled, mostly chickens, but also ducks, geese, Japanese quail, and some wild birdsb Table 6.1. Continued Number Affected With High Hemagglutinin Proteolytic Cleavage Mortality or Were Speciﬁ c No. Date Prototype AI Virus Subtype Site Deduced Amino Acid Sequence Depopulateda References

17 1997 A/chicken/New South H7N4 PEIPRKRKR/GLF 3 farms; 128,000 broiler 86, 104 Wales/1651/97 breeders, 33,000 broilers, 261 emu (Dromaius novaehollandiae) 18 1997 A/chicken/Italy/330/97 H5N2 PQRRRKKR/GLF 8 rural ﬂ ocks (hobby and 29, 59 backyard only); 2116 chickens, 1501 turkeys, 731 guinea fowl, 2322 ducks, 204 quail (species unknown), 45 pigeons (Columbia livia), 45 geese

132 (species unknown) and 1 pheasant (species unknown) 19 1999–2000 A/turkey/Italy/977/99 H7N1 PEIPKGR/GLF (LPAI virus) 413 farms: 8.1 million laying 17, 28 (LPAIV) chickens; 2.7 million meat A/turkey/Italy/4580/99 PEIPKGSRVRR/GLF (n = 360) and breeder turkeys; 2.4 (HPAIV) PEIPKGSRMRR/GLF (n = 3) million broiler breeders and PEIPKRSRVRR/GLF (n = 1) broilers; 247,000 guinea fowl; 260,000 quail, ducks, and pheasants; 1,737 backyard poultry and 387 ostriches 20 2002 A/chicken/Chile/ H7N3 PEKPKTR/GLF (LPAIV) 2 farms of 1 company, 66, 93, 115 176822/2002 (LPAIV) multiple houses; 617,800 A/chicken/ PEKPKTCSPLSRCRETR/GLF broiler breeders died Chile/4322/2002 (150,500) or destroyed, (HPAIV) 18,500 turkey breeders (2 A/chicken/ PEKPKTCSPLSRCRKTR/GLF houses) destroyed Chile/4325/2002 (HPAIV) 21 2003 A/chicken/Netherlands/ H7N7 PEIPKRRRR/GLF 255 infected ﬂ ocks, and 1381 9, 41, 49 621557/2003 commercial and 16,521 backyard/smallholder ﬂ ocks depopulated. 30 million died or depopulated: majority were chickens; backyard/smallholder: 175,035 birds, commercial: 25 million infected or preemptive culled birds, 4.5 million birds for welfare reasons; Belgium, 8 farms, 2.3 million chickens; Germany, 1 farm, 419,000

133 chickens 22 2004 A/Chicken/Canada/ H7N3 PENPKTR/GLF (LPAI virus) 42 commercial and 11 52, 74, 84 AVFV1/04 (LPAIV) backyard ﬂ ocks infected A/Chicken/Canada/ PENPKQAYRKRMTR/GLF (n = 3); (1.2 million poultry): AVFV2/04 (HPAIV) later HPAI isolates approximately 16 million PENPKQAYQKRMTR/GLF (n = 24), commercial poultry PENPKQAYKKRMTR/GLF (n = 4), depopulated, most were PENPKQAYHKRMTR/GLF (n = 3), chickens PENPKQAHQKRMTR/GLF (n = 1), PENPRQAYRKRMTR/GLF (n = 1), PENPKQACQKRMTR/GLF (n = 1) 23 2004 A/chicken/ H5N2 PQRKKR/GLF 1 noncommercial farm (6608 59, 78 Texas/298313/2004 chickens) and 2 live poultry markets infected and depopulated, 3 additional LPM voluntarily depopulated Table 6.1. Continued Number Affected With High Hemagglutinin Proteolytic Cleavage Mortality or Were Speciﬁ c No. Date Prototype AI Virus Subtype Site Deduced Amino Acid Sequence Depopulateda References

24 2004, 2006 A/ostrich/South H5N2 PQREKRRKKR/GLF 2004: 11 ostrich farms with 9, 77, 81, 82 Africa/2004 depopulation of 23,625 ostriches and 3,550 other poultry (chickens, turkeys, geese, ducks and pigeons); 2006: 2 farms, 7342 ostriches dead or culled 25 2005 A/chicken/North H7N7 PEIPKGRHRRPKRGLF 3 farms, 218,882 layer 78, 79, P. Selleck Korea/1/2005 chickens culled; number and H. Heine,

134 dead not reported personal communication, 2007 26 2007 NA H7N3 NA 1 farm, 48,560 broiler 82a breeders culled; 540 roosters died a Most outbreaks were controlled by “stamping out” or depopulation policies for infected and/or exposed populations of birds. Chickens, turkeys, and birds in the order Galliformes had clinical signs and mortality patterns consistent with HPAI, while ducks, geese, and other birds lacked or had low mortality rates or infrequent presence of clinical signs. b The H5 and N1 gene lineages have been maintained among the HPAI viruses from outbreaks in various Asian, African and European countries (1996– 2007). The six internal gene segments have undergone reassortment with other AI viruses in Asia. The initial H5N1 HPAI outbreaks were reported in China (1996) with three incursions in Hong Kong (1997, 2001, and 2002). This was followed by regional extension with outbreaks in 2003–2005 within Southeast Asia (South Korea, Vietnam, Japan, Indonesia, Thailand, Cambodia, Laos, China, and Malaysia). In mid-late 2005, outbreaks occurred in both wild birds and poultry in central Asia with extension to Eastern Europe and the Middle East by fall of 2005. In 2006, outbreaks were reported in Africa. Initially, chickens were the main species affected with disease and death, but in many of the outbreaks, domestic ducks have emerged to be a major species in maintenance and epidemiology of the viruses. Various wild birds have succumbed to infection. NA, Not available. 6 / The Global Nature of Avian Influenza 135 of a few weeks to 1 year, that is, initially detected been adapted to poultry and have been maintained as LPAI virus that mutated to HPAI virus (epidem- in village/backyard/hobby poultry and LPM systems ics 8, 14, 19, 20, 22, 23, and 26). In Chapter 2 before introduction into commercial poultry. Each (Molecular Determinants of Pathogenicity for Avian of the HPAI epidemics has involved different agri- Infl uenza Viruses), the mechanisms for the mutation cultural systems during the outbreaks. Some began of H5 and H7 LPAI viruses to HPAI viruses were as viruses in the LPM system such as the 1983–1984 discussed. The number of epizootics, number of H5N2 AI virus of the northeastern United States or cases (i.e., farms), and number of birds affected by the H5N1 in Hong Kong in 1997, or they began as HPAI has grown geometrically since 1959. From LPAI viruses in range-reared layers, as in the 2003 1959 to 1998, the number of birds affected in HPAI H7N7 Dutch outbreak, before spreading into com- outbreaks was calculated at 23 million, while from mercial poultry sectors (59, 139, 142). Others were 1999 to early 2004 over 200 million were involved detected in the LPM system and were eliminated (27). Through 2007, with completion of outbreaks before spreading to commercial poultry, such as in in Canada and North Korea and the expanding H5N1 the H5N2 HPAI virus in Italy during 1997 and the HPAI in Asia, Europe, and Africa, the latter number H5N2 HPAI virus in Texas during 2004. Other is now over 270 million (see Table 6.1). HPAI viruses appeared to have emerged after the Since 1959, the primary control method has been introduction of LPAI virus in commercial poultry, stamping-out, which has been documented with such as with H7N3 viruses in Chile during 2002 and eradication of the virus in 22 of the 26 epidemics in Canada during 2004. In other outbreaks, the lack (epidemics 1–13 and 17–26). In three outbreaks of good surveillance inhibits determination of initial (epidemics 14, 15, and 25), vaccination programs source of infections, but the commercial sectors are with some depopulation have eliminated the clinical more easily blamed because they have the majority HPAI disease, but demonstration of eradication by of the surveillance while the village/rural sector has surveillance programs was not completed. The the least. However, when AI infections do occur H5N1 HPAI that appeared in 1996 (epidemic 16) in commercial industries, they sometimes spread has become the largest HPAI outbreak of the past rapidly throughout the integrated system from farm- 50 years, exceeding 220 million birds affected by to-farm, resulting in epidemics of HP (see Table 5.3) the disease or culled. This epidemic has spread from or LPAI depending on how well the biosecurity its initial cases in China during 1996 to affecting measures contain the spread. poultry and wild birds in over 60 countries in Africa, The following chapters are dedicated to discus- Europe, and Asia (see Table 6.1). A few of the sion of the 26 HPAI epidemics: countries have conducted successful eradication campaigns, but the endemicity of the virus in village • Chapter 8, High Pathogenicity Avian Infl uenza in poultry and LPM systems in many countries (espe- the Americas cially in domestic ducks), the lack of movement • Chapter 9, Highly Pathogenic Avian Infl uenza controls on village poultry and LPM systems, and Outbreaks in Europe, Africa, and Asia since 1959, the infection of migratory waterfowl has created Excluding the Asian H5N1 Virus Outbreaks recurring outbreaks of disease within countries and, • Chapter 10, Avian Infl uenza in Australia in some instances, reintroduction into countries that • Chapter 11, Multicontinental Epidemic of H5N1 were declared free of HPAI in 2004 and 2005 (i.e., High Pathogenicity Avian Infl uenza Virus (1996– Japan, South Korea, and Thailand, late 2006 to early 2007) 2007) (53, 55). Wild aquatic birds are the primordial reservoirs CONCLUSIONS for all AI viruses, and these AI viruses or their genes AI virus is a global virus that knows no geographic have appeared in AI viruses that have infected boundaries, has no political agenda, and can infect domestic poultry and captive birds (see Chapter 3). poultry irrespective of their agricultural or other However, the immediate source of LP and HP epi- anthropocentric systems. AI viruses or evidence of demic viruses is not always determined as feral wild their infection has been detected in poultry and wild birds, captive wild birds, village poultry, commer- birds on all seven continents. However, the reported cial poultry, etc. Some LP viruses, though, have frequency of AI is greatly skewed by the availability 136 Avian Influenza of diagnostics, quantity and quality of surveillance 8. In 1970, the AGID serological test was intro- conducted, the type of birds and production sector duced, which allowed easy and rapid identifi ca- tested, the time of year, geographic location, cli- tion of AI virus–infected poultry fl ocks. matic conditions, and other undefi ned factors. The 9. In 1972, there were the fi rst isolations of LPAI greatest quantity of surveillance in domestic and viruses in asymptomatic wild birds: ducks in the wild birds has been conducted in North America and United States and shorebirds in Australia. Europe because of scientifi c interest, availability of 10. In 1981, the term “highly pathogenic avian virological and serological tests, and fi nancial infl uenza” was accepted as standard nomencla- resources. Because infl uenza is an international ture for fowl plague and related synonyms. problem, solutions will require international efforts 11. In 1983, LPAI virus was observed mutating to and cooperation. HPAI virus during LPAI fi eld outbreak, and Historically, three major scientifi c advances have specifi c genomic changes were identifi ed in the changed our defi nition of avian infl uenza and thus proteolytic cleavage site of the hemagglutinin impacted the reported frequency of AI in the world: responsible for this virulence change. (1) early diagnosis of fowl plague in domestic 12. In the late 1980s and early 1990s, molecular poultry limited to primarily clinical features, lesions, criteria were added to the defi nition for classify- and animal studies (2) recognition of LPAI viruses ing an AI virus as HPAI. and their infections in domestic poultry based on 13. In 2002, there were the fi rst reported infections serology and virus isolation, and (3) discovery of AI and deaths in a wide variety of wild bird species in asymptomatic wild bird reservoirs. However, from AI virus (i.e., H5N1 HPAI virus). some very specifi c discoveries have impacted our understanding of the pathobiology of AI and how to REFERENCES better control the disease at its source: 1. Alexander, D.J. 1981. Current situation of avian infl uenza in poultry in Great Britian. R.W. 1. From 1878 through 1955, fowl plague was Bankowski (ed.). Proceedings of the First Inter- described as a high mortality disease of poultry national Symposium on Avian Infl uenza, April in many countries throughout Europe, Asia, 1981, Beltsville, Maryland. Symposium on Avian North and South America, and Africa, and the Infl uenza, US Animal Health Association: Rich- etiology was proved to be a fi lterable virus. mond, VA, pp. 35–45. 2. During the 1930s through the 1950s, fowl 2. Alexander, D.J. 1982. Avian infl uenza. Recent plague disappeared as an endemic disease in developments. Veterinary Bulletin 52:341–359. most of the world. 3. Alexander, D.J. 1987. Avian infl uenza: historical aspects. In: B.C. Easterday and C.W. Beard 3. In 1949, the fi rst report of a low virulent dis- (eds.). Proceedings of the Second International ease in chickens caused by LPAI virus was Symposium on Avian Infl uenza, Georgia Center reported. for Continuing Education, the University of 4. In 1955, the etiology of fowl plague was deter- Georgia, Athens, Georgia, USA, September 3–5, mined to be an infl uenza A virus, which subse- 1986. Symposium on Avian Infl uenza, US Animal quently was identifi ed as the H7 subtype. Health Association: Richmond, VA, pp. 4–13. 5. In 1959, a “fowl plague-like” outbreak was 4. Alexander, D.J. 1993. Orthomyxovirus infec- described in chickens, which was the fi rst report tions. In: J.B. McFerran and M.S. McNulty (eds.). of fowl plague caused by a non–H7 AI virus Virus Infections of Birds. Elsevier Science: (i.e., fi rst fowl plague outbreak from H5 subtype London, pp. 287–316. of AI virus). 5. Alexander, D.J. 1996. Highly pathogenic avian infl uenza (fowl plague). In: OIE Manual of Stan- 6. In 1961, the fi rst wild bird infections and deaths dards for Diagnostic Tests and Vaccines. List A were reported in common terns of South and B Diseases of Mammals, Birds and Bees, 3rd Africa. ed. Offi ce International des Epizooties: Paris, 7. In 1966 and 1971, the fi rst H5 and H7 LPAI pp. 155–160. viruses, respectively, were identifi ed; prior to 6. Alexander, D.J. 2000. The history of avian this period, only HPAI viruses had H5 and H7 in fl uenza in poultry. World Poultry (November): subtypes. 7–8. 6 / The Global Nature of Avian Influenza 137

7. Alexander, D.J. 2003. Report on avian infl uenza infl uenza viruses in Italy. Archives of Virology in the Eastern Hemisphere during 1997–2002. 146(5):963–973. Avian Diseases 47(Suppl):792–797. 18. Barr, D.A., A.P. Kelly, R.T. Badman, A.R. 8. Alexander, D.J., W.H. Allan, D.G. Parsons, and Campey, M.D. O’Rourke, D.C. Grix, and R.L. G. Parsons. 1978. The pathogenicity of four Reece. 1986. Avian infl uenza on a multi-age avian infl uenza viruses for fowls, turkeys and chicken farm. Australian Veterinary Journal 63: ducks. Research in Veterinary Science 24:242– 195–196. 247. 19. Bashiruddin, J.B., A.R. Gould, and H.A. West- 9. Alexander, D.J., I. Capua, and G. Koch. 2008. bury. 1992. Molecular pathotyping of two avian Highly pathogenic avian infl uenza outbreaks in infl uenza viruses isolated during the Victoria Europe, Africa and Asia since 1959, excluding 1976 outbreak. Australian Veterinary Journal the Asian H5N1 virus outbreaks. In: D.E. Swayne 69:140–142. (ed.). Avian Infl uenza. Blackwell: Ames, IA, 20. Beard, C.W. 1970. Avian infl uenza antibody pp. 217–237. detection by immunodiffusion. Avian Diseases 10. Alexander, D.J., and R.E. Gough. 1978. A long- 14:337–341. term study of the pathogenesis of infection of 21. Beard, C.W. 1970. Demonstration of type-spe- fowls with three strains of avian infectious bron- cifi c infl uenza antibody in mammalian and avian chitis virus. Research in Veterinary Science sera by immunodiffusion. Bulletin of the World 24:228–233. Health Organization 42:779–785. 11. Alexander, D.J., S.A. Lister, M.J. Johnson, C.J. 22. Beard, C.W., and D.H. Helfer. 1972. Isolation of Randall, and P.J. Thomas. 1993. An outbreak of two turkey infl uenza viruses in Oregon. Avian highly pathogenic avian infl uenza in turkeys in Diseases 16:1133–1136. Great Britian in 1991. Veterinary Record 132: 23. Becker, W.B. 1966. The isolation and classifi ca- 535–536. tion of Tern virus: infl uenza A-Tern South 12. Alexander, D.J., and D. Spackman. 1981. Char- Africa—1961. Journal of Hygiene 64:309–320. acterisation of infl uenza A viruses isolated from 24. Brown, I.H., J. Banks, R.J. Manvell, S.C. Essen, turkeys in England during March–May 1979. W. Shell, M. Slomka, B. Londt, and D.J. Alexan- Avian Pathology 10:281–293. der. Recent epidemiology and ecology of infl u- 13. Anonymous. 1976. The outbreak of fowl plague enza A viruses in avian species in Europe and the in Victoria. In: Annual Report. Division of Middle East. Developments in Biologicals 124: Animal Health, Department of Agriculture: Vic- 45–50. toria, Australia, pp. 4–6. 25. Brugh, M. 1992. Re-evaluation of the pathogenic- 14. Bankowski, R.A. 1981. Introduction and objec- ity of A/chicken/Alabama/75 (H4N8) infl uenza tives of the symposium. In: R.W. Bankowski virus. Avian Diseases 36:968–974. (ed.). Proceedings of the First International Sym- 26. Burnet, F.M., and J.D. Ferry. 1934. The differen- posium on Avian Infl uenza, April 1981, Belts- tiation of the viruses of fowl plague and New- ville, Maryland. Symposium on Avian Infl uenza, castle disease: experiments using the technique of US Animal Health Association: Richmond, VA, the chorio-allantoic membrane of the developing pp. vii–xiv. hen’s egg. British Journal of Experimental Pathol- 15. Bankowski, R.A. 1981. In: R.W. Bankowski ogy 15:56–64. (ed.). Proceedings of the First International Sym- 27. Capua, I., and D.J. Alexander. 2004. Avian infl u- posium on Avian Infl uenza, April 1981, Belts- enza: recent developments. Avian Pathology ville, Maryland. Symposium on Avian Infl uenza, 33:393–404. US Animal Health Association: Richmond, VA, 28. Capua, I., S. Marangon, and F.M. Cancellotti. pp. 1–215. 2003. The 1999–2000 avian infl uenza (H7N1) 16. Banks, J., E.C. Speidel, J.W. McCauley, and D.J. epidemic in Italy. Veterinary Research Commu- Alexander. 2000. Phylogenetic analysis of H7 nications 27:123–127. haemagglutinin subtype infl uenza A viruses. 29. Capua, I., S. Marangon, L. Selli, D.J. Alexander, Archives of Virology 145(5):1047–1058. D.E. Swayne, M.D. Pozza, E. Parenti, and F.M. 17. Banks, J., E.S. Speidel, E. Moore, L. Plowright, Cancellotti. 1999. Outbreaks of highly pathogenic A. Piccirillo, I. Capua, P. Cordioli, A. Fioretti, avian infl uenza (H5N2) in Italy during October and D.J. Alexander. 2001. Changes in the hae- 1997–January 1998. Avian Pathology 28:455–460. magglutinin and the neuraminidase genes prior to 30. Chen, H., G.J.D. Smith, S.Y. Zhang, K. Qin, J. the emergence of highly pathogenic H7N1 avian Wang, K.S. Li, R.G. Webster, J.S.M. Peiris, and 138 Avian Influenza

Y. Guan. 2005. H5N1 virus outbreak in migratory 40. Eckroade, R.J., and L.A. Silverman-Bachin. waterfowl: a worrying development could help to 1986. Avian infl uenza in Pennsylvania. The spread this dangerous virus beyond its stronghold beginning. In: B.C. Easterday and C.W. Beard in southeast Asia. Nature (London) 436(7048): (eds.). Proceedings of the Second International 191–192. Symposium on Avian Infl uenza, Georgia Center 31. Cross, G.M. 1987. The status of avian infl uenza for Continuing Education, the University of in poultry in Australia. In: B.C. Easterday and Georgia, Athens, Georgia, USA, September 3–5, C.W. Beard (eds.). Proceedings of the Second 1986. Symposium on Avian Infl uenza, US International Symposium on Avian Infl uenza, Animal Health Association: Richmond, VA, pp. Georgia Center for Continuing Education, the 22–32. University of Georgia, Athens, Georgia, USA, 41. Elbers, A.R.W., T.H.F. Fabri, T.S. de Vries, J.J. September 3–5, 1986. Symposium on Avian de Wit, A. Pijpers, and G. Koch. 2004. The highly Infl uenza, US Animal Health Association: Rich- pathogenic avian infl uenza A (H7N7) virus epi- mond, VA, 96–103. demic in The Netherlands in 2003—lessons 32. Davison, S., R.J. Eckroade, and A.F. Ziegler. learned from the fi rst fi ve outbreaks. Avian Dis- 2003. A review of the 1996–1998 nonpathogenic eases 48(3):691–705. H7N2 avian infl uenza outbreak in Pennsylvania. 42. FAO. 2006. Summary of confi rmed HPAI out- Avian Diseases 47(Supplement):823–827. breaks in affected countries. FAO AIDE News AI 33. Downie, J.C., and W.G. Laver. 1973. Isolation of Bulletin 41:9–10. a type A infl uenza virus from an Australian 43. Fifth International Symposium on Avian Infl u- pelagic bird. Virology 51:259–269. enza. 2003. Recommendations of the Fifth Inter- 34. Dunn, P.A., E.A. Wallner-Pendleton, H. Lu, national Symposium on Avian Infl uenza. Avian D.P. Shaw, D. Kradel, D.J. Henzler, P. Miller, Diseases 47:1260–1261. D.W. Key, M. Ruano, and S. Davison. 2003. 44. Garcia, M., J.M. Crawford, J.W. Latimer, Summary of the 2001–02 Pennsylvania H7N2 M.V.Z.E. Rivera-Cruz, and M.L. Perdue. 1996. low pathogenicity avian infl uenza outbreak in Heterogeneity in the hemagglutinin gene and meat type chickens. Avian Diseases 47(Suppl): emergence of the highly pathogenic phenotype 812–816. among recent H5N2 avian infl uenza viruses from 35. Easterday, B.C., and C.W. Beard (eds.). Proceed- Mexico. Journal of General Virology 77:1493– ings of the Second International Symposium on 1504. Avian Infl uenza, Georgia Center for Continuing 45. Garnett, W.H. 1987. Status of avian infl uenza in Education, the University of Georgia, Athens, poultry: 1981–86. In: B.C. Easterday and C.W. Georgia, USA, September 3–5, 1986. Sympo- Beard (eds.). Proceedings of the Second Interna- sium on Avian Infl uenza, US Animal Health tional Symposium on Avian Infl uenza, Georgia Association: Richmond, VA, pp. 1–475. Center for Continuing Education, the University 36. Easterday, B.C. (ed.). Proceedings of the Third of Georgia, Athens, Georgia, USA, September International Symposium on Avian Infl uenza, 3–5, 1986. Symposium on Avian Infl uenza, US May 27–29, 1992, Madison, Wisconsin. Sympo- Animal Health Association: Richmond, VA, sium on Avian Infl uenza, US Animal Health pp. 61–66. Association: Richmond, VA, pp. 1–458. 46. Halvorson, D.A. 2002. Twenty-fi ve years of 37. Easterday, B.C., V.S. Hinshaw, and D.A. Halvor- avian infl uenza in Minnesota. In: Proceedings of son. 1997. Infl uenza. In: B.W. Calnek, H.J. the 53rd North Central Avian Disease Confer- Barnes, C.W. Beard, L.R. McDougald, and Y.M. ence. NCADC: Minneapolis, pp. 65–69. Saif (eds.). Diseases of Poultry, 10th ed. Iowa 47. Halvorson, D.A., D.D. Frame, K.A.J. Friendshuh, State University Press: Ames, IA, pp. 583–605. and D.P. Shaw. 1998. Outbreaks of low pathoge- 38. Easterday, B.C., D.O. Trainer, B. Tumova, and nicity avian infl uenza in U.S.A. In: D.E. Swayne H.G. Pereira. 1968. Evidence of infection with and R.D. Slemons (eds.). Proceedings of the infl uenza viruses in migratory waterfowl. Nature Fourth International Symposium on Avian Infl u- 219:523–524. enza, May 29–31, 1997, Athens, Georgia. Sym- 39. Easterday, B.C., and B. Tumova. 1972. Avian posium on Avian Infl uenza, US Animal Health infl uenza. In: M.S. Hofstad, B.W. Calnek, C.F. Association: Richmond, VA, pp. 36–46. Helmbolt, W.M. Reid, and H.W. Yoder, Jr. (eds.). 48. Halvorson, D.A., C.J. Kelleher, and D.A. Senne. Diseases of Poultry, 6th ed. Iowa State University 1985. Epizootiology of avian infl uenza: effect of Press: Ames, IA, pp. 670–700. season on incidence in sentinel ducks and domes- 6 / The Global Nature of Avian Influenza 139

tic turkeys in Minnesota. Applied and Environ- variant, A/turkey/Ontario/7732/66. Canadian mental Microbiology 49:914–919. Veterinary Journal 9:151–160. 49. Harder, T.C., and O. Werner. 2006. Avian infl u- 59. Lee, C.W., D.E. Swayne, J.A. Linares, D.A. enza. In: B.S. Kamps, C. Hoffmann, and W. Senne, and D.L. Suarez. 2005. H5N2 avian infl u- Preiser (eds.). Infl uenza Report. Available at enza outbreak in Texas in 2004: the fi rst highly http://www.infl uenzareport.com/ir/ai.htm. pathogenic strain in the United States in 20 years? 50. Hinshaw, V.S., and R.G. Webster. 1982. The Journal of Virology 79(6):3692–3702. natural history of infl uenza A viruses. In: A.S. 60. Lee, C., D.E. Swayne, J.A. Linares, D.A. Senne, Beare (ed.). Basic and Applied Infl uenza Research. and D.L. Suarez. 2005. H5N2 avian infl uenza CRC Press: Boca Raton, FL, pp. 79–104. outbreak in Texas in 2004: the fi rst highly patho- 51. Hinshaw, V.S., J.M. Wood, R.G. Webster, R. genic strain in the United States in 20 years? Deibel, and B. Turner. 1985. Circulation of infl u- Journal of Virology 79(17):11412–11421. enza viruses and paramyxoviruses in waterfowl 61. Lipkind, M., Y. Weisman, E. Shihmanter, and D. originating from two different areas of North Shoham. 1981. Review of the three-year studies America. Bulletin of the World Health Organiza- on the ecology of avian infl uenza viruses in Israel. tion 63:711–719. In: R.W. Bankowski (ed.). Proceedings of the 52. Hirst, M., C.R. Astell, M. Griffi th, S.M. Cough- First International Symposium on Avian Infl u- lin, M. Moksa, T. Zeng, D.E. Smailus, R.A. Holt, enza, April 1981, Beltsville, Maryland. Sympo- S. Jones, M.A. Marra, M. Petric, M. Krajden, D. sium on Avian Infl uenza, US Animal Health Lawrence, A. Mak, R. Chow, D.M. Skowronski, Association: Richmond, VA, pp. 69–78. S.A. Tweed, S. Goh, R.C. Brunham, J. Robinson, 62. Liu, J., H. Xiao, F. Lei, Q. Zhu, K. Qin, X.-W. V. Bowes, K. Sojonky, S.K. Byrne, Y. Li, D. Zhang, X.-L. Zhang, D. Zhao, G. Wang, Y. Feng, Kobasa, T. Booth, and M. Paetzel. 2004. Novel J. Ma, W. Liu, J. Wang, and G.F. Gao. 2005. avian infl uenza H7N3 strain outbreak, British Highly pathogenic H5N1 infl uenza virus infec- Columbia. Emerging Infectious Diseases tion in migratory birds. Science 309(5738):1206. 10(12):2192–2195. 63. Lvov, D.K., S.S. Yamnikova, I.T. Fedyakina, 53. Hugita, H. 2007. Highly pathogenic avian infl u- V.A. Aristova, D.N. Lvov, N.F. Lomakina, E.S. enza, Japan. WAHID Interface—OIE World Petrova, V. Zlobin, I, M.A. Khasnatinov, E.A. Animal Health Information. Available at http:// Chepurgina, A. Kovtunov, I, A.F. Djarkenov, www.oie.int/wahid-prod/public.php?page=single_ M.N. Sankov, G.N. Leonova, D. Maslov, V, M. report&pop=1&reportid=4522. Accessed on Y. Shchelkanov, E.A. Nepoklonov, and T. Aliper, March 13, 2007. I. 2004. Ecology and evolution of infl uenza 54. Jackwood, M.J.P., and D.E. Swayne. 2007. viruses in Russia (1979–2002). Voprosy Viruso- Pathobiology of Asian H5N1 avian infl uenza logii 49(3):17–24. virus infections in ducks. Avian Diseases 64. Manvell, R.J., P. McKinney, U. Wernery, and 51(Suppl):250–259. K.M. Frost. 2000. Isolation of a highly patho- 55. Kim, C.S. 2006. Highly pathogenic avian infl u- genic infl uenza A virus of subtype H7N3 from a enza, Korea (Rep. of). WAHID Interface—OIE peregrine falcon (Falco peregrinus). Avian World Animal Health Information. Available at Pathology 29:635–637. http://www.oie.int/wahid-prod/public.php?page= 65. Marangon, S., and I. Capua. 2006. Control of single_report&pop=1&reportid=4514. Accessed avian infl uenza in Italy: from stamping out to on March 13, 2007. emergency and prophylactic vaccination. Devel- 56. Krauss, S., D. Walker, S. P. Pryor, L. Niles, L. opment in Biologicals 124:109–115. Chenghong, V.S. Hinshaw, and R.G. Webster. 66. Max, V.K., J.R. Herrera, R.Z. Moreira, and H.O. 2004. Infl uenza A viruses of migrating wild Rojas. 2007. Avian infl uenza in Chile: a success- aquatic birds in North America. Vector-Borne ful experience. Avian Diseases 51(Suppl):363– and Zoonotic Diseases 4(3):177–189. 365. 57. Krohn, L.D. 1925. A study on the recent outbreak 67. McFadyean, J. 1908. The ultravisible viruses. of a fowl disease in New York City. Journal of Journal of Comparative Pathology and Therapeu- the American Veterinary Medical Association tics 21:58–242. 20:146–170. 68. McNulty, M.S., G.M. Allan, R.M. McCracken, 58. Lang, G., O. Narayan, B.T. Rouse, A.E. Fergu- and P.J. McParland. 1985. Isolation of a highly son, and M.C. Connell. 1968. A new infl uenza A pathogenic infl uenza virus from turkeys. Avian virus infection in turkeys II. A highly pathogenic Pathology 14:173–176. 140 Avian Influenza

69. Mohler, J.R. 1926. Fowl Pest in the United States. 82. OIE. 2006. Highly pathogenic avian infl uenza in Journal of the American Veterinary Medical South Africa. Follow-up report No. 2. OIE Association 21:549–559. Disease Information, 19(31). Available at http:// 70. Morgan, I.R., and H.A. Westbury. 1981. Virologi cal www.oie.int/eng/info/hebdo/AIS_07. studies of Adelie penguins (Pygoscelis adeliae) HTM#Sec4. OIE: Paris. Accessed on September in Antarctica. Avian Diseases 25:1019–1026. 8, 2006. 71. Mullaney, R. 2003. Live-bird market closure 82a. Offi ce Internationale des Epizooties. 2007. Highly activities in the Northeastern United States. Avian pathogenic avian infl uenza, Canada. Immediate Diseases 47(Suppl):1096–1098. notifi cation (9/28/2007). Accessed on October 72. Naeem, K. 1998. The avian infl uenza H7N3 out- 27, 2007. break in South Central Asia. In: D.E. Swayne and 83. Olsen, B., V.J. Munster, A. Wallensten, J. Wal- R.D. Slemons (eds.). Proceedings of the Fourth denstrom, D.M.E. Osterhaus Albert, and A.M. International Symposium on Avian Infl uenza, Fouchier Ron. 2006. Global patterns of infl uenza May 29–31, 1997, Athens, Georgia. Symposium A virus in wild birds. Science 312(21):384–388. on Avian Infl uenza, US Animal Health Associa- 84. Pasick, J., K. Handel, J. Robinson, J. Copps, D. tion: Richmond, VA, pp. 31–35. Ridd, K. Hills, H. Kehler, C. Cottam-Birt, J. 73. Naeem, K., and M. Hussain. 1995. An outbreak Neufeld, Y. Berhane, and S. Czub. 2005. Inter- of avian infl uenza in poultry in Pakistan. Veteri- segmental recombination between the haemag- nary Record 137:439. glutinin and matrix genes was responsible for the 74. OIE. 2004. Highly pathogenic avian infl uenza in emergence of a highly pathogenic H7N3 avian Canada. Follow-up report no. 7 (fi nal report). OIE infl uenza virus in British Columbia. Journal of Disease Information, 17(30). Available at http:// General Virology 86(Pt 3):727–731. www.oie.int/eng/info/hebdo/AIS_35.HTM#Sec6 85. Perdue, M.L., M. Garcia, D. Senne, and M. Fraire. . OIE: Paris. Accessed on May 17, 2005. 1997. Virulence-associated sequence duplication 75. OIE. 2004. Highly pathogenic avian infl uenza in at the hemagglutinin cleavage site of avian infl u- Pakistan. OIE Disease Information, 17. enza viruses. Virus Research 49:173–186. 76. OIE. 2004. Highly pathogenic avian infl uenza in 86. Perdue, M.L., D.L. Suarez, and D.E. Swayne. Pakistan. Follow-up report No 1. OIE Disease 2000. Avian infl uenza in the 1990s. Avian and Information, 17. Poultry Biology Reviews 11(1):1–20. 77. OIE. 2004. Highly pathogenic avian infl uenza in 87. Pereira, H.G., B. Tumova, and V.G. Law. 1965. South Africa. Follow-up report No. 3. OIE Avian infl uenza A viruses. Bulletin of the World Disease Information, 17(44). Available at http:// Health Organization 32:855–860. www.oie.int/eng/info/hebdo/AIS_21. 88. Perkins, L.E.L., and D.E. Swayne. 2001. Patho- HTM#Sec2. OIE: Paris. Accessed on May 17, biology of A/chicken/Hong Kong/220/97 (H5N1) 2005. avian infl uenza virus in seven gallinaceous 78. OIE. 2004. Highly pathogenic avian infl uenza in species. Veterinary Pathology 38:149–164. the United States of America. OIE Disease Infor- 89. Perkins, L.E.L., and D.E. Swayne. 2002. Patho- mation, 17(9). OIE: Paris. Available at http:// genicity of a Hong Kong-origin H5N1 highly www.oie.int/eng/info/hebdo/AIS_56.HTM#Sec4. pathogenic avian infl uenza virus for emus, geese, Accessed on May 17, 2005. ducks, and pigeons. Avian Diseases 46(1):53–63. 79. OIE. 2005. Avian infl uenza in Korea (Democratic 90. Perroncito, E. 1878. Epizoozia tifoide nei gallina- People’s Republic of -). OIE Disease Informa- cei. Annali della Reale Accademia d’Agricoltura tion, 18(14). Available at http://www.oie.int/ di Torino 21:87–126. eng/info/hebdo/AIS_76.HTM#Sec4. OIE: Paris. 91. Petek, M. 1981. Current situation in Italy. In: Accessed on May 17, 2005. R.W. Bankowski (ed.). Proceedings of the First 80. OIE. 2006. Avian infl uenza. International Animal International Symposium on Avian Infl uenza, Health Code—2006. Available at http://www.oie. April 1981, Beltsville, Maryland. Symposium on int/eng/normes/mcode/en_chapitre_2.7.12.htm. Avian Infl uenza, US Animal Health Association: OIE: Paris. Accessed on September 8, 2006. Richmond, VA, pp. 31–34. 81. OIE. 2006. Highly pathogenic avian infl uenza in 92. Rohm, C., J. Suss, V. Pohle, and R.G. Webster. South Africa. OIE Disease Information, 19(27). 1996. Different hemagglutinin cleavage site Available at http://www.oie.int/eng/info/hebdo/ variants of H7N7 in an infl uenza outbreak in AIS_11.HTM#Sec6. OIE: Paris. Accessed on chickens in Leipzig, Germany. Virology 218(1): September 8, 2006. 253–257. 6 / The Global Nature of Avian Influenza 141

93. Rojas, H., R. Moreira, P. Avalos, I. Capua, and ridge, and M. Peiris. 2003. An update on avian S. Marangon. 2002. Avian infl uenza in poultry in infl uenza in Hong Kong 2002. Avian Diseases Chile. Veterinary Record 151(6):188. 47(Suppl):1083–1086. 94. Salem, M. 1995. Avian infl uenza in Mexico. In: 104. Sims, L.D., and A.J. Turner. 2008. Avian infl u- Proceedings of the 30th National Meeting on enza in Australia. In: D.E. Swayne (ed.). Avian Poultry Health and Processing, 30th ed. Ocean Infl uenza. Blackwell: Ames, IA, pp. 239–250 City, MD, pp. 99–105. 105. Slemons, R.D., D.C. Johnson, J.S. Osborn, and F. 95. Schafer, W. 1955. Vergleichende sero-immuno- Hayes. 1974. Type-A infl uenza viruses isolated logische untersuchungen uber die viren der infl u- from wild free-fl ying ducks in California. Avian enza unf klassichen gefl ugelpest. Zeitschrift fur Diseases 18:119–124. Naturforschung 10B:81–91. 106. Smithies, L.K., F.G. Emerson, S.M. Robertson, 96. Selleck, P.W., L.J. Gleeson, P.T. Hooper, H.A. and D.D. Ruedy. 1969. Two different type A Westbury, and E. Hansson. 1997. Identifi cation and infl uenza virus infections in turkeys in Wiscon- characterization of an H7N3 infl uenza A virus sin. II. 1968 outbreak. Avian Diseases 13:606– from an outbreak of virulent avian infl uenza in 610. Victoria. Australian Veterinary Journal 75(4):289– 107. Spackman, E., D.A. Senne, S. Davison, and D.L. 292. Suarez. 2003. Sequence analysis of recent H7 97. Senne, D.A., J.C. Pedersen, and B. Panigrahy. avian infl uenza viruses associated with three dif- 2005. Live-bird markets in the Northeastern ferent outbreaks in commercial poultry in the United States: a source of avian infl uenza in com- United States. Journal of Virology 77(24):13399– mercial poultry. In: R.S. Schrijver and G. Koch 13402. (eds.). Avian Infl uenza. Prevention and Control, 108. Stallknecht, D.E. 1998. Ecology and epide- Springer: Dordrecht, pp. 19–24. miology of avian infl uenza viruses in wild bird 98. Senne, D.A. 2004. Avian infl uenza. Proceedings populations: waterfowl, shorebirds, pelicans, cor- of the United States Animal Health Association morants, etc. In: D.E. Swayne and R.D. Slemons 108:545–547. (eds.). Proceedings of the Fourth International 99. Senne, D.A., T.J. Holt, and B.L. Akey. 2005. Symposium on Avian Infl uenza, May 29–31, An overview of the 2002 outbreak of low- 1997, Athens, Georgia. Symposium on Avian pathogenic H7N2 avian infl uenza in Virginia, Infl uenza, US Animal Health Association: Rich- West Virginia and North Carolina. In: R.S. Schri- mond, VA, pp. 61–69. jver and G. Koch (eds.). Avian Infl uenza. Pre- 109. Stallknecht, D.E., and S.M. Shane. 1988. Host vention and Control, Springer: Dordrecht, pp. range of avian infl uenza virus in free-living birds. 41–47. Veterinary Research Communications 12:125–141. 100. Senne, D.A., B. Panigrahy, Y. Kawaoka, J.E. 110. Stallknecht, D.E., S.M. Shane, P.J. Zwank, D.A. Pearson, J. Suss, M. Lipkind, H. Kida, and R.G. Senne, and M.T. Kearney. 1990. Avian infl uenza Webster. 1996. Survey of the hemagglutinin viruses from migratory and resident ducks of (HA) cleavage site sequence of H5 and H7 avian coastal Louisiana. Avian Diseases 34:398–405. infl uenza viruses: amino acid sequence at the HA 111. Stubbs, E.L. 1926. Fowl pest. Journal of the cleavage site as a marker of pathogenicity poten- American Veterinary Medical Association 21: tial. Avian Diseases 40(2):425–437. 561–569. 101. Senne, D.A., J.E. Pearson, and B. Panigrahy. 112. Stubbs, E.L. 1948. Fowl pest. In: H.E. Biester and 1992. Live poultry markets: A missing link in the L.H. Schwarte (eds.). Diseases of Poultry, 2nd ed. epidemiology of avian infl uenza. In: B.C. Easter- Iowa State University Press: Ames, IA, 603– day (ed.). Proceedings of the Third International 614. Symposium on Avian Infl uenza, May 27–29, 113. Suarez, D.L., M.L. Perdue, N. Cox, T. Rowe, C. 1992, Madison, Wisconsin. Symposium on Avian Bender, J. Huang, and D.E. Swayne. 1998. Com- Infl uenza, US Animal Health Association: Rich- parison of highly virulent H5N1 infl uenza A mond, VA, pp. 50–58. viruses isolated from humans and chickens from 102. Shortridge, K.F. 1982. Avian infl uenza A viruses Hong Kong. Journal of Virology 72(8):6678– of southern China and Hong Kong: ecological 6688. aspects and implications for man. Bulletin of the 114. Suarez, D.L., and C.S. Schultz. 2000. Immunol- World Health Organization 60:129–135. ogy of avian infl uenza virus: a review. Develop- 103. Sims, L.D., Y. Guan, T.M. Ellis, K.K. Liu, K. mental and Comparative Immunology 24(2–3): Dyrting, H. Wong, N.Y.H. Kung, K.F. Short- 269–283. 142 Avian Influenza

115. Suarez, D.L., D.A. Senne, J. Banks, I.H. Brown, 126. D.E. Swayne, and R.D. Slemons (eds.). 1998. S.C. Essen, C.W. Lee, R.J. Manvell, C. Mathieu- Proceedings of the Fourth International Sympo- Benson, V. Moreno, J.C. Pedersen, B. Panigrahy, sium on Avian Infl uenza, May 29–31, 1997, H. Rojas, E. Spackman, and D.J. Alexander. Athens, Georgia. Symposium on Avian Infl uenza, 2004. Recombination resulting in virulence shift US Animal Health Association: Richmond, VA, in avian infl uenza outbreak, Chile. Emerging pp. 1–401. Infectious Diseases 10(4):693–699. 127. Swayne, D.E., and D.L. Suarez. 2000. Highly 116. Suarez, D.L., P.R. Woolcock, A.J. Bermudez, pathogenic avian infl uenza. Revue Scientifi que et and D.A. Senne. 2002. Isolation from turkey Technique Offi ce International des Epizooties breeder hens of a reassortant H1N2 infl uenza 19(2):463–482. virus with swine, human, and avian lineage genes. 128. Swayne, D.E., and D.L. Suarez. 2003. Proceed- Avian Diseases 46(1):111–121. ings of the Fifth International Symposium on 117. Swayne, D.E. 2000. Understanding the ecology Avian Infl uenza. Avian Diseases 47(Suppl):783– and epidemiology of avian infl uenza viruses: 1267. implications for zoonotic potential. In: C.C. 129. Tang, Y., C.W. Lee, Y. Zhang, D.A. Senne, R. Brown and C.A. Bolin (eds.). Emerging Diseases Dearth, B. Byrum, D.R. Perez, D.L. Suarez, and of Animals. ASM Press: Washington, D.C., Y.M. Saif. 2005. Isolation and characterization of pp. 101–130. H3N2 infl uenza A virus from turkeys. Avian Dis- 118. Swayne, D.E. 2007. Proceedings of the Sixth eases 49(2):207–213. International Symposium on Avian Infl uenza. 130. Turner, A.J. 1976. The isolation of fowl plague Avian Diseases 51(Supplement):157–514. virus in Victoria. Australian Veterinary Journal 119. Swayne, D.E., and B. Akey. 2005. Avian infl u- 52:384. enza control strategies in the United States of 131. U.S. Department of Agriculture. 1999. Agricul- America. In: R.S. Schrijver and G. Koch (eds.). tural Statistics 1999. USDA: Washington D.C., Avian Infl uenza. Prevention and Control. pp. 1–485. Springer: Dordrecht, pp. 113–130. 132. United States Animal Health Association. 1985. 120. Swayne, D.E., and D.J. Alexander. 1994. Confi r- Report of the Committee on Transmissible Dis- mation of nephrotropism and nephropathogeni- eases of Poultry and Other Species. Proceedings city of 3 low-pathogenic chicken-origin infl uenza of the United States Animal Health Association viruses for chickens. Avian Pathology 23:345– 89:296–305. 352. 133. United States Animal Health Association. 1994. 121. Swayne, D.E., and D.A. Halvorson. 2003. Infl u- Report of the Committee on Transmissible Dis- enza. In: Y.M. Saif, H.J. Barnes, A.M. Fadly, eases of Poultry and Other Avian Species. Crite- J.R. Glisson, L.R. McDougald, and D.E. Swayne ria for determining that an AI virus isolation (eds.). Diseases of Poultry, 11th ed. Iowa State causing an outbreak must be considered for erad- University Press: Ames, IA, pp. 135–160. ication. Proceedings of the United States Animal 122. Swayne, D.E., and D.A. Halvorson. 2008. Infl u- Health Association 98:522. enza. In: Y.M. Saif, J.R. Glisson, A.M. Fadly, 134. United States Animal Health Association. 1995. L.R. McDougald, and L. Nolan (eds.). Diseases of Report of the Committee on Transmissible Dis- Poultry, 12th ed. Blackwell: Ames, IA, in press. eases of Poultry and Other Avian Species. Pro- 123. Swayne, D.E., M.J. Radin, T.M. Hoepf, and R.D. ceedings of the United States Animal Health Slemons. 1994. Acute renal failure as the cause Association 99:550–588. of death in chickens following intravenous inocu- 135. United States Animal Health Association. 2006. lation with avian infl uenza virus A/chicken/ Report of the Committee on Transmissible Dis- Alabama/7395/75 (H4N8). Avian Diseases 38: eases of Poultry and Other Avian Species. Avail- 151–157. able at http://www.usaha.org/committees/pad/pad. 124. Swayne, D.E., and R.D. Slemons. 1990. Renal shtmlUSAHA. Richmond, VA. pathology in specifi c-pathogen-free chickens 136. U.S. Department of Agriculture. 1925. Source inoculated with a waterfowl-origin type A infl u- of fowl pest in introduced virus. U.S. Dep- enza virus. Avian Diseases 34:285–294. artment of Agriculture Offi cial Record 4(10):1– 125. Swayne, D.E., and R.D. Slemons. 1995. Com- 2. parative pathology of intravenously inoculated 137. Villareal, C.L. 2006. Control and eradication wild duck- and turkey-origin type A infl uenza strategies of avian infl uenza in Mexico. Develop- virus in chickens. Avian Diseases 39(1):74–84. ments in Biologicals 124:125–126. 6 / The Global Nature of Avian Influenza 143

138. Villareal, C.L., and A.O. Flores. 1998. The for the presence of low pathogenic avian infl u- Mexican avian infl uenza (H5N2) outbreak. In: enza virus infections. Avian Pathology 33:565– D.E. Swayne and R.D. Slemons (eds.). Proceed- 570. ings of the Fourth International Symposium on 143. Wood, G.W., J. Banks, I. Strong, G. Parsons, and Avian Infl uenza, May 29–31, 1997, Athens, D.J. Alexander. 1996. An avian infl uenza virus of Georgia. Symposium on Avian Infl uenza, US H10 subtype that is highly pathogenic for chick- Animal Health Association: Richmond, VA, ens, but lacks multiple basic amino acids at the pp. 18–22. haemagglutinin cleavage site. Avian Pathology 139. Webster, R.G., W.J. Bean, Y. Kawaoka, and D. 25(4):799–806. Senne. 1986. Characterization of H5N2 infl uenza 144. Wood, G.W., J.W. McCauley, J.B. Bashiruddin, viruses from birds in live poultry markets in USA. and D.J. Alexander. 1993. Deduced amino acid Proceedings of the United States Animal Health sequences at the haemagglutinin cleavage site of Association 90:278–286. avian infl uenza A viruses of H5 and H7 subtypes. 140. Wells, R.J.H. 1963. An outbreak of fowl plague Archives of Virology 130:209–217. in turkeys. Veterinary Record 75:783–786. 145. Xu, X., K. Subbarao, N.J. Cox, and Y. Guo. 1999. 141. Westbury, H.A. 1998. History of highly patho- Genetic characterization of the pathogenic infl u- genic avian infl uenza in Australia. In: D.E. enza A/Goose/Guangdong/1/96 (H5N1) virus: Swayne and R.D. Slemons (eds.). Proceedings of similarity of its hemagglutinin gene to those of the Fourth International Symposium on Avian H5N1 viruses from the 1997 outbreaks in Hong Infl uenza, May 29–31, 1997, Athens, Georgia. Kong. Virology 261(1):15–19. Symposium on Avian Infl uenza, US Animal 146. Ziegler, A.F., S. Davison, H. Acland, and R.J. Health Association: Richmond, VA, pp. 23–30. Eckroade. 1999. Characteristics of H7N2 (non- 142. Wit, J.J., G. Koch, T.H.F. Fabri, and A.R.W. pathogenic) avian infl uenza virus infections in Elbers. 2004. A cross-sectional serological survey commercial layers, in Pennsylvania, 1997–98. of the Dutch commercial poultry population Avian Diseases 43(1):142–149. 7 The Beginning and Spread of Fowl Plague (H7 High Pathogenicity Avian Infl uenza) Across Europe and Asia (1878–1955)

Erhard F. Kaleta and Catherine P. A. Rülke

INTRODUCTION 75, 83, 84, 116, 120, 130, 159). Other authors were Fowl plague is not a new disease in domestic poultry confi dent that the new disease represents defi nitely and free-living birds. Perroncito (1878) (224) and, a disease entity that was never seen before (152, later, other contemporary Italian veterinarians 158, 167, 173, 176, 177, 180, 196, 214, 224, 229, observed in northern Italy a “malattia gravissima” 249). In addition, since the fi rst description of New- (a most severe disease) that was never seen before castle disease (ND) (Doyle, 1927) (69), additional among chickens, turkeys, and other birds. The confusion became obvious as to the etiology of ND observed disease started with a rather mild course in compared with fowl plague. some villages and changed rather suddenly to a very Questions arise in relation to fowl plague—why virulent form that eliminated almost all chickens did fowl plague occur at this particular period of of domestic and foreign origin of affected fl ocks. time, and why in this part of the Italian country? Affected also were turkeys, geese, and some species Further, is Perroncito’s publication a historical curi- of free-living birds following natural infections. osity, or is there a sound and explainable background Seldom were signs seen in domestic ducks. Perron- in terms of historical developments that led directly cito mentioned also that the course of the disease and inevitably to the “new” disease, and is it a major changed back again to a milder form. scientifi c achievement of Perroncito to recognize the The World Organization of Animal Health (Offi ce disease as a really “new” entity? Answers to these Internationale des Epizooties [OIE]) was founded in questions require a brief historical excursus to the 1925. In the following years, many countries joined political, economic, and scientifi c background of the OIE as offi cial or associated members and tried Perroncito’s time. to comply with the rules and regulations as lined out This contribution attempts to review the history in the “Terrestrial Animal Health Code.” The fi rst of fowl plague between 1878 and 1955 with a special statistics on reported epidemic animal diseases are focus on outbreaks in European and Asian countries. listed some years after the foundation of the OIE in Numerous reports and textbooks were published in the annual “Reports on Animal Health Status.” a variety of European languages. However, the Thus, detailed fi gures are not available from the OIE majority of publications were written in Italian and in the early period of fowl plague (298). The authors German languages. In addition to these, texts in of most of the fi rst reports on fowl plague were not English, French, and Dutch were published. All certain if the disease under study was indeed a “new” these sources were examined in detail in an attempt disease or a variant form of fowl cholera (20, 37, 57, to gain reliable information on the fi rst appearance

Avian Influenza Edited by David E. Swayne 145 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 146 Avian Influenza of fowl plague as well as its modes and routes of languages were given in a similar mode but also in spread to avian and mammalian hosts in various an attempt to differentiate fowl plague from other countries. The issue of transmissibility of the puta- diseases of dramatic devastating nature. Many of the tive agent was followed in detail in order to gain other names are listed in Table 7.1. The desire of so information on the host range. Also described are many authors to coin a meaningful name reﬂ ects the available means for diagnosis and on the dif- both, the anticipation of a “new” disease and to ferentiation from similar diseases. express the unusual severity of the disease. The original name “epizoozia tifoide nei gallina- SYNONYMS AND ACCESS TO “OLD” cei” that Perroncito (224) introduced was not used by LITERATURE other Italian workers. Initial reports appeared under Many different names were introduced in the past to “epizoozia dei polli” (20, 32, 83). The name “peste denote severity and predominant hosts of the “new” aviaria” was coined by Centanni and Savonuzzi (40). disease. Perroncito (224) used the term “epizoozia In adaptation to the often seen violet to dark-blue tifoide nei gallinacei” (epizootic typhoid of fowl). discoloration of the comb and wattles of chickens, Subsequent publications in many different national Lode and Gruber (175) introduced the name “kyanol-

Table 7.1. Terminological designations for the disease and for the causative agent.

Cited publications Author(s)/ Names of the Year country Names given to the disease causative agent

1878 Perroncito/Italy Epizoozia tifoide nei gallinacei None 1880 Rivolta and Delprato/Italy Tifo essudativo None 1899 Belfanti and Zenoni/Italy Epizoozia dei polli None 1899 Foá and Cesaris-Demel/Italy Epizoozia dei polli None 1901 Greve/Germany Braunschweiger Hühnerseuche None 1901 Jess/Germany Braunschweiger Hühner-und None Putenseuche 1901 Krausz/Hungary Pseudohühnercholera, None Hühnerepizootie, Hühnerseuche 1901 Lode and Gruber/Austria Cyanolophia Virus 1901 Scheurlen and Buhl/Germany Seuchenhafte None Bauchfellentzündung, Peritonitis epizootica 1902 Enders/Germany Phasianidenseptikämie None 1902 Künnemann/Germany Vogelpest None 1908 Giemsa and Prowazek/Germany Hühnerpest Virus 1908 Freese/Germany Hühnerpest, Gefl ügelpest None 1908 Kraus/and/Schiffmann/Austria Hühnerpest Virus 1909 Eggebrecht/Tsingtau, China Italienische Hühnerseuche Virus 1919 Brieg/Denmark Hühnerpest Virus 1925 Beaudette/NJ, USA Fowl plague None 1925 Boughton and Tunnicliff/IL, USA European fowl pest None 1925 Johnson/MI, USA European fowl pest Virus 1926 Gerlach and Michalka/Germany Gefl ügelpest None 1926 Miessner and Berge/Germany Gefl ügelpest Virus 1931 Doerr et al./Germany Hühnerpest oder Gefl ügelpest Virus 1946 Jungherr et al./MA, USA Fowl plague Virus 1955 Schäfer/Germany Klassische Gefl ügelpest, Virus Classical fowl plague 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 147 ophiea gallinarum” (Greek for violet comb). How- and additional contributions increased. As a result, ever, this term was not adopted by other authors. The thousands of exploited people left their homes and outbreak in and after the poultry exhibition in the emigrated, mainly to the United States of America city of Braunschweig (engl. Brunswick) led to the and other Western countries (14). The remaining name “Braunschweiger Hühnerseuche” (Brunswick enduring population formed various associations to chicken epizootic) as proposed by Greve (102). Jess organize resistance and protest against their gov- (129) expanded the name to “Braunschweiger ernments. The latter alternative resulted into new Hühner- und Putenseuche” (Brunswick chicken and legislation that provided for all people more self- turkey epizootic) that included chickens and turkeys. determination, freedom of press, free choice of Even further went Centanni (41) who introduced the movement, and other rights. These newly gained term “Vogelpest,” which means “bird pest.” Enders civil rights led to greater access to public education, (75) worked with ring-necked pheasants and con- well-paid work, and increased incomes. Liberty, sequently proposed a name “Phasianidenseuche, democracy, and the absence of economic restrictions Phasianidenseptikämie, Darmseuche” (phasianid paved the way to more and better education at all epizootic, phasianid septicemia, intestinal epizootic), levels. As a result, the total population increased which delineates the susceptibility of ring-necked enormously and the fl ourishing economy created the pheasants to fowl plague virus. Kaupp (139) in the backbone for higher demands for food (14, 51, 267). United States adopted the name “fowl pest” and Higher education and passionate research at uni- “pestis avium” in his textbook. In the German lan- versities and in the growing industry acquired high guage, the term “Hühnerpest” (chicken pest) was priorities. In retrospective, the relatively short period used by Freese (84) and Dinter (58), expressing the of time between 1880 and 1914 revolutionized all severity of the disease in analogy to other devastating fi elds of science, industry, and trade. This period of diseases. Daubney et al. (52) introduced in their time was probably the most fruitful and progressive publication the term “fowl plague.” In an attempt to phase in many fi elds of sciences, social life, and, in distinguish ND from fowl plague, the term “classical particular, emerging microbiology. To name a few, fowl plague” was introduced for the latter (263). electricity, gasoline-operated engines, and airplanes Despite the existence of publications in a large were developed and introduced, as well as preserva- number of different languages that appeared in many tion of human food by refrigeration and pasteuriza- textbooks and journals, attempts were made to tion and fundamental hygienic measures such as collect and evaluate the content of all these com- community water supply and waste water disposal munications and to relate their content to data in in underground pipes. other languages and countries. It needs to be admit- In medical and microbiological terms, around ted that not all publications in all languages could 1850–1870, the theory of disease development was be studied. Also, some scientifi c journals and books still based on the action of an ill-defi ned “miasma” are no longer available due to various historical and “contagium animale” that are acting on humans reasons such as war, fi re/confl agration, and disloca- and animals, respectively. Gradual changes in tion of libraries by various armies. The authors science and technology started with the construction regret these shortcomings, which could not be of the fi rst high-resolution microscopes (the compa- entirely avoided, and apologize for any publication nies Zeiss and Leitz), development of differential that escaped our detection and citation. staining methods (e.g., Gram,* Giemsa†), and detec- The authors of this chapter prefer in this retro- tion of many small bacteria and their culturing spective communication to use the historical term (Pasteur,‡ Koch§). These dramatic developments “fowl plague.” This term denotes the severity of the made it possible to come to new concepts to explain disease and gives credit to the historical authors. * Hans Christian Gram developed the Gram stain in SELECTED HISTORIAL ASPECTS Copenhagen, Denmark, in 1884. † Gustav Giemsa described his Giemsa staining method Napoleon Bonaparte introduced major civil rights for protozoa in 1902 in Hamburg, Germany. (Codex Civil) during his regency. After the end of his ‡ Louis Pasteur cultured bacteria that are now known empire in 1815, a period of suppression and restora- as Pasteurella multocida in 1880 in Paris, France. tion dominated in many European countries. Human § Robert Koch discovered Bacillus anthracis in 1880 rights were narrowed or suspended completely; taxes in Berlin, Germany. 148 Avian Influenza

Table 7.2. Major achievements of research on fowl plague in Europe. Year Author(s) Country Achievements

1878 Perroncito Italy First description of fowl plague that is different from fowl cholera 1880 Rivolta and Delprato Italy Additional report on fowl plague in chickens and contact transmission to turkey, goose, duck 1901 Centanni and Savunozzi Italy Demonstration of a ﬁ lter-passing virus 1901 Greve; Jess Germany Poultry exhibition in Brunswick and subsequent Horizontal spread to neighbouring countries 1902 Ostertag and Wolffhügel Germany A few recovered chickens are immunized against reinfection with fowl plague virus. Serum from re- convalescent birds protects against challenge 1902 Dubois Belgium Fowl plague is due to invisible microbes 1903 Maggiora and Valenti Italy Expansion of the host range and experimental transmission with various tissues, blood, and feces to avian but not mammalian hosts 1906 Stazzi Italy Fowl plague in psittacine birds 1908 Freese Germany Fowl plague in geese 1908 Giemsa and Prowazek Germany Filtration experiments with fowl plague virus 1934 Burnet and Ferry England Replication of virus in embryonated chicken eggs 1927 Doyle England First description of Newcastle disease (ND) in Europe 1948 Chu England Agglutination and elution of red blood cells by fowl plague virus 1949 Daubney et al. Egypt Protection of chickens against fowl plague with inactivated vaccines 1949 Dinter Germany Isolation of the ﬁ rst variant of fowl plague virus 1955 Schäfer Germany Differentiation of fowl plague from ND

the occurrence of diseases and helped to develop assisted Pasteur by providing a specially designed means for their prevention. glass fl ask for culturing bacteria in bouillon medium Within a very short period of time, many major that we still use under the name “Roux bottle.” discoveries were made (Table 7.2). Indeed, a tre- Gustav Giemsa, working in the Institute for Tropical mendous number of discoveries were made in Diseases in Hamburg, published his work in 1902 several European countries; examples of scientifi c and 1904 on a staining procedure for fl agellates, progress in relation to fowl plague and other dis- blood cells, and bacteria, which is now almost ubiq- eases during this fruitful period of science are uitously used as the “Giemsa stain.” This stain was presented. soon commercially available by the pharmaceutical Due to the pioneering work of Louis Pasteur in company in Darmstadt that was founded by Heinrich France (218–220), bacteria were cultured for the Emanuel Merck in 1827. Visualization of bacteria fi rst time in human history in bottles, and Robert was made possible in a better mode than at any time Koch in Germany discovered that bacteria grow in before due to the development of powerful micro- single colonies on agar plates. Simultaneously, scopes by the two companies Carl Zeiss in Jena Julius Richard Petri, a co-worker of Koch in Berlin, (302) and Ernst Leitz in Wetzlar; both companies invented a glass plate that is suitable for culturing still exist in Germany. of bacteria (and other microorganisms), now well Unfortunately for the brilliant research at that known as the “Petri dish.” Pierre Emile Roux time, all attempts to visualize the virus of fowl 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 149 plague met with complete failure. The various iso- Savonuzzi in 1901 (40), who were probably aware lated bacteria from cases of fowl plague were not of the fi ltration experiments that were published by able to induce a disease that would be similar to the Loeffl er and Frosch (1898) (179) with the virus of spontaneous cases of fowl plague. The huge amount foot and mouth disease in bovines. Consequently, of work that was devoted to culturing virulent bac- the agent of fowl plague became the more precise teria (20, 32, 39, 70, 75, 116, 120, 264, 277) from “ultravisible and fi lter-passing” agent or virus. birds that succumbed to fowl plague failed com- In agriculture, especially in poultry, the traditional pletely. These rather disappointing results provided way of poultry keeping provided insuffi cient eggs at least circumstantial evidence for the fact that the and meat for the growing human population (15, new disease is distinguishable from fowl cholera. 23). No more than 200 chickens were kept on one Perroncito cites (224–227) a large number of authors farm since Roman times. All these small farms were who describe fowl cholera as a severe disease of all located within the walls of tiny villages and all birds domestic bird species that included chickens and were well attended by elderly women and children turkeys but also ducks, geese, pigeons, peafowl, and (46, 235, 289; see 135). guinea fowl. In addition to the causative bacteria of To comply with the increased demand for eggs fowl cholera, many other bacteria were cultured and and meat, farmers in northern Italy were the fi rst in described but none of these were able to induce fowl Europe who improved herd book breeding that was cholera following infection of chickens. The dis- not entirely based on judgments of the exterior but crepancy between various cultured bacteria and the on economic traits such as egg production records, failure to associate disease manifestations with them food intake, and survival rates for laying chickens prompted Jakob Henle (119), Koch et al. (147), and (51). Due to the growing demand for well-perform- Koch and Rabinowitsch (148) to formulate funda- ing Italian White Leghorn laying chickens (23), the mental principles for a causal relationship between fl ock size increased to several thousand head. a newly discovered microorganism and a studied Detailed records on performance were kept and the disease that are presently known as the “Henle-Koch forerunners of contemporary egg contests were postulates.” introduced to compare productivity between new Circumstantial evidence indicated quite clearly breeds. Indeed, Italian layers of Leghorn type per- again and again that the new outbreaks of fowl formed much better than any other breed in Europe plague that were predominantly seen in chickens and the demand for these birds was high throughout and other gallinaceous bird species can be transmit- Europe (23, 30, 51, 68, 267). ted by direct contact between birds and indirectly The enlarged farms could not be allocated within by materials that were in close proximity to diseased the narrow spaces of villages and had to be built or dead chickens. The attempts to visualize and outside; for fresh water supply and waste water culture the virus continued but did not succeed. For drainage, they were quite frequently built on the the time being, the contagiousness of the new disease banks of rivers, ponds, and lakes. Poultry farms are had to be demonstrated by transmission experiments indeed frequently seen in northern Italy along the using tissues, blood, feces, or swabs from affected open waters of the river Po and its tributaries. In chickens as an inoculum and healthy-appearing parallel to these changes in the maintenance of chickens and other birds as recipients of the poultry, fowl plague appeared as a recurrent disease inocula. in this area (93). The location of large poultry farms Fortunately, the breweries regularly used ceramic with highly productive fl ocks of about 1000 chick- fi lters to remove the yeast (Saccharomyces cerevi- ens in close proximity to open waters is now con- siae) from the produced beer. The application of sidered a major prerequisite for effective transmission such fi lters to remove bacteria and yeast from tissue of pathogens from waterfowl to chickens (283). homogenates provided strong evidence that the The Roman writer Columella (53 ad) described causative virus does pass these bacteria- and yeast- in great detail the construction of chicken houses removing fi lters and the fi ltrates were still powerful and the proper feeding and care of chickens, geese, enough to cause a disease that was indistinguish- and pigeons. He wrote, “A number of 200 chickens able from spontaneous cases of fowl plague. This is large enough and can be well attended by a dili- major achievement was obtained by Centanni and gent, elderly woman and a young boy to guard them 150 Avian Influenza and to prevent roaming about of chickens in a way and Andalusia (Spain) that seem to be less suscep- that the chickens become victims of malice humans tible to AI virus infections than Italian breeds; or (3) or beasts of prey.” However, this maximum number allocation of chicken farms outside the boundaries of birds per farmer was probably not frequently of villages, which made intrusion of waterfowl reached. The order “Capitulare Villis” that was likely. Enders (75) infected chickens of different issued by Carolus Magnus (approx. 800 ad) demands breeds such as Minorka Italian and common land that larger agricultural enterprises have to maintain chickens orally and via subcutaneous injection and at least 100 chickens and 30 geese, whereas smaller noted negligible differences in incubation periods properties should have at least half this number (22 to 60 hours) and times of mortality post infection (cited in 105). Exact numerical data on fl ock sizes (45 to 60 hours). This suggests that only minor dif- are not available for the Middle Ages and later time ferences in susceptibility might have existed between periods until about 1880. However, it can be deduced breeds. Furthermore, because farms of 8 to several from published results of the number of dead in hundred chickens were affected, the advent of fowl relation to surviving birds following fowl plague plague as a new disease may be most closely associ- that fl ocks were rather small. As examples, Perron- ated with housing that is close to waterfowl. cito (224, 225) mentions two fl ocks in which 8 of 8 A number of clinical signs, modes of spread, and and 4 of 33 chickens died. Lode and Gruber (175) macroscopic lesions in diseased poultry of the fi rst summarize losses due to fowl plague in Austria. In and second epizootic bear obvious similarities to 300 farmsteads, 2304 of a total of 2560 chickens contemporary fi ndings in poultry with infections of died, which would mean an average fl ock size of highly pathogenic avian infl uenza viruses. Conse- only 9 chickens. In another case, only 2 of a fl ock quently, it makes sense to reexamine these early of 80 chickens survived. Lode and Gruber also state reports on fowl plague that were published between that farms in Italy that produced chickens for export 1878 and 1955. It appears to be worthwhile also to were much “larger” than those in Austria. Brieg (30) consider the means of origin, modes of spread, and in Denmark mentioned 45 farmsteads with a total of the observed spontaneous extinction of such his- 2772 chickens and 73 farmsteads with 7886 chick- torical outbreaks. For comparison to contemporary ens. This would mean an average fl ock size of 62 measures, it seems reasonable to examine how pre- and 108 chickens, respectively. Baudet (16) in the vious outbreaks were handled by governments, vet- Netherlands mentions fowl plaque–affected chicken erinarians, and farmers. fl ocks in the range of 100 to 200 birds. Gratzl and The authors of these early publications had not Köhler (100) group fl ock sizes into three historical yet means for (1) advanced histopathology, (2) virus phases: (1) until World War (WWI), only small- isolation and characterization, and (3) reproduction scale poultry farming existed in central Europe, with of the originally seen disease under well-controlled fl ock sizes below 100 birds; (2) after WW I, poultry experimental conditions (139). Consequently, these farms developed with around 1000 chickens; and (3) numerous initial reports provide more or less cir- after WW II, large farms were created that had more cumstantial evidence for the existence of outbreaks than 10,000 birds. These examples indicate that that were caused by infl uenza A viruses. Unfortu- fl ocks of chickens more than 100 years ago were nately, none of the tissues or other materials that rather small compared with contemporary fl ocks contained the transmissible agent survived until (4, 47). present times. The oldest samples that contain infl u- The description of fowl plague by Perroncito enza A viruses stem from outbreaks in later epidem- (224) is, therefore, not a historical curiosity that ics between 1924 to 1930. These old viruses carry more than 100 years ago the altered locations of the designations “Brescia,” “Dutch,” “Rostock,” housing of chickens increased the likelihood for “Wien,” and “Paris.” Using the contemporary World transmission of an infectious agent like fowl plague Health Organization (WHO)-recommended methods virus from waterfowl to chickens. Thus, the report and terminology, they were typed as H7N1 and of Perroncito could be interpreted as a consequence H7N7 infl uenza A viruses (9, 252, 283). of the following: (1) increased demand for eggs and Table 7.2 presents a selection of major achieve- meat, which led to larger farms with higher levels ments and discoveries of fowl plague (AI) in chron- of productivity; (2) chickens imported from Sumatra ological order. Obviously, the fi rst step is the detailed 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 151 description of the disease by Perroncito in 1878 in scientifi c contributions from ancient China and chickens and turkeys and the visible signs over a India. given time and the gross pathology. Second, the sources and the modes of transmission of the likely Ancient Greek and Roman Writers causative organism to chickens, turkeys, geese, and Hippocrates in ancient Greece and Galen later (cited ducks were studied and described by Rivolta and in 212) differentiated diseases of humans and Delprato (249). Third, some of the inherent proper- animals into (1) sporadically occurring diseases, ties of the agent in various tissues such as blood, which affect few individuals in almost constant internal organs, and feces were examined (224, frequency over long periods, and (2) pandemic 249). These studies resulted in the postulation of an diseases, which are seen in large quantities of the invisibly small organism that can be transmitted to population. The latter category is better subdivided susceptible hosts. Fourth, this organism is able to in (2a) endemic diseases, which occur not only pass fi lters that retain bacteria, fungi, and parasites locally but also constantly in certain restricted areas, and can cause the disease under study (40). Fifth, and (2b) epidemic diseases that affect many indi- demarcation criteria for closely related microorgan- viduals and show a pronounced tendency for large- isms were formulated and the Henle-Koch postu- scale spread. Many major observations supported lates fulfi lled. This step was cumbersome and took the view that pandemic diseases appear more or less a long time to achieve. Whereas differentiation of suddenly and do vanish after some time. fowl plague from fowl cholera was possible by bac- Written historical records say that chickens—after terial cultures that were positive only for cholera their long westward journey from Asia to Europe— bacterium (Pasteurella multocida), the demarcation were bred for more than 2000 years in south within the growing group of viruses, including ND European countries (135), especially Thrakia (now viruses, took a long time and was fi nally obtained Ukraine), Greece, and Macedonia. The ancient by Schäfer and his colleagues (259, 261–263). Greek writers (Aristotle, Pythagoras, Demokrit, and others, cited in 105 and 212) and Roman writers (46, Ancient Asian Writers on Poultry Diseases 235, 289) describe in detail good husbandry condi- The original homeland of chickens and their places tions, behavioral characteristics including cock of domestication are southern China and northeast fi ghting, anatomical structures, and versatile utiliza- India (296). It is generally accepted that domesti- tion of various breeds of chickens and their eggs. cated chickens were moved westward to Europe However, these authors remain silent on topics like along the ancient Silk Road (51) that led from south- diseases and losses (31, 135). ern Asia, past southeastern European countries like Russia, Ukraine, Romania, and Bulgaria, and reached Writers and Artists on Fowl of the Past Ten the Mediterranean basin through Turkey and Greece. Centuries It is very likely that all infectious agents are the Colored paintings, belletristic books, and naturalis- result of co-evolution of contemporary vertebrates tic sculptures that date back to the early Middle including chickens, turkeys, and many other avian Ages unequivocally proved that the chicken was a species. However, if these statements on westward frequently seen bird and constituted a highly migration and co-evolution of diseases are true, the welcome part of rural and urban life. Paintings and very fi rst cases of fowl plague (and also likely other sculptures depict many different breeds of chickens diseases) should have occurred for the fi rst time in but also geese, white-feathered ducks, various breeds these Asian countries. of pigeons, and peacocks (31, 97). Unfortunately, It is an intriguing question whether ancient authors diseases and their treatments are not mentioned in saw fowl plague many centuries before it was present books, nor were meaningful illustrations of sick in Italy. Unfortunately, the authors of this contribu- birds depicted. Thus, we failed to gather information tion were not able to locate such ancient descriptions on any disease from these sources in this period of either with the help of librarians or through reading time. contributions in the languages of these countries. The situation changed gradually after the times of Because references and translations are not available Napoleon Bonaparte, some 200 years ago. Volumi- to us, we believe that we may have missed major nous books were published in Dutch, English, 152 Avian Influenza

French, German, Italian, and many other languages and gross and histological lesions, including immu- on good housekeeping practices, care of farm nity and means for control. animals, and their versatile use (91, 280). Unfortu- nately, these books focus on horses, cattle, and, to DETAILED DESCRIPTIONS OF FOWL some extent, pigs and dogs. Little, if any, space was PLAGUE left in these books for poultry (275). Consequently, During the period of time between 1878 and 1910, only marginal information is available on any of the numerous publications dealt with anamnestic data, likely ailments of domestic birds. For example, clinical signs, and gross and, to a much lesser extent, some of these books make mention of “pip,” which histological pathology. A precise diagnosis of fowl is described as an occlusion by dried mucus in the plague and its reliable comprehensive differential nostrils of chickens due to undetermined causes. diagnosis are essential if the detection of spread The resulting diffi culties in breathing caused the to other locations and neighboring countries is tongue to become very dry and solid, and this attempted. However, the available diagnostic means dryness is known as “pip” (139). A treatment con- of fowl plague were in the early phase. The follow- sisted of removal of the dried tip of the tongue with ing section outlines in more detail the characteristics a sharp knife and subsequent repeated application of the outbreaks of fowl plague that were primarily of hydrogen peroxide followed by potassium chlo- provided by Italian and German authors (see later rate. As an example of confusion due to imperfect and Table 7.2). terminology is the fact that van Heelsbergen (118) relates the “pip” to a chronic infl ammation, enlarge- Anamnesis ment, and hardening of the uropygeal gland of An astonishing and sudden increase in mortality in chickens. adult chickens, turkeys, and pheasants, but less so in To our knowledge, the fi rst textbook that is geese and even less in ducks, is constantly commu- entirely devoted to diseases of poultry was published nicated. Further details on location and size of by Rivolta and Delprato under the title fl ocks, types or breeds of birds, and commercial “L’Ornithoiatria” in Italian in Pisa, Italy, in 1880 and/or personal connections between owners of the (249). The fi rst book on domestic poultry, with a affected fl ocks are nearly completely lacking. Lateral major emphasis on parasites that were divided into spread to neighboring farms or other species was “entozoa,” “epizoa,” and “entophyta” (bacteria, rarely mentioned. The likely role of free-living birds fungi, and molds), was published by F. A. Zürn including waterfowl has never been described. It (1882) (305), a parasitologist in Dresden, Germany. appears that the majority of the authors did not visit Soon thereafter, an additional book on “Gefl ügel- farms to collect additional data on the history and Krankheiten” was issued by Robert Klee (144), current situation of diseased fl ocks. working as a medical assessor in the Veterinary Clinic in Jena, Germany. As far as we know, the fi rst Natural and experimental hosts comprehensive book in English language on all Besides the domestic chicken and turkey, a large known poultry diseases that was highly appreciated number of birds can be successfully infected and the by European poultry specialists was published by B. observed signs and lesions fi t well into current views F. Kaupp (139), a pathologist and poultry investiga- of susceptibility or resistance. Actually all authors tor in North Carolina (United States). A very com- used the chicken as “gold standard” for the monitor- prehensive book on poultry diseases in German was ing of the infectivity and virulence of the inocula. edited in 1929 by the Dutch scientist T. van Heelsber- The avian and mammalian species that were tested gen (118), a bacteriologist at the University of are summarized in Table 7.3. Enders (75) transmit- Utrecht, the Netherlands. Contemporary knowledge ted fowl plague virus via injection or feeding of is provided on “fowl pest (pestis avium)” by Kaupp infectious tissues and obtained mortality within 1 to (139) and on “Gefl ügelpest” (fowl plague) by van 2 days in blue tits (Parus coeruleus), canaries Heelsbergen (118). Both authors describe a highly (Fringilla canaria), linnet (F. cannabia), chaffi nch contagious, frequently lethal disease that is caused (F. coeleps), house sparrow (Passer domesticus), by an ultravisible, fi lter-passing virus. Still fragmen- guinea fowl (Numida meleagridis), golden pheasant tary details are mentioned on epidemiology, signs, (Phasianus pictus), and peafowl (Pavo cristatus). Table 7.3. Natural and experimental host range of fowl plague. Cited publications Year Authors Susceptible species Resistant species

1878 Perroncito Chicken, turkey 1880 Rivolta and Delprato Chicken, turkey, goose, duck 1882 Perroncito Chicken, turkey 1894 Perroncito Chicken, turkey, young pigeon 1901 Greve Chicken, house sparrow Adult pigeon 1901 Jess Chicken Adult pigeon 1901 Lode and Gruber Chicken Adult pigeon, duck 1901 Scheurlen and Buhl Chicken Pigeon, duck, mouse, Guinea pig, rabbit 1902 Enders Chicken, golden pheasant, Pigeon, white and grey mouse, turkey, Guinea fowl, blue Guinea pig, dog, cat, rabbit, tit, canary, sparrow, fox chafﬁ nch, linnet 1902 Künnemann Chicken Pigeon 1903 Maggiora and Valenti Chicken, turkey, sparrow, Pigeon, domestic and wild starling, goldﬁ nch, falcon, duck, mouse, Guinea pig, little owl, sparrow hawk rabbit 1904 Marcone Pheasant 1905 Kleine and Möllers Chicken, goose 1906 Stazzi Psittacine birds (cockatoo, pappagallus, budgerigar, love bird, partridgea) 1906 Rosenthal Chicken, goose 1906 Schiffmann Chicken, goose 1908 Freese Chicken, goose, pheasant, Duck, mouse, Guinea pig, sparrow rabbit 1908 Kraus and Schiffmann Chicken, goose 1912 Von Ostertag Chicken, turkey, pheasant, blackbird, owl, parrot, sparrow 1915 Kraus and Loewy Chicken, goose, young pigeon Duck, adult pigeon 1917 Kaupp Chicken, psittacine birds 1925 Boughton and Tunnicliff Chicken Rabbit 1926 Gerlach and Michalka Chicken, goose, turkey, sparrow 1926 Miessner and Berge Chicken, goose Mouse, Guinea pig, rabbit 1927 Van Heelsbergen Chicken Young chick, duck, pigeon 1927 Doyle Chicken, young pigeon Adult pigeon 1931 Doerr et al. Chicken Mouse, Guinea pig, rabbit, frog 1942 Nakamura and Iwasa Chicken, domestic cat a Cockatoo—Eolophus roseicapillus; budgerigar—Melopsittacus undulatus; love bird—Agapornis swinderianus; pappagallus—Ara ambigua; partridge—Alectoris spp.

153 154 Avian Influenza

Künnemann (159) found the common carrion crow whereas nestlings and young pigeons may develop (Corvus corone) susceptible. Maggiora and Valenti signs and recover within some days (41; see detailed (185, 186) published voluminous materials on the review in 136). host range and found, besides the “gold standards” All mammals (dogs, rabbits, mice, guinea pigs, chicken and turkeys, the common house sparrow cat, and fox but also the frog) were resistant to (Passer domesticus), the European starling (Sturnus disease (29, 65–67, 84, 127, 128, 131, 185, 201, 224, vulgaris), the goldfi nch (Carduelis carduelis), a 264). In contrast, only Nakamura and Iwasa (207) falcon (Astur nisus), a little owl (Strix passerina), report on diseased domestic cats following natural and a sparrow-hawk (Accipiter nisus) susceptible. and experimental fowl plague infections. Trans- Also, von Ostertag (213) mentioned the transmis- mission studies with brain of a cat that had devel- sion of fowl plague to ring-necked pheasants (Pha- oped nervous lesions and died after several days of sianus colchicus), blackbirds (Merula merula), owls severe illness to other cats resulted in rapid death (Strix spp.), parrots, and house sparrows (scientifi c of these cats. The same brain tissue was injected names of birds were not provided by the author). into chickens, which died under signs of fowl Stazzi (277) transmitted fowl plague virus to a cock- plague. This is the fi rst published evidence that the atoo (Cacatua spp.). The tests for the susceptibility domestic cat is susceptible to fowl plague virus. of domestic pigeons yielded erratic or contradictory Although no hemagglutinin subtype was provided results (see Table 7.3). The ring-necked pheasant by Nakamura and Iwasa (207), it is likely that it was (Phasianus colchicus) was shown to be susceptible of the prevailing H7 subtype. Similar data on the to disease by several investigators (75, 84, 213). susceptibility of domestic cats were obtained in Signs and lesions correspond well to those seen in recent years in Asia and confi rmed in Europe using chickens. the Asian H5N1 virus, which proves again that cats The fi rst reports on sporadic and mostly endemic are susceptible to some infl uenza A viruses (160, cases in young domestic geese were published by 248, 273). Kleine (145) and Kleine and Möllers (146). Adult The virus of fowl plague multiplies in cells of the geese seem to be resistant to disease (146, 201). The central nervous system of mice following intracer- incubation period of young geese following intra- ebral inoculation and in the testicles of rats follow- muscular injection of blood from sick chickens is ing intratesticular inoculation (45, 65). Back-passages approximately 5 days. The most prominent signs to chickens confi rm the presence of pertained viru- consist of central nervous system disorders. The sus- lence of the inoculated virus. ceptibility of young but not adult geese was subse- Perroncito (224) mentioned humans who con- quently confi rmed by Maue (198), Rosenthal (253), sumed processed meat of fowl plague–infected Schiffmann (265, 266), Kraus and Schiffmann (154), chickens without any subsequent health problems. Freese (84), Doerr and Pick (63), Kraus and Loewy None of the other European investigators reported (157), Brieg (30), Miessner and Berge (201), Gerlach on any unintended transmission of fowl plague virus (92), and Komarov (149). For 4 to 5 days post infec- to scientifi c or technical staff members who handled tion, intramuscularly infected young geese contain diseased birds. fowl plague virus in their blood that is lethal to chickens. No signs are seen in infected chickens if Clinical Signs blood from infected geese is transmitted to chickens The fi rst signs in a fl ock consisted of sudden onset after 5 days (155). Brain tissue and the spinal cords of complete anorexia but increased thirst was con- of infected sick geese contain virus until their death, stantly seen. Vocalization ceased either completely, which might take several weeks (201). or sudden cries with unusual sounds were noticed. In contrast to geese, the domestic white Pekin Locomotion was greatly reduced, and upon pushing duck and other domestic duck breeds seem to be of sick birds, only minor movements were seen. Egg rather resistant to disease development following laying and fertilization of cockerels stopped com- natural or experimental exposure (16, 40, 75, 84, pletely. The eyes were closed as in photophobia. The 175, 185, 264). birds of affected fl ocks were sitting in a resting posi- The adult domestic pigeon seems to be rather tion on the fl oor, did not perch, and displayed ruffl ed resistant to infection and disease development, feathers. Other signs are frequently absent. 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 155

The appearance of individual sick chickens that the body and straddled with their legs. It was never were submitted for investigation was described in reported that diseased birds recovered completely great detail by many investigators (see later). Signs and resumed egg laying (92, 175, 224, 231). are generally more frequent and more pronounced For ducks, in contrast to chickens and turkeys, the in experimentally infected chickens than in sponta- disease was minor or nonexistent. In geese, the incu- neous cases (231). The estimates of the incubation bation period following natural exposure—in most period following natural infections range from 1 or cases due to proximity to sick chickens—was longer 2 days to 1 week. The uniform prevailing view was than in chickens. The course of the disease is less that adult chickens and turkeys are most severely severe. and equally affected. Later, only one report notes Spread among geese was slow and the rate of that young chicks were less susceptible than adults mortality lower (146, 154, 201, 249). Prominent to disease development (117). No differences in ﬁ ndings consisted of nervous signs such as lame- signs existed between female and male birds. The ness, drooping of wings, tremor of the heads and body temperature of sick chickens increased up to necks, and atypical vocalization. Feces were dark- 43.5º C during sickness and decreased rapidly just green and covered with increased amounts of urates. prior to death. At this stage—after 1 to 2 days of The duration of the disease in geese could take illness—many birds started dying. Many of the several weeks if sick birds were not culled. Lateral remaining birds had swollen violet to dark-blue spread from geese to chickens and other terrestrial combs and wattles, edema of the face, and conjunc- bird species was noticed but not described in tivitis (Table 7.4). Diarrhea was occasionally seen detail. and discharges were liquid to watery and of dark- greenish color. Some chickens had blood-stained Gross Pathology feces. Foamy liquid protruded from the beak and Chickens and turkeys are at necropsy (due to the nostrils, which resulted in laborious breathing. very short duration of the disease) in good bodily However, distinct respiratory signs were never condition with fully developed muscles and fat reported. After 2 to 5 days of severe illness, the tissue under the skin and in the body cavity. A very complete ﬂ ock was eliminated. In some other cases, detailed, quantitative study on the grossly altered few survivors resumed consumption of food and organs was provided by Pfenninger and Metzger water. These few birds displayed at intervals for (231), who examined 40 cases of natural and 69 some weeks head tremors, shaky movements of the experimentally infected chickens in an attempt to head and neck, stiff walking, and backward move- compare signs and lesions in relation to the mode of ments of the body. Some birds laid on one side of infection with fowl plague at the University of

Table 7.4. Clinical signs of fowl plague in chickens following spontaneous and experimental infections. Frequency of clinical signs (%) Signs Spontaneous infection Experimental infection

Cyanotic comb 30 82 Cyanotic wattles 25 74 Edema of the face 28 14 Conjunctivitis 13 36 Edema of the throat 3 10 Petechiae in skin around sternum 3 6 Hyperemia of skin 8 6 Experimental intramuscular infection was done with 0.5 ml of heart blood from a spontaneously fowl plague infected chicken that died just prior to blood sampling (231). 156 Avian Influenza

Table 7.5. Gross pathology of chickens that died following spontaneous or experimental infections with fowl plague virus. Frequency of pathological lesions in per cent Gross lesions Spontaneous infection Experimental infection

Petechias on Epicardium 48 57 Hydropericardium 20 30 Laryngitis, tracheitis 18 19 Edema in lung 40 32 Enlarged, hyperemic spleen 15 58 Hemorrhage in proventriculus 60 84 Hemorrhage in gizzard 50 59 Hemorrhage in intestines 35 58 Lesions in pancreas 0 1 Hyperemic kidney 25 48 Hyperemic ovary 13 41 Hyperemic uterus 15 42 Source of virus, see Table 7.4 (231).

Zurich, Switzerland (Table 7.5). Obvious differ- mucosa contained hemorrhage. Kidneys were also ences between spontaneous and experimental infec- enlarged and the ureters were fi lled with urates. The tions of chickens were not noted. The most frequently spleen was in most cases normal or only slightly seen lesions in both groups of chickens were hemor- enlarged. The reliability of kind and extent of path- rhages in the mucosa of the proventriculus, ven- ological changes are frequently uncertain and require triculus (gizzard), duodenum, and on the ovarian isolation of the causative organisms for a defi nite follicles. About one third of all chickens displayed diagnosis (85). petechia on the serosa and mucosa of the distal intes- Geese were generally in poor bodily condition tine. Rather surprising—if compared with more due to the longer duration of the disease. Dead geese recently published data—lesions in the pancreas had less-pronounced lesions that consisted of some were noted only once by Pfenninger and Metzger hemorrhage on the serosa and mucosa of the prov- (231). entriculus. Lesions of the respiratory tract, fi brinous The upper respiratory tract, trachea, and lung in pericarditis, and perihepatitis were never mentioned. chickens and turkeys were occasionally congested Ducks remained unaffected in all experimental but otherwise unchanged. Lesions of severe pneu- infections (84, 125, 146, 201, 265). monia like in cases of fowl cholera were not reported. Females that stopped egg laying had an extensive Histopathology fi brinous peritonitis. Fibrin-like masses may contain It should be kept in mind that histological methods yolk with albumen, possibly due to retroperistaltic and equipment were still in their infancy at the time motility of the uterus (so-called “egg peritonitis”). of the fi rst outbreaks of fowl plague. For example, Ovarian follicles were shrunken and covered with in 1858 Rudolf Virchow published his concept on bloodspots. Prominent extensive or pinpoint hemor- cellular pathology that successfully replaced the rhages were noticed on the epicardium and serosa of traditional humoral pathology (292). Consequently, the body cavity. Frequently seen were serous or morphological studies on cellular level were initi- fi brinous pericarditis with accumulation of yellow- ated. However, histology needs a number of prereq- ish liquid, and perihepatitis of the enlarged liver. Not uisites. The necessary fi xation of tissues by formalin constantly was the mucosa of the proventriculus was introduced into histology by Blum (1893) (26) covered with hemorrhage and blood clots. The wall and the usefulness of the hematoxylin stain was fi rst of the duodenum was constantly enlarged and the published by Boehmer (1865) (27). Another staining 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 157 method like trichrome staining with orange-eosin- smears (Hans Christian Gram, pathologist in Copen- toluidin stains for differentiation of neural tissues hagen, Denmark, published his staining method in was published in 1894 by Mann (190). Additional 1884) provided evidence for the presence of numer- methods for the examination of various fi xed tissues ous bacteria of different size, shape, and tinctorial came into use even later (140). Higher microscopic properties (99). These fi ndings were initially confus- magnifi cations required excellent optical systems ing. Pasteur (218–220) published his studies on fowl and a powerful light source; both developments cholera bacteria that are visible in blood, liver, and were initiated by Abbe, beginning in 1869 (3). lung smears in huge numbers; are morphologically The fi rst study on histological lesions of fowl uniform; have the shape of small rods; and are bipo- plague were published by Kleine (145) and Kleine larly stained. These fi ndings made a distinction and Moellers (146), who examined the brain of between Pasteur’s cholera bacteria and all of the experimentally infected young geese. These authors many others possible and helped greatly to confi rm noted accumulations of small, round cells adjacent or to exclude the presence of fowl cholera. Fioren- to blood vessels in the brain. They detected small tini (81) described a lethal hemorrhagic disease in bodies especially in ganglial cells that they described swans (Cygnus olor) and Egyptian geese (Anser as similar in size and internal structure to Negri aegyptiacus) that died in large numbers in a city bodies. Negri published his work on rabies in 1903 park of Milano, Italy. Other species of waterfowl that resulted in the detection of eosinophilic inclu- and gallinaceous birds remained unaffected. Signs sions in the cytoplasma of cells in the ammon horn, and lesions in the examined dead birds were sugges- which were subsequently termed “Negri bodies” tive of fowl cholera or fowl plague. The subcutane- (208). Similar bodies were seen by Rosenthal (253). ous injection of a few drops of cultured bipolar He concluded that the frequently seen clinical simi- staining short rod-like bacteria to chickens, geese, larities in terms of nervous system disorders in geese ducks, and pigeons resulted in death within 8 to 10 and in foxes and dogs are the result of an identical hours. However, cultures that were stored for 1 etiology, such as lyssa (rabies). Prowazek (237) and month at room temperature under atmospheric con- Schiffmann (266) were unable to detect such inclu- ditions induced only intermittent signs of disease but sions. Lépíne and Haber (168), Levaditi and Haber failed to cause mortality in chickens. These results (171, 172), Lépíne et al. (169) and Lépíne and remain open to interpretation of whether fowl plague Sautter (170) detected intranuclear inclusions in leu- virus in addition to the bacteria was involved. kocytes that they considered as nonspecifi c for fowl Because the mortality was seen very shortly after plague. injection and because overwhelming evidence exists Freese (84) noticed in brain tissue and in internal that the shortest known incubation period is around organs (heart, liver, and spleen) of naturally and 1 day, it appears that the disease seen by Fiorentini experimentally infected chickens extensive hemor- (81) is more likely due to unknown toxic effects and rhage, infl ammatory reactions, and congestion but not due to fowl plague virus. no cellular inclusions. These fi ndings were subsequently confi rmed by Gerlach and Michalka (93), DIFFERENTIAL DIAGNOSES Pfenninger and Finik (232), and Findlay and Mac- At least fi ve major diseases of different etiologies kenzie (80). In more protracted cases, foci of necro- have similar signs and gross lesions as fowl plague. sis and granulocytic leukocytes were observed (92). This emphasizes the importance of ruling out other It was shown much later using electron micros- diagnoses such as fowl cholera, intoxications, septi- copy by Sterz and Weiss (278) that cultured throm- cemic spirochetosis, ND, and infectious laryngotra- bocyces can phagocytize and replicate fowl plague cheitis. Initially, the differentiation of these diseases virus. was primarily based on clinical and pathological grounds and later also on histological and microbio- Bacteriological Examinations logical grounds. In addition to histopathology, bacteriology was still poorly developed at the beginning of the fowl plague Fowl Cholera era. Direct examination of unstained smears yielded Fowl cholera was widespread in the nineteenth no uniform populations of bacteria. Gram-stained century in many European countries (55, 139, 144, 158 Avian Influenza

216, 219, 220, 226). Severe forms of fowl cholera Intoxications were frequently seen in terrestrial birds and in water- The intoxication by phosphorous-containing com- fowl that were associated with high losses (159). pounds was common in this period of time. These Table 7.6 details signs and gross pathology that were were intoxications by metallophosphide-containing used as aids to differentiate fowl plague and fowl compounds that were in widespread use as rodenti- cholera. As a consequence of recurrent and severe cides, in particular to reduce the abundant accumula- outbreaks, fowl cholera was classifi ed around 1900 tions of fi eld mice (Mus musculus) and voles as a notifi able disease in many European countries (Microtus sp.) but also to reduce the large numbers (31). of crows (Corvus corone) on agricultural fi elds and Due to the development of effective drugs and in the proximity of villages (24, 250, 259). In the inactivated adjuvant vaccines and along with major past, the very cheap and highly effective zinc phos- improvements in housing and hygienic feed and phide and arsenic preparations were widely and water supplies, notifi cation ceased in Germany in repeatedly used for the destruction of all kinds of 1994. In the past, fowl cholera was especially wide- vermin (24). Pest Offi cers of the Department of spread in many Mediterranean countries and was Agriculture in Scotland and War Agricultural Exec- responsible for repeated and severe outbreaks in utive Committees in England, as well as communal chickens and turkeys but also in ducks and geese offi cers in Germany, laid baits containing 5% zinc (84, 219, 220, 226, 227). Pasteur cultured the caus- phosphide that accidentally resulted in heavy losses ative organism and tried to develop an attenuated when consumed by free-range domestic poultry. vaccine (221). His early attempts to prevent fowl Uptake of these compounds by chickens and espe- cholera by vaccination with a suspension containing cially by ducklings resulted in signs and macro- modifi ed bacteria met with limited success (221). A scopic lesions that were consistent with acute fi rm differentiation of fowl plague from cholera was poisoning and fowl plague (24). Poisoning was asso- obtained by the successful bacteriological cultures ciated with depression, weakness, trembling, thirst, of internal organs and by subsequent biochemical and sometimes diarrhea and fi nally resulted in death. and serological analyses of the bacteria that are now Macroscopic lesions consist of hemorrhagic lesions named in honor of Pasteur’s pioneering work, Pas- and deep erosions in the proventriculus. Presump- teurella multocida (98). tive diagnosis was based on anamnestic reports,

Table 7.6. Differentiation of fowl plague from fowl cholera. Indicative for Parameter Fowl cholera Fowl plague

Disease occurrence at time of the Hot summer Late autumn, winter year Natural hosts All domestic species Predominantly chickens and turkeys Transmission with blood to pigeons Easily possible Only young squabs sick Incubation period Twelve to 48 hours One to 3 days Duration of disease Two days Two to 4 days Rate of mortality Very high All sick birds die Enteritis and droppings Watery Greenish with blood clots Pneumonia Frequent Rather rare Spleen Enlarged Seldom changed Liver Multiple necroses Enlarged Microscopy of stained blood Numerous bacteria No bacteria Effects of fi ltrates to chickens No signs Severe disease and losses Based on References 92, 98, 139, and 281. 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 159 gross pathology, and the characteristic smell of the monic for ND. Thus, confusion persisted until virus content of crop and gizzard. The defi nite diagnosis isolation and differentiation between fowl plague required a chemical analysis of intestinal content and ND viruses became general practice (6–8, 38, and internal organs for zinc phosphide (281). 56, 269, 288, 300). Quite convincing evidence is at hand to correctly Septicemic Spirochetosis state that the fi rst cases of a hitherto unrecorded new Septicemic spirochetosis (borreliosis) was an impor- disease that was named fowl plague have their origin tant disease especially in Mediterranean countries in northern Italy in 1878 (224–227), whereas the (118, 139, 144). This disease affected primarily fi rst cases of ND are described approximately 50 chickens and turkeys but occasionally also water- years later in Southeast Asia (150, 151, 234, 251). fowl (Table 7.7). Only nonspecifi c signs and The viruses that were studied by Perroncito and his enhanced rates of losses are seen in natural cases of contemporary colleagues are not available for char- avian septicemic spirochetosis. Rapid detection and acterization with current methods. Therefore, the differentiation from other bacteria are possible by rather philosophical theory can be posed that the the microscopic examination of Giemsa-stained virus of the fi rst Italian epidemic around 1878 was blood smears that contain characteristic long spirilla- not infl uenza A but paramyxovirus. The fi rst Italian like bacteria in large amounts (103, 283). investigators, including Doyle (69) later, used for differentiation anamnestic data such as outbreaks in Newcastle Disease chickens and other birds that were not traded within ND needed differentiation from fowl plague after its Italy. Commercial trade between Asian and Euro- fi rst description in Europe by Doyle (1927) (69) and pean countries used two routes: the well-known his- soon in Asia under the name “Ranikhet disease” torical silk route passing through continental Asia (49). Confusion existed initially during the fi rst out- and reaching the Black Sea, Turkey, and Greece. If breaks of ND due to similar signs and gross pathol- ND viruses would have arrived along the silk route, ogy (Table 7.8). None of the case histories and none the fi rst cases of disease should have been noted in of the signs and the gross pathology are pathogno- southeastern Europe and not in Italy. Obviously, no

Table 7.7. Differentiation of fowl plague from spirochetosis (syn. Borreliosis). Indicative for Parameter Spirochetosis Fowl plague

Disease occurrence at time of the year Hot summer Late autumn, winter Geographic distribution Tropical and subtropical World wide Tick-borne disease Yes Not essential Natural hosts Young domestic species Predominantly chickens and turkeys Transmission with blood to pigeons No signs Only young squabs sick Incubation period Three to 8 days One to 3 days Duration of disease Three to 5 days Two to 4 days Rate of mortality From 1% to 90% All sick birds die Enteritis and droppings Rare Greenish with blood clots Pneumonia Rare Rather rare Spleen Enlarged, necrotic Seldom changed Liver Enlarged, necrotic Enlarged Microscopy of stained blood Spiral-like bacteria visible No bacteria Effects of ﬁ ltrates on chickens None Severe disease and losses Antibacterial drugs (Atoxyl, Salvarsan) Prevents disease None effective Based on References 92, 103, 118, 139, and 282. 160 Avian Influenza

Table 7.8. Differentiation of fowl plague from Newcastle disease. Indicative for Parameter Newcastle disease Fowl plague

Disease occurrence at time of the Hot summer Late autumn, winter year Natural hosts Many avian species Predominantly chickens, including pigeons almost not pigeons Transmission from chicken to Frequent After close and prolonged chicken direct contact Transmission to antibody free Rarely signs in individual If any, only young squabs pigeons adult pigeons get sick Incubation period Four to 7 days Less than one to three days Duration of disease in chickens Five to ten days Less than one to four days Respiration Laborious Not frequently changed Rate of mortality Moderate to high Almost all sick birds die Infectivity of blood Very low Extremely high Intestine and droppings Watery with blood clots Greenish with blood clots Pneumonia Frequent Rather rare Spleen Enlarged Seldom changed Liver Enlarged Enlarged Microscopy of stained blood smears No bacteria visible No bacteria visible Effects of blood fi ltrates to chickens Disease mainly during Chickens die during viremia viremia at the early phase of the disease Based on References 8, 69, 115, 118, 133, 234, and 261. such reports exist from Ukraine, Romania, Turkey, Tyne in the United Kingdom maintained a large and Greece. The second important trading route harbor at the beginning of the nineteenth century, leads westbound from Asia around the Cape of which makes repeated imports of live chickens from Good Hope of Africa to Europe. It is well known Asia likely, as pointed out by Doyle in his lengthy that the crews of sailing clippers and later steam paper. ships liked to have chickens on board for fresh meat Several biological criteria support historical dif- and egg supply but also fi ghting cocks for entertain- ferentiation of fowl plague and ND viruses. First, a ment. Ships arriving in Europe after a long journey major repeatedly demonstrated criterion for differ- seldom or never had chickens left. The opening of entiation of both viruses was the resistance of adult the Suez Canal in 1869 dramatically shortened the pigeons and ducks to fowl plague virus. However, time needed for the journey from Asia to Europe. pigeons are not completely resistant to all fowl Interestingly, only 8 years elapsed between the com- plague viruses, including some AI viruses of the pletion of the passage through the Suez Canal in subtype H7N7. At least some young pigeons can be 1869 and the fi rst cases of the “tifo exsudativo” in successfully infected and develop nervous signs, Perroncito’s report in 1878 (224). Such a period is whereas adult pigeons remain unaffected. Thus, the obviously long enough for ND virus–carrying chick- frequently used pigeons must be regarded as a rather ens (fi ghting cocks and other breeds) to also arrive weak criterion for the differentiation of fowl plague in Italy. The problem is that trade connections and ND viruses. Second, the incubation periods are between ND-infected Asian countries and northern different with a 1- to 2-day incubation period for Italy were not reported. This fact makes a direct link fowl plague and 4 to 5 days for ND. Gross pathology unlikely. In contrast, the city of Newcastle upon may help in differentiation if the frequently observed 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 161

fi brinous pericarditis and egg peritonitis in fowl causative organism of fowl plague had to prove the plague cases are used as criteria. Third, several following: reports provide evidence that young geese can be infected with fowl plague virus, whereas ND viruses Contagiousness hardly induce disease in geese of any age (28). It became soon apparent to Perroncito (224) and Fourth, Doyle (69) himself used a cross-immuniza- many others later that diseased chickens were able tion test in chickens to differentiate these two dis- to infect others if the two groups of birds were in eases, but the frequent lack of immune chickens close physical contact to each other for prolonged made the feasibility of this test diffi cult. times (20, 32, 37, 40, 57, 70, 72, 84, 92, 102, 116, More recently, Hirst (1941) (122) detected the 120, 215, 217). Fowl plague virus is excreted in agglutination of erythrocytes by infl uenza viruses. large amounts with feces (92). Intramuscular injec- Later, Lush (1943) (183) introduced the hemagglu- tion of fecal suspensions results constantly in disease. tination and hemagglutination inhibition tests for the It was also observed that other chickens—although differentiation of fowl plague and ND viruses. Chu healthy appearing—were able to transmit the disease (44) detected discrepancies in hemagglutination to contact chickens under fi eld condition (64). It was titers if erythrocytes of different animal species were also seen that chickens can occasionally become used; avian erythrocytes agglutinated to almost infected through bites of the tick Argas persicus identical titers, whereas erythrocytes derived from (121). Centanni (41) investigated the role of the red cats and horses failed to agglutinate fowl plague mite (Dermanyssus avium, now known as D. galli- virus. Chu also noted that fowl plague virus eluted nae) in horizontal spread of fowl plague virus and rapidly from hemagglutinated cells. Besides hemag- tends to deny transmission of the virus. glutination, the complement fi xation test was pro- Direct physical contact of orally infected chickens posed for the differentiation of ND and fowl plague to penmates does not constantly result in disease in viruses (209, 210, 299, 304). Differentiation of ND the latter (64, 69, 111). Contact birds of intramuscu- and fowl plague viruses was possible after clarifi ca- larly infected chickens died in 25% of the cases tion of the different etiologies of the two epidemic (239). In laboratory conditions, Doerr and Zdansky diseases (101, 263). (64) and Doyle (69) reported that only 15% of orally ND remained a health problem but also a trade- infected chickens developed signs of fowl plague. In associated problem, particularly in chickens and contrast, conjunctivally, intramuscularly, and subcu- turkeys, until widespread vaccination with live and taneously infected chickens transmit the virus to in- inactivated adjuvant vaccines became available for contact chickens, resulting in disease, virus shedding, the effective control of ND some 50 years ago. and mortality (63, 77, 78, 195). Domestic geese seem to be relatively resistant to Infectious Laryngotracheitis natural infection by fowl plague virus that is excreted Less often is infectious laryngotracheitis (ILT) men- from infected chickens. Several investigators pub- tioned as a differential diagnosis (115, 117, 216). lished data on signs and lesions of naturally and Head edema, severe respiratory rales, drops in egg experimentally infected geese. Pekin ducks appear production, and enhanced losses are nonspecifi c to be completely resistant (see later). indicators for many diseases. Histopathology and Experimental transmission of fowl plague virus to the demonstration of intranuclear inclusion bodies adult domestic pigeons failed in almost all instances, were initially used to demonstrate the presence of whereas young pigeons were only partially suscep- ILT (271). tible (5, 39, 45, 69, 136, 146, 158, 159, 176, 178, 191–193, 200, 224, 239, 264, 281, 303). Fowl plague SEARCH FOR THE CAUSATIVE AGENT OF virus is closely attached to red blood cells and can FOWL PLAGUE be transmitted with purifi ed erythrocytes of diseased Since the pioneering work of Pasteur (219–221), chickens to immunologically naïve chickens (66). evidence is available that microscopically visible, on artifi cial media and outside the natural host, replicat- Experimental Transmission ing microorganisms are able to cause diseases in at Because neither the exact location of the contagium least some species. Dedicated search for a single animatum nor its quantity in a diseased bird was 162 Avian Influenza known, a large variety of materials were used to prevent accidental exposure to other agents were not examine the important issue of transmissibility. All used. It needs to be admitted that obligatory rules investigators used blood, brain, spinal cord, and for conduct and recording of precise animal experi- internal organs (mainly liver, heart, feces, and ments were not yet developed and consequently not others) for their transmission studies (75, 164, 138, known to the authors. Such guidelines came into 175, 185, 186, 187, 224, 237). It became also clear use much later as reviewed by Pearson (223) and during experimentation that the materials used must Wiegers (297, OIE Manual). be removed soon after death of donors and must be applied in a “fresh” stage to chickens or other birds. Means that Infl uence Experimental A large variety of experimental animals were used Transmission for these studies, which provided evidence for dif- It soon became evident to the Italian researchers that ferent levels of susceptibility in different hosts. The fresh blood derived from a sick or moribund chicken conclusion from these transmission experiments yields the most reproducible results, whereas other provided evidence that not all species of animals can materials such as internal organs or feces on occa- be equally well infected. And, also very important, sion provided erratic results. This observation led to each animal can be successfully infected only once. the conclusion that blood is rich in the contagium A second infection of a recovered chicken with and that the property to induce disease is vanishing similar material resulted in no signs and no mortal- with time and elevated temperature of storage. To ity. The idea that such resistance was the result of overcome these phenomena, glycerine was added to acquired immunity was developed at a later date. the blood as a stabilizer (93, 175). Galliforme birds, mainly chickens and turkeys, Although not expressed in these terms in the early served as “gold standard” for the verifi cation of papers, evidence accumulated that the results of transmissibility of the inocula. These birds died con- transmission experiments depend largely on the viral sistently after a short period of severe illness. Fol- content that varies from organ to organ. The effi - lowing injections or feeding of blood, liver, brain, ciency of an inoculum is also dependent on the time spinal cord, or intestines, birds of prey, owls, crows, after collection. Storage at low temperatures can pre- and many different species of passerine birds also serve infectivity in the inocula. Addition of glycerine died (Table 7.3). In contrast, very minor or no signs to blood stabilizes the infectivity (92, 93, 175). Pfen- were observed in pigeons, ducks, and laboratory ninger and Metzger (231) demonstrated that serial mammals such as mice, rats, guinea pigs, and rabbits. passage of virus contained in blood from chicken to Thus, the host range of fowl plague virus bears great chicken did not alter the course of fowl plague. similarities to ND viruses (134, 137). Virus Replication in an Infected Host Reevaluation of Transmission Experiments Direct evidence of the inherent properties of the From today’s view, most of the results of initial contagium animatum was not available to workers transmission experiments seem to be conclusive and prior to 1900. Indirect proof for replication was fi t into contemporary concepts on the susceptibility obtained by dilution of the inoculum in “water” and versus resistance of animals used by Perroncito injection of the obtained dilutions into chickens. The (175, 185, 224). If criticism is applied to the methods recovery of blood from the inoculated bird, which and types of animals used in these studies, it becomes was diluted again and injected into another bird, obvious that (1) the various inocula were more or resulted in disease and death (175, 185). Repetition less crude preparations derived from dead or mori- of this procedure for up to 10 serial passages led to bund birds; (2) the inocula were not subjected to any the conclusion that there must be a replication of the further characterization and quantifi cation of the contagium to explain its effi cacy to kill the infected viral content, (3) nothing was done to exclude the birds even after 10 serial passages (175, 213). presence of concurrent concomitant extraneous All authors of the cited studies performed their agents, (4) the origin, the number, and the status of work on transmission and host range with juvenile infection and/or immunity of animals were not to adult birds. Only Centanni (41) inoculated embry- assessed prior to infection, (5) recovery of the virus onic chicken eggs prior to incubation and used was not attempted, and (6) isolation facilities to infected chickens as positive control of the virulence 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 163 of his inoculum. Following incubation of inoculated or with palladium at an angle of 25 degrees and a eggs, he found embryo mortality and concluded that 10,600- to 15,000-fold primary and 30,000-fold total the embryos were susceptible to fowl plague virus. magnifi cation. The depicted particles are spherical However, he generated no idea in the direction of and of slightly different diameter (124). Surface the paramount value of embryos as a relatively easy- structures are hardly visible. A detailed electron to-handle tool to isolate, propagate, and quantify microscopic study on virus morphology and initial fowl plague viruses. This major achievement must and late stages of virus replication in ultrathin sec- be credited to Burnet and Ferry (33) and Burnet tions of infected chicken embryo fi broblast cultures (34–36). Burnet (36) also used egg-grown virus for was published by Reinacher and Weiss (244). This his studies to immunize ferrets and mice with prom- study shows that viral particles enter cells via pino- ising results. Haber (104) and later Ambrosioni (10) cytosis and replicate inside the cells and that newly used extensively 10-day-old chicken embryos and formed virions leave the cells by budding through inoculation of the chorioallantoic membrane for the outer cell membrane. isolation and titration of suspensions of infl uenza Early fi ltration experiments were performed (1) to viruses of avian and mammalian origin. Quantifi ca- remove bacteria from organ suspensions that cause tion of virus content was achieved by counting the fowl cholera and (2) to demonstrate that the obtained number of “pox-like” lesions on the chorioallantoic bacteria-free fi ltrates indeed cause fowl plague when membrane. used in various transmission experiments. Such Marchoux (194, 195) and Hallauer (107) made experiments were fi rst published by Centanni and fi rst attempts to grow fowl plague virus in artifi cial Savonuzzi (40) and almost simultaneously by Lode media that contained gelatine, salts, and chicken red and Gruber (175). Soon thereafter, Maggiora and blood cells. These attempts were repeated by Land- Valenti (185, 186) and other authors (11, 41, 96, steiner and Berliner (165). These trials did not yield 162, 173, 202, 258, 295) confi rmed that the infectiv- evidence for virus multiplication. ity passed Berkefeld fi lters that were produced from Egg transmission of fowl plague virus was never heat-treated diatomaceous earth, which is a fi ne reported. It can be assumed that the virus also white powder composed of the siliceous skeletons reaches the ovary during viremia and could pass to of diatoms. Baudet (16) used ceramic Chamberland egg follicles. Further survival of the virus in eggs is fi lters with variable success. Although not outlined likely to be limited due to the strong antiviral activ- specifi cally in these publications, the constantly ity of an inhibitor that is present in egg white (166). retained virulence in the fi ltrates after fi ltration through Berkefeld fi lters provided vague estimates The Ultravisible and Filter-Passing Virus on the small size of the causative virus (Wilkinson Many attempts were made with various preparations and Waterson, 1975, cited in 6). from succumbed chickens to visualize the fowl plague agent under the most powerful microscopes Survival of the Virus Inside and Outside of an (92). All these experiments failed completely. Infected Host Because these materials were able to cause disease, Perroncito (224) was convinced that a very poison- the researchers came to the conclusion that the fowl ous agent caused the outbreaks in chickens and plague contagium is extremely small and even the turkeys. At his time, details on viruses, their distri- best microscopes were not suited to reveal any struc- bution in the various compartments of the body, and tures that could be associated with the agent. their stability inside and outside the body were not Information on size, shape, and internal structures known. However, it was a practical experience that of the agent became available after invention and any material for intended use as inoculum must be technical improvements of the electron microscope “fresh.” The infectivity of fowl plague virus in blood by Ernst Ruska (256) and fi ltration technology (19). or other tissues was maintained to some extent by Dawson and Elford (54), Elford et al. (74), Schäfer the addition of glycerine. and Schramm (259), and Schäfer et al. (262) published electron micrographs of the strains “Brescia” Biological Properties of Fowl Plague Viruses and “Rostock,” respectively. The viral particles In view of current knowledge on methods and prac- were visualized following contrast either with gold tical experience with virulent infl uenza A viruses, 164 Avian Influenza much of the data on viral properties provided by containing liquids is much higher than similar liquids earlier investigators cannot be confi rmed today and with ND virus. are no longer available. However, it should be men- The early investigators have also shown that a tioned that more than 100 years ago, researchers large variety of avian species can successfully be tried to collect information on issues like stability of infected while mammals survive subcutaneous or the infectivity in different environments such as intramuscular injections (40, 175, 185). Doerr et al. blood, meat, feces, and other materials. Purchase (65) infected guinea pigs and mice via the intracer- (240) was able to demonstrate infectious virus in ebral route, which resulted in severe neurological carcasses after a period of more than 1 year at chill- disorders and death. ing temperature. He infected fowls by feeding infectious blood while orally giving virulent muscle and Descriptions of Spread of Fowl Plague failed to evoke mortality. The resistance of fowl Based on available publications and on a few gov- plague virus against different denaturalizing agents ernment records, the spread of fowl plague between in vitro was investigated by Schweizer (270). 1878 and 1959 in Europe is divided into three major Attempts to quantify the virus content in blood of epidemics (Fig. 7.1). dead donors by injections of serial dilutions yielded numbers of virus particles far beyond the number The First Epizootic in Italy that would fi t into one milliliter of inoculum. Such from 1877 to 1880 errors could be interpreted as a result of dilution To our regret, we do not currently have the possibil- deviations during pipetting of the samples (65). ity of confi rming the very early outbreaks as factual Doyle (69) injected different dilutions of peritoneal cases of fowl plague, nor can we verify even earlier fl uids derived from chickens that died of fowl reports that may represent descriptions of the same plague and found that the infectivity of such virus- disease. Examples of such early accounts remain

Number of outbreaks/ publications 5

0 1876-1881-1886-1891-1896-1901-1906-1911-1916-1921-1926-1931-1936-1941-1946-1951- 1880 1885 1890 1895 1900 1905 1910 1915 1920 1925 1930 1935 1940 1945 1950 1955 AI outbreaks 61025318134510000 AI research papers 0 0 0 1 1 3 14 13 6 5 17 17 12 9 14 11 Period of outbreaks/ publications

Figure 7.1. Graphic presentation of three epidemics of fowl plague in Europe. The fi rst epidemic occurred in Italy from 1877 to 1880; the second epidemic mainly took place in Italy, Austria, and Germany from 1895 to 1906; and the third epidemic spread over Europe from 1919 to 1931. For comparison, the number of publications on fowl plague is shown. 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 165 open to interpretation. Such papers were provided by Ercolani in 1861 (76) and Piana in 1876 (233), both Italian workers, and by the German veterinarian Straub in 1877 (279). Ercolani (76) describes variable forms of a disease with an acute course of 2 to 12 hours’ duration under the title “epizoozie tifi che.” The observed clinical signs were dejection, loss of appetite, and fl abby and blue discolored comb. Breathing was diffi cult due to mucus and secretions of mucohemorrhagic nature. Ercolani (76) and Piana (233) also observed in chickens nervous signs such as trembling of the head and convulsions. Gross pathological lesions consisted essentially of petechial and ecchymotic hemorrhages on the intestines and softening of the liver. Such alterations may also be associated with fowl cholera. Figure 7.2. Edoardo Perroncito (1847–1936), Straub (279) gives a report of an outbreak among full professor of pathology and parasitology poultry in Baden-Wuerttemberg, in southwestern from 1874 to 1923 at the Veterinary Faculty of Germany. Chickens as well as ducks and geese the University in Torino, Italy. He succeeded Prof. Sebastiano Rivolta, a highly respected appeared to be sleepy, lost their appetite, and secreted parasitologist (fi rst description of slime from the bill and the cloaca. The comb showed Trichomonas gallinae) and pathologist. a dark red or blue color and the birds had diffi culty Perroncito published his fi rst comprehensive breathing. The duration of the illness was described paper on a severe disease in chickens and as less than 1 day in general. Gross pathological turkeys that he termed “epizoozia tifoide nei fi ndings consisted of livid coloration of the skin, gallinacei,” currently known as highly redness of the intestines, enlarged and fragile liver, pathogenic avian infl uenza. Further blood-fi lled lungs, and petechial hemorrhage on the publications deal with the pathogenesis and heart. According to Depperich (57) similar cases prevention of Ancylostoma duodenalis, a occurred in southwestern Germany in 1880/1881 roundworm that lives in the intestines of and 1888 to 1897 in chickens, turkeys, and Califor- humans and causes anemia and high rates of mortality. Additional studies, some together nia quail (Callipepla californica). He attributes the with Louis Pasteur in France, focused on fowl frequent appearance of this highly contagious and cholera (Pasteurella multocida) and on lethal disease to imports of poultry from Italy. The diseases of honeybees and silkworms. true etiology of these cases cannot retrospectively Original photograph was reproduced with the be verifi ed solely on the basis of the interpretation kind written permission of dellÁrchivio of the texts. Storico per Utilizzo Fototeca, Torino. The report of Hungarian ornithologist Petényi in 1833 (228) deals most likely not with a case of fowl plague. The descriptions given by Petényi on the clinical signs and the gross pathological lesions are most severe disease) was provided by Professor irresolute and he mentions pigeons as frequently Edoardo Perroncito (Fig. 7.2). He was a highly affl icted birds, whereas chickens are relatively appreciated member of the Veterinary Faculty of the resistant. These fi ndings are not compatible with University of Torino in northwestern Italy. His main fowl plague. However, Manninger (191–193), interests were pathology and parasitology. Perron- another Hungarian scientist, is of the opinion that cito has seen a highly lethal disease in chickens, Petényi’s publication represents the fi rst recording turkeys, and birds of prey in late fall in 1877. He of fowl plague. termed the disease “epizoozia tifoide nei gallinacei” Today, it is generally accepted that the fi rst (engl., epizootic typhoid in fowl) in an article that detailed description of a “gravissima malattia” (ital., was published in the highly ranked journal Giornale 166 Avian Influenza della Reale Accademia d’Agricoltura di Torino “tifo essudativo” (exudative typhoid) as a separate (1878). His long and detailed report on 40 printed disease entity. They mention the great losses caused pages with two colored illustrations provides deep by the disease among poultry and other birds. The insight into the new types and scales of extensive authors describe the clinical signs and gross patho- poultry holdings and trade in live birds. In his view, logical lesions on the basis of data that they derived the new disease that was studied in the regions of from papers of Perroncito (224) and Longo (180). Lombardia, Parma, Bolognia, and Reggio is differ- They also regard the descriptions of Piana (233) and ent from fowl cholera, which occurred simultane- Nosotti (211) as likely cases of fowl plague. ously at that time in chickens, turkeys, and waterfowl The case of “tifo acutissimo” (peracute form of (1, 2, 32, 226, 227). The incubation period of the typhoid) recorded by Longo (180) happened in 1878 new disease in experimentally infected chickens was in the environs of Torino in northwestern Italy. The often less than 1 day. Prominent clinical signs con- epidemic was confi ned to chickens, turkeys and sisted of somnolence, closed eyes, anorexia, high pheasants, whereas pigeons and ducks were spared. fever (43º to 43.5º C), and foamy saliva in some Mammals resisted the inoculation as well. Longo cases. Just prior to death, the comb and wattles turn stated the duration of illness with 1 to 2 days in into violet to dark blue. Some birds develop nervous general. Frequent clinical signs were somnolence, signs such as head tremor and bending the head anorexia, high fever, diarrhea, and diffi culties in down to the legs. The duration of the disease is very walking. In the gross pathological examination, he short and often less than 1 day, occasionally 2 to 3 found a general emaciation, pale skin and muscles, days. It is said that some chickens walk a few steps hyperemia of the intestines, especially of the duode- and fall dead like they were struck by lightning. num, and exudates in the body cavities. The gross lesions of succumbed naturally and It is concluded that these publications clearly experimentally infected chickens and other birds are document the fi rst peak of outbreaks of fowl plague variable and depend largely on the duration of the (see Fig. 7.1). All these reports denote the astonish- illness. Most of the examined chickens were in good ing severity and rapid spread of the disease within bodily condition but appeared to have signs of water fl ocks. Spreading from Lombardia to adjacent prov- deprivation. The duodenal loop was enlarged and inces of northern Italy was not reported. The com- covered with ecchymotic to pinpoint hemorrhages. plete loss of all birds per fl ock had a self-limiting The duodenum contained food mixed with coagu- effect of this fi rst well-documented outbreak. lated blood. The serosa of the abdominal cavity was Because there are no references available on new frequently covered with hemorrhages and the lumen outbreaks for the years 1880 to 1899, it seems that contains fi brinous material, especially around the the outbreaks that were described by Perroncito heart, on serosal surfaces and among the intestines. (224) and reviewed by Rivolta and Delprato (249) The livers were pale and the gall bladder distended were restricted to that period of time and to Italy. with dark-green bile. The kidneys were enlarged These outbreaks must be considered as the fi rst epi- and covered with whitish fi brinous material. The demic. However, the poultry farmers in northern mucosa of the proventriculus appeared reddish due Italy were struck again with a new and severe wave to hemorrhages. of outbreaks of fowl plague some 20 years later Retrospectively, the disease seen by Perroncito (20, 83). It cannot be excluded that at least some (224, 226) is likely to be identical to what is pres- of the many described outbreaks of the second ently termed “highly pathogenic avian infl uenza period are due to bacterial infections like cholera (HPAI).” This—at that time—apparently new or salmonella. disease was named by Perroncito (224) “epizoozia tifoide nei gallinacei” and was also described by The Second Epizootic in Italy during several other researchers in different locations of 1895 to 1906 northern Italy (50, 180, 211, 249). After approximately 20 years of absence of case Rivolta and Delprato (249) defi ne in their exten- reports on fowl plague, new and detailed publica- sive work Lòrnitojatria o la Medicina Degli Uccelli tions appeared beginning in 1899 that describe forms Domestici e Semidomestici (engl. The Medicine of of disease in chickens and other birds with striking Domesticated and Partly Domesticated Fowl) the similarities to the original publication of Perroncito 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 167

(224). Around 1899 and in the following years, addi- these species, see “Natural and Experimental tional reports appeared in Italy (20, 83, 185, 186, Hosts”]) developed signs of dullness, anorexia, and 199, 227) that confi rm the observed characteristics central nervous system disorders and died within of the “epizoozia tifoide” as a transmissible disease less than 1 day to 3 days, whereas eight pigeons, two and its local spread with trade in live chickens. The domestic ducks, and one wild duck survived the reemerging epizootic was again highly contagious, infection and remained unaffected. All similarly and losses were extremely high, leaving some vil- inoculated mammals—two guinea pigs, two white lages in northern Italy without any chickens. mice, and three rabbits—remained healthy until the In contrast to the fi rst epizootic, the second wave end of the experiments. Unlike the current outbreaks of major outbreaks reached many European coun- due to H5N1 infl uenza A virus in some of the Asian tries and was also responsible for the outbreaks in countries, involvement in disease manifestations of Tyrol, Austria, and for the huge outbreak following mammals and humans was never reported during the the poultry exhibition in the city of Brunswick, early phases of fowl plague in Europe. Germany (see later). Several authors (20, 32, 81, 83, 174, 199, 226, Spread of fowl plague to Tyrol, Austria, in 1901 286) report also on the simultaneous isolation of Lode and Gruber, working at the University of Inns- various bacterial microorganisms. Surprisingly, all bruck, Tyrol, Austria, received in July 1901 infor- these authors used for transmission experiments mation through an article in a Tyrolian newspaper crude blood, liver, brain, and other tissue prepara- on a violent epidemic among chickens. These birds tions. Unfortunately, there is no material left for had direct contact to chickens that were imported by investigations with modern methods; therefore, it a dealer from Italy. About 90% to 95% of the chick- remains rather uncertain which of the described ens died. Prior to death, these chickens displayed cases were really fowl plague, fowl cholera, or ruffl ed feathers, and the comb and wattles showed mixed infections. Yet, all these Italian authors stress dark blue discoloration. Respiratory rales and severe the extremely high losses within short times, typical enteritis were seen in almost all birds. The course of of fowl plague (2, 20, 32, 39, 40, 42, 83, 185, 186, the disease was very rapid, and some chickens came 199, 227, 277). to death during oviposition. Inquiries made by Lode It is concluded that most of these publications and Gruber (175) provide information that a similar clearly document the second peak of outbreaks of disease was seen in more than 300 farms of 121 fowl plague in northern Italy (see Fig. 7.1). In con- villages in Tyrol. To illustrate the origin and course trast to the fi rst epizootic outbreaks between the of the disease, Lode and Gruber observed that an years 1877 and 1880, the second epizootic resulted Italian poultry dealer lodged in a guest house for 3 in major spread of fowl plague from northern Italy nights; he brought with him a hand-pulled vehicle to numerous European countries (see later). that was loaded with chickens and geese from the Maggiora and Valenti (185) published the results region of Padua, Italy. Three days after his departure of numerous transmission experiments and found all chickens kept in the guest house became sick; 80 many different bird species susceptible, whereas died and only 2 survived. The same dealer sold nine rodents were resistant and did not develop obvious geese to another person. Later, two of the geese were signs (Table 7.3). The primary aim of Maggiora and found dead. In a neighboring village, 24 chickens Valenti (185) was to demonstrate unequivocally the were sold by the same dealer to an innkeeper’s wife; transmissibility of the ultravisible, fi lter-passing all of these died within the next days. Domestic virus to various avian and mammalian hosts. Suc- ducks and pigeons remained healthy, although they cessful transmission was obtained by oral, intramus- were kept intermingled with diseased chickens. cular, intraperitoneal, and subcutaneous routes with Noteworthy was the lack of mortality among free- tissues such as blood, bone marrow, brain, intes- living “small birds.” tines, and feces or by feeding the dead chickens to Lode and Gruber (175) used for their transmis- these birds. Most avian species (e.g., 18 chickens, sion studies small pieces of liver from an infected one turkey, eight house sparrows, nine starlings, one chicken that was killed in moribund stage. Approx- European goldfi nch, three falcons, four little owls, imately 1 day after subcutaneous, intramuscular, or and three sparrow-hawks [for scientifi c names of oral infections, chickens became listless, showing 168 Avian Influenza somnolence, drooping wings, and closed eyes. Feces other domestic birds from all parts of Germany but were liquid and blood stained. also from surrounding countries, including Italy, Gross pathology of the succumbed birds revealed were presented in long rows of cages. Their exterior pale necrotic tissue on the site of intramuscular was individually judged with passionate scrutiny, injection, with ecchymotic hemorrhages on the and prizes in three categories for the most beautiful serosa in the body cavity and on the heart. Fibrinous birds were granted (159). Unfortunately, chickens pericarditis and perihepatitis were frequently seen. became sick soon after arrival and started dying. The The lungs were in most cases hyperemic but other- organizers of the fair decided—in an attempt to wise normal. Bacteriological examinations of tissues prevent further spread among the exhibited poultry— of succumbed chickens on agar or gelatin plates that to fi nalize the fair immediately and to send all birds were enriched with either cattle, calf, or chicken with their exhibitors back home (102, 129). meat water were completely negative. Gram-stained Dr. Greve, state veterinary offi cer in the city of smears were free of detectable bacteria. Oldenburg, provided a detailed record of his obser- Filtrates of homogenized and in water-suspended vations of the fate of chickens that were sent by six livers were passed through Berkefeld or Chamber- exhibitors to the Brunswick exhibition (Table 7.9). land fi lters. Intramuscular injection of freshly pre- The fate of a total of 34 chickens that were submit- pared fi ltrates into adult chickens resulted in signs ted by six breeders to the exhibition was recorded that were similar to the observed signs and lesions (102). Of these 34, 11 birds did not return due to of spontaneous cases. However, the duration of the death or culling, 22 arrived at the homes of the incubation period was extended to 3 to 7 days. Ten exhibitors and 1 chicken escaped during rail trans- serial passages through chickens with liver suspen- port to Brunswick. Of the 22 chickens that returned, sions for each transfer resulted invariably in death 9 arrived sick and 4 were dead on arrival. During of the chickens within 2 to 7 days post infection. The the next few days, nine chickens were found dead, authors used extremely high dilutions of their liver and only two birds survived. These two birds were homogenates that they calculated as being in the of a different breed and were placed on a different range of approximately 10−2 to 10−17 of the original site at the exhibition. Two other breeders that did liver tissue. These experiments yielded in all infected not attend the exhibition kept their chickens in close chickens similar results in terms of incubation proximity, but separated by a road, to the affected period, signs, and gross lesions. returned chickens. They lost all of their birds Three adult pigeons were also inoculated with within a few days upon arrival of the chickens from similar homogenates derived from a liver and from Brunswick. intestines of dead chickens. Two of these pigeons Initially, there was much uncertainty regarding developed tonic-clonic nervous signs, especially in the possible causes of the mortality (102, 116, 129). the muscles of the neck; two pigeons died and one Starvation during transport and/or at exhibition was survived. However, guinea pigs, rabbits, and mice the fi rst assumption. Another idea related the casual- did not develop any signs of disease. ties to poisoning by arsenic or other toxic com- Lode and Gruber (175) rejected the name “fowl pounds. Greve (102) explains the losses on the basis pest” or “bird pest” that was suggested by Centanni of a putative virulent agent that might be present in (41). They proposed for the disease under their study wild birds and subsequent transmission by free- the newly coined name “kyanolophiea” because this living birds, probably sparrows. name refers to the almost constantly seen dark-blue Jess (129) then advanced the idea of a cholera-like discoloration of the comb and wattles of diseased sickness on the basis of the detection of some ques- chickens. However, this new name did not obtain tionable bacteria that were detected in cultures after acceptance by other authors (92, 118, 139). 24 hours of incubation. These smears were prepared from blood-agar cultures that were streaked with The poultry exhibition in Brunswick in 1901 blood from the heart of a few dead chickens. These On February 1 through 3, 1901, the Federation of nonmotile bacteria revealed a bipolar appearance German Poultry Breeders held its annual large exhi- following staining with crystal violet and carbol bition of poultry in the city of Brunswick in northern fuchsin and looked very much like cholera bacteria. Germany. At this poultry fair, chickens, turkeys and The city of Brunswick was in 1901 a part of the state 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 169

Table 7.9. Fate of chickens of six breeders that attended the 1901 poultry exhibition in Brunswick. Number of chickens Returned to On arrival at owner home Breeder Sent to Dead on the code exhibition Noa Yes Sick Dead next days Survivors

Js. 9 5 4 1 0 3 0 Kn. 4 1 3 2 1 0 0 Ko. 2 0 2 2 0 2 0 Kü.b 403210 0 St. 6 0 6 0 1 3 2c To. 9 5 4 2 1 1 0 Total 34 11 22 9 4 9 2 From Greve (102). a Birds were not returned to the breeder due to death. b One chicken escaped during transport to exhibition. c Two birds of a different breed that were placed on a different site of the exhibition. of Prussia, which had prepared legal options to pay tions. Both the accumulation of many birds of dif- compensation for chickens that succumbed to fowl ferent origins on the fair grounds and the sudden cholera but not yet for fowl plague. Because signs fi nalization of the poultry fair resulted in the spread and lesions were at least suggestive of cholera (or of the fowl plague virus over Germany (57, 116, could not defi nitely exclude cholera), the demonstra- 145, 182, 214, 257, 264) and to a spillover into tion of cholera-like bacteria would open the way for neighboring countries like Belgium (70, 117), France indemnity payment for the exhibitors (129). (167, 194), Austria (177, 265), the Netherlands Experimental transmission studies with fi ltered (117) Denmark (30), and possibly other countries blood derived from some dead chickens to healthy that did not publish information on outbreaks. chickens resulted in total mortality of all injected Some of the federal states of Germany had in birds within few days. In contrast, the same fi ltrates 1901 legislation that declared fowl cholera to be a did not affect adult pigeons. This result is suggestive notifi able disease. An attempt to apply this legisla- of fowl plague according to the well-known experi- tion to the outbreak in the exhibition was not seri- mental data of Perroncito (224). The demonstra- ously initiated on the basis of a fowl “cholera-like” tion of a viral etiology precluded any payments disease. The responsible offi cial veterinarians forced on the basis of the assumed cholera-like disease the organizers of future exhibitions to apply for per- (102, 129). Still, Jess (129) claimed that the high mission and to take signifi cant precautions to reduce losses are likely to be due to a “symbiosis” of the likelihood of lateral spread and to make use of cholera-like bacteria and a yet unknown additional reasonable disinfection. microorganism. The disaster during and after the Brunswick Quite obviously, the decision to return all chick- poultry exhibition is a convincing example of errors, ens to their owners created a huge disaster among mismanagement, and faulty interpretation of facts chickens in various parts of Germany and elsewhere. that should never happen again. Greve (102) con- Quite a number of birds were infected at the site of cluded from his observations that (1) poultry exhibi- the exhibition and became sick or dead during rail- tions need state veterinary surveillance, (2) exhibition road travel to their home cities. After arrival in their cages must be constructed in a way that allows sep- poultry houses at home, transmission continued to aration of birds from different origin, (3) thorough resident chickens that reached unbelievable propor- disinfection of all cages is necessary, and (4) it is 170 Avian Influenza obligatory for breeders to keep birds that are returned ens. Within 1 to 2 days following direct contact from exhibitions separately from other poultry that between infected and noninfected chickens, the fi rst did not attend an exhibition for at least 5 days. signs of disease developed that are always followed Despite the drama of Brunswick, rumors from by enhanced losses. It was noteworthy that contact Germany about the appearance of the excellent transmissions between larger numbers of chickens Italian layer chickens reached farmers in France, the occur at farm level more frequently than under lab- Netherlands, Belgium, France, and Denmark (213). oratory conditions. Doyle (69) noted that chickens Very soon thereafter, Italian layers were again infected with the fowl plague virus strain “Paris” did exported from Italy to these countries, and as a fatal not transmit the disease very easily if infected and consequence, new cases of fowl plague were seen in noninfected chickens were placed in the same cage these countries. These observations indicate that with a wire fl oor. A very large amount of excreted healthy-appearing chickens served as infected carri- virus was necessary for successful transmission. The ers and transported the disease-causing agent over result with this virus strain questions the effi cacy of long distances. However, communication and trans- sentinel birds. (2) At least anecdotal evidence sug- port at that time were relatively slow and the number gests that contaminated personal and equipment that and dimension of outbreaks were rather small. No was used in infected farms serves as mechanical exact fi gures are available on the number of affected vectors between chickens on different farms. It took, fl ocks and birds. It appears retrospectively that all according to van Heelsbergen (117, 118), 13 to 21 outbreaks resulted in almost complete losses of all days for transmission between the originally infected birds in all fl ocks and the few remaining chickens farm and two adjacent farms. (3) Wild birds that were immediately destroyed and burned or buried. seek food and shelter in infected farms may serve as living vehicles between neighboring farms. Spar- The Third Epizootic of Fowl Plague in rows may become infected, develop signs, and die Europe from 1919 to 1931 (92). This statement is underlined by the fact that After a period of absence of fowl plague for about other investigators demonstrated under experimen- 15 years, new and devastating outbreaks (see Fig. tal conditions that sparrows, blackbirds, starlings, 7.1) were seen again in European countries (16, 30, goldfi nches, and even a little owl were susceptible 69, 78, 92, 93, 117, 132, 181, 197, 201, 230, 234, to infection and may come down with disease (21, 245–247, 284). These authors incriminated as the 48, 84, 93, 185, 213). Transmission experiments source of the virus various imports of chickens from with tissues from these small birds resulted in death northern Italy. Signs, losses, and pathology did not of chickens, which proves that these birds suc- differ from those of the fi rst and second epizootics. cumbed to fowl plague. (4) Running water in creeks Due to the larger fl ock number and fl ock size, that passed very close to poultry farms may have the losses were higher than in the previous two served as a vector if the water was contaminated. epizootics (92). The water-borne spread was cited by van Heelsber- After several decades of absence of fowl plague gen (117), who mentioned a number of outbreaks in in Europe, again outbreaks were recorded in Italy, farms along a small creek that passed several other- the Netherlands, Belgium, and Germany (see Chapter wise nonconnected farms. 9, Highly Pathogenic Avian Infl uenza Outbreaks in The numerous outbreaks of fowl plague in coun- Europe, Africa, and Asia since 1959, Excluding the tries of destination following purchase of live chick- Asian H5N1 Virus Outbreaks). ens required an attempt for explanation. Two hypotheses seem likely and need consideration. GENERAL MODES OF SPREAD OF FOWL First, the very few chickens that recovered from PLAGUE VIRUS fowl plague remained shedders and infected the It is very clear that the major mode of spread was resident chickens in recipient countries. This hypoth- the commercial trade in live infected chickens. The esis appears to be unlikely in view of the small most common modes of spread are detailed by van number of birds that usually survived outbreaks and Heelsbergen (118): (1) The most effective way of the large number of traded chickens. The second spread was quite obviously mutual physical contact hypothesis implies that chickens in the country of between infected and immunological naïve chick- origin were initially infected with a fowl plague 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 171 virus of low pathogenicity that resulted in at least Austria, and Germany, including Prussia, which partial immunity. An assumed subsequent infection today partially belongs to Poland, Belgium, and with HPAI virus would not result in numerous France, are stated by Hoare (123) in the beginning losses. Because such dually infected chickens remain of the 20th century. The disease spread in these clinically normal, they can be traded in large countries after the poultry show in Brunswick in numbers. However, such chickens will be shedders 1901 and by direct importation from Italy and pos- and thus transmit fowl plague virus to immunologi- sibly also from Sudan and Egypt (123). cally naïve birds. However, direct proof for this According to the information provided by Gerlach hypothesis is not evident from the literature because (92), the outbreaks in Tyrol, France, and Belgium avirulent AI viruses had not been discovered and caused the death of approximately 200,000 chick- described at that time. The presented hypothesis of ens; cases in Austria involved 16 districts with the putative existence of low pathogenicity infl uenza 121 villages and about 300 farms. Although exact viruses around 1900 cannot retrospectively be fi gures are not available, Gerlach (92) and van proved. However, it seems likely that low patho- Heelsbergen (118) state that losses were particularly genicity viruses were present during the fi rst and high during the outbreaks in the year 1925 in the second outbreaks in Italy as has been shown to countries Italy, Tyrol, Austria, Germany, Switzer- reoccur in northern Italy in recent times (1999– land, Czechoslovakia, Hungary and Romania. Other 2006). If so, it could easily explain the recurrent and not mentioned countries might have also had out- large numbers of outbreaks in importing countries breaks but written accessible information was not following the purchase of numerous chickens. available. Few records are published on further outbreaks SPREAD OF FOWL PLAGUE TO OTHER between 1912 and 1920. After this time, new intro- EUROPEAN COUNTRIES ductions of fowl plague occurred due to repeated The additional outbreaks around 1900 resulted in imports of live chickens from northern Italy. These multiple introductions of fowl plague into neighbor- outbreaks continued for several years. Zschokke ing countries. Fröhner (86) states that almost all (303) reports an outbreak in chickens sent from German states recognized outbreaks of severe nature Hungary to Switzerland by railway. Just 1 of 34 (159). The total losses during 1899 comprised chickens of this shipment survived. In Hungary, a 33,000 poultry (86). It is quite obvious from the weak contamination is mentioned in offi cial reports published information that the only source of fowl of 1907 to 1912 (71). Again, detailed fi gures on the plague virus was the poultry population in northern number of outbreaks per year are not available. Italy. From the numerous chickens of the layer type In the following years until 1925, spontaneous, and other domestic poultry that crossed the Alps (92, mostly single, outbreaks in different central and 213), some were sold in Tyrol, Austria, and caused eastern European countries were observed and many outbreaks among alpine fl ocks and in other described. These countries include Hungary, Czech- parts of Austria (93, 175). Some chickens arrived in oslovakia, Romania, and possibly others. Cases of southwestern Germany, which was followed by out- fowl plague in Bulgaria causing the death of thou- breaks there (213); some chickens were sent east and sands of poultry are mentioned by Bittner (22). arrived in Hungary (106, 158). Further imports of Perhaps these have to be attributed to fowl cholera, chickens from Italy reached the Netherlands (117), because later authors (12, 222) state fowl plague Great Britain (247), and Denmark (30). Outbreaks outbreaks had not been seen in this country. The due to imports from Italy (200) were repeatedly occurrence of fowl plague in Romania is reported recorded in France (69, 167), Belgium (70), and the by Eckert (71) in 20 cases in 1924. In opposition to Netherlands (118). According to Rasch (243), the this information, Cernaianu and Popovici (43) fowl plague virus was additionally carried from Italy declare Romania to be free from fowl plague since after 1898 to the Czech Republic and Romania (290, 1906 besides a single outbreak in 1932 in poultry cited in 71). The fowl plague case reported by Kün- imported from Italy. Vianello (291) and Pop et al. nemann in 1902 (159) occcurred in Breslau, in that (236) regard the epidemic beginning in 1941 as fowl time a city in Germany but today part of Poland plague, while others (126, 161) state the ND virus (there named Wroclaw). Outbreaks in northern Italy, to be the causative agent. 172 Avian Influenza

Unfortunately, exact data on country, year, dimen- a total of nine chickens died during January and sion of the outbreaks, and the involved avian species April 1949. The last one was submitted for virus are not available to the authors in many cases. isolation attempts. The observed signs in sick birds However, it appears from citations (30, 92, 118) that of the original fl ock were suggestive of fowl plague, losses were usually high for the individual farmer but the rate of mortality and the course of the disease but of little signifi cance for the national economy of within this fl ock and the known lack of virus spread these countries. Chicken meat and chicken eggs to neighboring fl ocks did not fi t into the current were at that time rather seasonal foods, and losses knowledge on fowl plague (62). in chickens were easily compensated by pork and The inoculation of pooled tissue homogenates into beef meat or vegetarian foods. the allantoic cavity of 10-day-old embryos resulted During 1925 to 1927, again a large epidemic of in embryo mortality at times similar to the strain fowl plague started in northern Italy and spread “Brescia,” a highly virulent virus of fowl plague that through commercial trade in live chickens to many Dinter obtained from Dr. C. Hallauer, University of European countries (see Fig. 7.1). Announcement of Bern, Switzerland (114). The harvested allantoic fowl plague in Czechoslovakia had been made to the fl uids of Dinter’s new virus agglutinated chicken red OIE in 1932 to 1934 (71). blood cells to high titers. Hemagglutination inhibi- According to Stubbs (281), fowl plague had also tion (HI) tests with the new isolate were done with been found in Russia. Unfortunately, he does not serum samples obtained from hens of the original give information about the year or circumstances of fl ock in Bavaria, Germany, and with sera prepared the outbreaks. Eckert (71) was not able to obtain against the “Brescia” strain and “Herts” virus, an ND extended information about outbreaks of fowl plague virus that Dinter obtained from Dr. F. D. Asplin, in the Soviet Union from the OIE. Lithuania and Weybridge, England. The results of the HI tests with Latvia have not been concerned according to the the new virus yielded high HI homologous titers, information Eckert (71) was able to collect. In 1905 whereas all tests with allantoic fl uids containing and 1909, the introduction of fowl plague by poultry either Brescia or Herts viruses were constantly nega- imports from Russia is reported in offi cial statistics tive (hence Dinter coined the tentative name “N” for (71); thus, the disease must have been present there. the virus, which stands for Negative). Tumova (287) records a number of outbreaks in Transmission of “N” virus to 8-week-old and eastern European countries including the Soviet adult chickens produced no obvious clinical signs, Union during 1972 and 1985, but she does not but all infected chickens seroconverted. Cross- mention cases during earlier times. transmission studies using these “N” virus–infected, The third epizootic of fowl plague was the last seropositive chickens and the Brescia and Herts one for a long time to come. It is believed that the viruses as inocula resulted in severe signs and mor- newly introduced ND “replaced” fowl plague in tality with both viruses. Thus, the antibodies against continental Europe. The initial cases of ND were virus N were unable to protect against challenge detected and investigated in Great Britain (69). with the virulent fowl plague and ND viruses. Iden- Almost uncontrolled spread occurred during World tical results were subsequently obtained in cross- War II as a result of major movements of military neutralization tests in embryonated chicken eggs and civilians (293). (60). Repeated egg passages of “N” virus resulted in THE FIRST “MILD” CASE OF FOWL rather erratic embryo mortality. While all inoculated PLAGUE, THE N VIRUS embryos died at predictable times during the fi rst fi ve Dinter (59, 60) isolated a hemagglutinating virus passages, the “N” virus did not cause consistent from a pool of liver, peripheral blood and brain of a embryonic death at higher passages (61). Thus, it is dead chicken in embryonated chicken eggs. That clear from the experiments that the hemagglutinating dead chicken originated from a Bavarian fl ock of 48 “N” virus was distinct from fowl plague and ND viruses adult chickens of the Italian light leghorn breed. and that serial passage of virus N in eggs altered Some of the birds of this fl ock had diarrhea, dull- inherent viral properties such as embryo mortality. ness, and central nervous system disorders. At A defi nite clarifi cation of the position of “N” virus approximately 2-week intervals, only one bird and within the infl uenza A viruses came 10 years later 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 173 with studies of Rott and Schäfer (254), who were The fi rst and very detailed paper on a highly able to assign virus N to the infl uenza A viruses and infectious disease among chickens and other birds thus solve the puzzle. in Asia was published by Kranefeld in 1926 (151) The “N” virus is now considered as the fi rst isolate and Rodier in 1928 (251). Both authors used the of infl uenza A viruses of low virulence that is not a name “Philippine fowl disease.” Soon later Farinas, member of the HPAI viruses of the H5 and H7 working in Manila, Philippines, described in very subtypes. Under current terminology and nomencla- detail a disease in chickens under the name “avian ture, virus N is now designated A/chicken/Germany/ pest” (79). Farinas compares his own virus isolate N/49 (H10N7) (283). with those which he obtained from Dr. T. M. Doyle, Weybridge, UK; Dr. W. K. Picard, Buitensorg, Java, SPREAD OF FOWL PLAGUE ACROSS ASIA Indonesia; and Dr. E. A. Rodier, Manila, Philip- The occurrence of fowl plague in China was men- pines. Farinas concludes that all of the isolates were tioned by Eggebrecht (72), Gerlach (92), Mrowka serologically and—as tested by cross-challenge (203), and Stubbs (281) in a Chinese region termed experiments—identical to each other. Thus, these Tsingtau, which is presently named Qingdao. Tsing- viruses are indeed ND viruses and completely dif- tau was under German law between 1897 and 1914. ferent from European fowl plague. No written evidence is available that could construct All at similar time, published articles by Japanese a relationship between cases of fowl plague in authors such as Nakamura (204–206), but also Tsingtau and imports of chickens or other goods Fukushima and his co-workers (88–90), Ochi and from Germany. Thus, the origin of the fowl plague Hashimoto (1929, cited in 88), and Cooper (49) refer virus in Tsingtau remains unknown (281). Accord- to a disease under the names “Hühnerpest,” “Korean ing to Eggebrecht (72), epidemic deaths occurred in chicken epizootic,” and “Chosen Keieki.” All these chickens in Tsingtau every year during the rainy descriptions refer to a disease that has some clinical season and caused great losses in the chicken yards. and pathological similarities to fowl plague. How- Mrowka (203), likewise working in Tsingtau, iso- ever, cross-transmission and cross-immunization lated a virus from a sick turkey that was sent to his studies performed by Farinas (79) prove unequivo- institute and used it for his experiments. He con- cally that all viruses are identical to each other and cluded that the causative agent of fowl plague was represent ND viruses. Only Fukushima (88) com- fi lter passing and has properties of a globulin. pares different strains of Japanese and Korean ND Records of fowl plague from other large countries viruses with a virus termed Chiba. He fi nds marked like India, Indonesia, and Philippines are not avail- differences in clinical, gross, and histological pathol- able for the period of time to be covered in this ogy. However, Nakamura et al. (206) proved the contribution. Some publications might describe “Chiba” strain to be immunologically indifferent cases that could be suggestive of fowl plague. from the virus of Chosen disease. The well-known However, a concise differentiation from ND and fowl plague strain “Chiba” is still preserved in labo- fowl cholera is not possible solely on the basis of ratories. Unfortunately, Fukushima gives no infor- published material. Reports describing well estab- mation about the origin of this strain. Chiba is the lished evidence for fowl plague appeared much later name of a city and region in Japan. in these countries and constitute the current infl u- Todd and Rice (285) and Stubbs (281) write on enza crisis [see Chapter 11, Multicontinental Epi- fi ndings of fowl plague in Japan. Nakamura et al. demic of H5N1 High Pathogenicity Avian Infl uenza (1934, cited in 268) mention a serologic study with Virus (1996–2007)]. a fowl plague–related virus in their country. In con- In contrast to the reports from Italy and other trast, Nakamura and Iwasa (207) in Japan describe European countries, no publications are available to “fowl plague” in chickens that died within 1 to 2 document fowl plague in Asian countries corre- days following natural or experimental exposure. sponding to the early period after the publication of Mice, rabbits, and guinea pigs survived the infec- Perroncito’s article. National accounts were not pro- tion. Interestingly, a cat that was kept in their labo- duced in these Asian countries and the likely written ratory to control wild rats in the foodstore cottage documentations by the former colonial powers were developed nervous disorders and died suddenly. A not accessible to the authors. tissue emulsion prepared from the brain of the cat 174 Avian Influenza was injected into chickens and other cats. The four of communicable disease exists (25). Quarantine is infected chickens died within 40 to 65 hours. The aimed to detect infected poultry and simultaneously very short incubation period and the observed pre- preventing further lateral transmission to susceptible mordial signs of the chickens and their macroscopic resident species or at least at reduction of the risk of lesions are indistinguishable from those animals that infection. Over the years, the concept of creating and died previously and are consistent with current maintaining a quarantine was expanded to many epi- knowledge on fowl plague. The experimentally demic diseases and adopted to special fi eld situa- infected four cats developed nervous signs that were tions. Retrospectively, three major developmental indistinguishable from those that the naturally phases of quarantine are obvious. These are phases infected cat showed and died after a period of illness I, II, and III. of 7 to 13 days. The naturally infected cat seems to have acquired Phase I the virus from chickens that were experimentally The observation of newly purchased poultry (mainly infected by the authors. It appears that this is the fi rst chickens, turkeys, and geese but also other birds) report on the susceptibility of house cats to fowl and their strict separation from resident birds were plague virus. The typing of this virus was later done advised by Rivolta and Delprato (249). The recom- and found to be of the subtype H7N7 (Kunio Satou, mended period of time was initially 40 days (in National Institute of Animal Health, Tsukuba, Japan, Italian language “quaranta giorni”). The popular personal communication in 2006). currently used term “quarantine” was derived from quaranta—forty—days. Presently, this term is in use PREVENTION AND CONTROL for any reasonable period of time and does not The outbreaks of fowl plague during the early part adhere to the originally recommended 40 days. of the 20th century were quite often self-limiting due The phase I quarantine consists of separation and to the extremely high mortality and culling of the observation of newly acquired birds and remained few remaining sick birds. A government imposed the method of choice from the very beginning of slaughter policy came in effect later (see later). fowl plague epizootics until direct assays for the Although epidemic diseases like rinderpest in Italy causative viruses and/or their serum antibodies were controlled since 1711 by clubbing of diseased became available to confi rm fowl plague–negative cattle (301), similar drastic measures were initially status. not imposed on fowl plague due to almost complete losses. Phase II The phase II quarantine contains, in addition to Trade and Quarantine separation and observation, the random testing of National and international trade in live poultry, selected newly obtained poultry for antibodies and/ hatching eggs, and day-old chicks was essential for or viruses using established laboratory methods. The the equal distribution of highly performing poultry, duration and the level of separation are variable and for the supply of the growing world population with depend on national legislation and recommendations valuable poultry meat and table eggs, and conse- of international bodies such as OIE, FAO, and quently for a fl ourishing poultry industry. However, WHO. Phase II testing for infl uenza A viruses is trade in live poultry especially has on occasions applied in low-risk situations and in surveillance been associated with the risk of spreading infectious studies of domestic and free-living birds. agents. Commercial trade in live infected but healthy-appearing poultry was recognized during Phase III the fi rst outbreaks of fowl plague as the predominant Phase III represents an additional expansion of mode of spread. As an initial countermeasure, quar- methods and aims. This was done in view of the antine of newly purchased poultry was recommended limitations in sensitivity and specifi city of testing (224, 249). The term “quarantine” is defi ned as (1) procedures and the a priori unknown prevalence a place or period of detention of animals coming rates of infectious agents in suspected poultry. The from infected or suspect sources and (2) restrictions sophistication consists of the addition of known placed on entering or leaving premises where a case antibody- and virus-free marked chickens to suspi- 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 175 cious fl ocks. For such chickens or other avian toxic compounds such as mercury chloride, phenols, species, the term “sentinels” was coined. The senti- and formaldehyde were used in the past without nels are intermingled with birds of doubtful origin sincere regard to environmental issues. in an attempt that the introduced birds pick up even low quantities of fowl plague virus of any subtype Attempts to Control Fowl Plague by and any level of virulence. The sentinel birds are Vaccination swabbed and bled at intervals. These samples will The practical value or nonvalue of any form of vac- test positive after some time if virus is present. The cination against fowl plague was extensively debated evaluation of the samples is assessed by recom- by Kitt (141–143), a professor of hygiene and mended laboratory methods. Phase III testing is pathology at the University of Munich, Germany. mandatory in situations where subclinical infections He concluded that vaccination should never be used with fowl plague viruses of low virulence are likely. and eradication should be the primary option to Also, virulent fowl plague viruses can be detected control fowl plague. Despite Kitt’s opinion, many in previously AI-vaccinated fl ocks. different attempts were made to provide protection to the highly susceptible chickens and turkeys. Cleansing and Disinfection The fi rst attempts to control fowl plague by vac- Contaminated cages and stables can be the source of cination were described by Maue (198) working in infection for poultry (117, 139, 224). Greve (102) Koch’s Institute of Hygiene in Berlin, Germany, and and Jess (129) recommended that cages be thor- by Kraus and Schiffmann (154) and Kraus et al. oughly washed and disinfected. The careful use of a (156), who worked in the serotherapeutic institute in 0.1% solution of mercury chloride is proposed by Vienna, Austria. These authors used spinal cord Krausz (158) for disinfection of fl oors, and a 5% tissues, which they obtained from experimentally carbol solution is proposed for contaminated walls. infected young geese. This tissue was dried over Krausz also recommends hot steam for cages. The potassium hydroxide for 2 to 10 days and thereafter disinfectants containing mercury chloride or carbol intramuscularly injected into susceptible young solution are also proposed by Künnemann (159) and geese. These “immunized” geese survived intramus- by von Ostertag (213). The application of a 1% solu- cular challenge with fully infectious untreated virus, tion of sulfuric acid, a 2% suspension of bleaching whereas young geese without such “immunization” powder, and 50% ethanol for 10 minutes at room died on fowl plague. To our knowledge, these exper- temperature completely destroys the infectivity of iments were never repeated or confi rmed by other fowl plague virus (17, 176, 242). investigators. Erdmann (77, 78), Jouan and Staub Due to the extreme toxicity of mercury chloride (132), Lagrange (163), Purchase (238), Smith et al. for animals and humans, this compound is presently (272), Spears (274), and Staub (276) conducted classifi ed as a major environmental hazard and com- vaccine experiments to protect chickens. A large pletely banned, whereas carbol solutions are occa- volume of 10 ml of blood or tissue homogenates was sionally still in use to destroy the highly resistant kept for 3 days at temperatures of 46º to 47º C and eggs of intestinal worms and coccidian oocysts. afterward injected into chickens or adult geese. The Weidenmüller (294) made comparative disinfection obtained serum samples were injected into suscep- experiments with the strain Herts, an ND virus, and tible chickens. Subsequent challenge with highly “N” virus, an LPAI virus of the subtype H10N7. The virulent blood demonstrated that the chickens sur- best inactivation results were obtained with 2% vived. Pfenninger (230) “mitigated” whole blood sodium hydroxide for 20 minutes at room tempera- with carbolic acid but did not gain promising ture. Less effective under these conditions were 70% rates of protection. Doerr and Zdansky (64) have or 95% ethanol, 3% phenol, and 1% to 2% formalin. shown that oral ingestion of large quantities of fowl The results of these tests indicate that the strain plague virus did not protect against intramuscular Herts is slightly more resistant to inactivation than challenge. “N” virus (294). The currently applied disinfectants Doyle (69), working in the Veterinary Laboratory contain chlorine, oxygen, or organic acids and are in Weybridge, United Kingdom, used formalized considered effective and safe and represent no homogenized spleen tissue from experimentally hazard to the environment. In retrospective, highly infected chickens for the induction of immunity in 176 Avian Influenza chickens. Such material was obviously effective in Beginning in 1909, any attempts to vaccinate or protecting chickens against severe challenge (108– to treat infected chickens or any other bird species 110). were illegal. This legislation was actually the end of Daubney et al. (52), working in the Serum Insti- any further vaccine development and testing for tute in Cairo, Egypt, started their vaccination exper- many decades to come. iments with formalized suspensions of embryos, embryonic membranes, and allantoic fl uids derived CONCLUSIONS from fowl plague virus–inoculated embryonated This review of fowl plague in Europe and Asia chicken eggs. The formalin-treated fl uids were between 1878 and 1955 is entirely based on publica- mixed with either aluminium hydroxide gel or tions in scientifi c journals and offi cial governmental mineral oil as adjuvants. Protection against statistical documents. In an attempt to place the challenge was obtained in 50 of 52 immunized spread of fowl plague in a historical perspective, the chickens. background of knowledge in natural sciences and The fi rst observations on possible variations or of microbial and other poultry diseases and the differences in virulence of fowl plague virus were early developments of breeding and trade of poul- published by Kraus and Loewy (153) and by Mac- try are briefl y described. At the second half of kenzie and Findlay (184) and subsequently con- the 19th century, fowl cholera (now Pasteurella fi rmed by Daubney and Ishak (53) and Hallauer multocida) was the predominant avian disease. (112, 113). Von Magnus (188, 189) was able to alter Perroncito (1878) studied a disease that was differ- viral properties during multiplication in embryo- ent from fowl cholera due to its very short incu- nated eggs. However, none of these studies led to bation period of only 1 day, due to the extremely the development of attenuated live virus vaccines. high rates of losses in chickens and turkeys at almost 100% level, and by its gross lesions of internal Legislation organs. The resistance of adult pigeons following The legislation on the control of fowl plague and infection with fowl plague virus was extensively fowl cholera has been in effect since 1909 in Austria used as a criterion to differentiate fowl cholera and Germany and at later times in almost all coun- from fowl pest. It was shown that blood of dead tries (31, 92, reviewed in 255). The aims of all chickens is more infectious than any other tissue. legislation were (1) detailed monitoring and mean- Using the rather crude criteria of resistance of ingful prevention of further spread, (2) burial or pigeons and the high infectivity of blood, many burning of carcasses and cleansing and disinfection investigators concluded that fowl cholera and fowl under veterinary surveillance, and (3) estimation of plague have different etiologies. Healthy chickens the level for compensation. were generally used as “gold standard” to measure Such legislation required immediate reporting of the infectivity of fowl plague virus in blood, internal questionable cases in poultry, diagnosis and subse- organs and feces. The conduct of in vivo experimen- quent stamping out of all diseased and contact fl ocks, tation but also histopathological, bacteriological, stand-still of all kinds of transport to and from and virological methods were at the beginning of the infected farms, burning of equipment, burying or fowl plague era still in its infancy. Consequently, burning of carcasses, and thorough disinfection of clearcut differentiation of fowl plague from fowl cages vehicles used for transport (31, 82, 85, 86). cholera and other diseases was occasionally diffi cult In Germany, fowl plague was declared to be a to obtain. notifi able disease by the chancellor of the German The chronologically grouped publications provide Empire in 1903. He was authorized to this tempo- evidence for the existence of three epidemics of fowl rary step, which included substantial measures for plague in Europe between 1878 and 1955. The fi rst control, by a special paragraph of the legislation of epidemic occurred between 1877 and 1880 and 1880. The addition of fowl plague to the existing list seemed to be restricted to northern Italy. In contrast of notifi able diseases was done with the reorganiza- to practices in previous centuries, the size of chicken tion of the German legislation in 1909. Practically fl ocks was expanded and farms were located outside all countries that were affected by fowl plague fol- of the boundaries of villages and in many cases close lowed similar rules (6). to open waters in the northern part of Italy. Thus, 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 177 intrusion of free-living birds and especially water- minium hydroxide gel or mineral oil as adjuvants. fowl was likely. Further development and testing of effi cacy of inac- The second epidemic between 1899 and 1906 tivated vaccines were not pursued after adopted started again in northern Italy and spread to a large legal requirements outlawed vaccination in favor of number of European countries. The demand for eradication. Italian white leghorn chickens was still high due to their excellent high performance in egg production. ACKNOWLEDGMENTS Fowl plague–infected chickens from Italy attended The authors thank Frau Sabine Sommer of the the poultry exhibition in Brunswick, Germany, library of the Stiftung Tierärztliche Hochschule which resulted in major lateral spread from infected Hannover, Prof. Dr. Lothar H. Wieler, director of to immunologically naïve birds and subsequently to the Institute of Microbiology and Epizootics, Vet- major outbreaks in Austria, Germany, and neighbor- erinary Faculty, Free University Berlin, and the staff ing countries. Experimental transmissions proved of the Library of the University of Frankfurt for their that chickens, turkeys, geese, and a large number of generous support during literature retrival. Sincere other avian species, including songbirds, birds of thanks go to Prof. Dr. Antonio Zanella, Brescia, prey, and owls, are susceptible to fowl plague virus Italy, Dr. Lorenzo Crosta, Como, Italy, Dr. D. and die after a period of less than 1 to 3 days. Young Demenechi, University of Milano, Italy, and Dr. pigeons develop transient forms of nervous disor- Bela Lomniczi, Veterinary Medical Research Insti- ders but recover, whereas adult pigeons are resistant tute of the Hungarian Academy of Sciences, Buda- to disease. Domestic ducks were described as com- pest, Hungary, for their generous support during the pletely resistant to infection. An overspill from dis- search for historical publications. We appreciate eased poultry to free-living birds was not recorded. the obtained curriculum vitae and the photograph The third epidemic between 1919 and 1931 had of Professor Edoardo Perroncito provided by Dr. again its index case in northern Italy and spread Stefano Benedetto and Adriana Viglino, Archivio from there due to trade with live chickens and pos- Storico della Cittá di Torino, Italy. We thank Prof. sibly contaminated equipment to other European Dr. Yoshihiro Kawaoka, University of Tokyo, Japan, countries. Dominant routes of spread at national and Dr. T. S. Tsim and Dr. W. H. Lee, Kowloon, Hong international levels were the abundant trade in live Kong SAR, Dr. Yu Kangzhen, Beijing, and Dr. poultry. At local level, the modes of spread include Zhiming Pan, Yangzhou, PR China, for their advice besides trade in live poultry also contaminated on obtaining Japanese and Chinese references. equipment, vehicles (including railroad transport), We wish to thank also Dr. Katja Trinkaus for her vermin and rodents, and, in some instances, open skillful translations of publications from Italian to waters and small rivers. German language and Dagmar Sommer for proof- Records on the detection of fowl plague in Asian reading of the manuscript. countries are sparse. Only one publication from Japan in 1942 that affected chickens and acciden- REFERENCES tally also domestic cats was observed. The discovery 1. Abba, F. 1899. Tifo essudativo. Giornale della of ND in 1927 resulted in widely present confusion Reale Accademia di Medicina di Torino 62:182– with fowl plague. 189. During all three European epidemics, attempts 2. Abba, F. 1901. Comunica di avere isolato da due were made to reduce the risks of further spread by polli il bacillo del colèra dei polli. Giornale della culling of the remaining few survivors of outbreaks Reale Accademia di Medicina di Torino 64:15– and disinfection of premises and equipment using 16. 3. Abbe, E. 1869. Kondensor. Available at http:// mercury chloride, ethanol, phenols, and carbolic www.zeiss.de/de/home.nsf. Carl Zeiss AG, Jena, acid. Also, fi rst vaccination trials were described. Germany. Accessed in August 2006. Initially, dried or heat-treated virus-containing 4. Achilles, W. 1993. Die deutsche Agrargeschichte tissues (mainly liver and blood) derived from suc- im Zeitalter der Reformen und Industrialisierung. cumbed chickens were used. Around 1930, the fi rst Eugen Ulmer Verlag: Stuttgart. inactivated vaccines were produced with formalized 5. Aggazoni, A. 1901. Infl uenza della razza sulla allantoic fl uids of infected chicken embryos and alu- resistenza dei colombi alle depressioni barometri- 178 Avian Influenza

che. Comptes Rendus du Congrès Internationale Federviehzucht vom wirtschaftlichen Stand- de Physiologie de Turin. punkte, Schönfeld’s Verlagsbuchhandlung: Dresden, 6. Alexander, D.J. 1981. Current situation of avian pp. 193–194. infl uenza in Great Britain. In: R.W. Bankowski 16. Baudet, E.A.R.F. 1924. Hoenderpest. Tijdschrift (ed.). Proceedings of the First International Sym- voor Diergeneeskunde 51:791–793. posium on Avian Infl uenza, April 1981, Belts- 17. Baumann, R. 1928. Desinfektionsversuche bei ville, Maryland. Symposium on Avian Infl uenza, Gefl ügelpest mit Sulfoliquid DS. Archiv für US Animal Health Association: Richmond, VA, Wissenschaftliche und Praktische Tierheilkunde pp. 35–45. 57:110–112. 7. Alexander, D.J. 1986. Avian infl uenza—histori- 18. Beaudette, F.R. 1925. Observations upon fowl cal aspects. In: B.C. Easterday and C.W. Beard plague in New Jersey. Journal of the American (eds.). Proceedings of the Second International Veterinary Medical Association 67:186–194. Symposium on Avian Infl uenza, Georgia Center 19. Bechhold, H., and M. Schlesinger. 1931. Zentri- for Continuing Education, the University of fuge und Filter zur Bestimmung der absoluten Georgia, Athens, Georgia, USA, September 3–5, Größe von subvisiblen Erregern (Pockenvakzine 1986. Symposium on Avian Infl uenza, US Animal und Hühnerpest). Zeitschrift für Hygiene und Health Association: Richmond, VA, pp. 4–13. Infektionskrankheiten, Medizinische Mikrobi- 8. Alexander, D.J. 2003. Newcastle disease. In: ologie, Immunologie und Virologie 112:668– Y.M. Saif, H.J. Barnes, J.R. Glisson, A.M. Fadly, 679. L.R. McDougald, and D.E. Swayne (eds.). Dis- 20. Belfanti, S., and C. Zenoni. 1899. Sulla recente eases of Poultry, 11th ed. Iowa State University epizoozia dei polli in Lombardia. Giornale della Press: Ames, IA, pp. 64–87. Reale Accademia di Medicina di Torino 62:533– 9. Allan, W.H. 1981. Uncomplicated infection with 543. virulent strains of avian infl uenza viruses. In: R. 21. Belfanti, S., and A. Ascoli. 1916. Spigolature W. Bankowski (ed.). Proceedings of the First nella peste aviaria e nell’ “afta.” La Clinica Vete- International Symposium on Avian Infl uenza, rinaria, Milano 39:577–597. April 1981, Beltsville, Maryland. Symposium on 22. Bittner, H. 1926. Tierärztliche Tätigkeit und Avian Infl uenza, US Animal Health Association: tierärztlicher Stand in Bulgarien. Berliner Tierärz- Richmond, VA, pp. 4–7. tliche Wochenschrift 42:565–568. 10. Ambrosioni, P. 1938. Coltura del virus della peste 23. Blancke, B. 1921. Geschichtliches. In: B. Blancke aviare sulla “membrana chorion-allantoidea” dell (ed.). Unser Hausgefl ügel, 4th ed. Verlag Fritz embrione di pollo. La Clinica Veterinaria, Milano Pfenningstorff: Berlin, pp. 1–6. 61:539–541. 24. Blaxland, J.D., and R.F. Gordon. 1945. Zinc 11. Andriewsky, P. 1914. L’ultrafi ltration et les phosphide poisoning in poultry. The Veterinary microbes invisible. I. Communication: La peste Journal 101:108–110. des poules. Zentralblatt für Bakteriologie, Para- 25. Blood, D.C., and V.P. Studdert. 1988. Bailliére’s sitenkunde und Infektionskrankheiten. I. Abteilung, Comprehensive Veterinary Dictionary. Bailliére Originale 75:90–93. Tindall: London. 12. Angeloff, O.V. 1939. Das Veterinärwesen 26. Blum, F. 1893. Der Formaldehyd als Härtungsmit- und die Tierseuchenbekämpfung in Bulgarien. tel. Zeitschriftfür Wissenschaftliche Mikroskopie Deutsche Tiers¨rztliche Wochenschrift 47:292– 10:314–315. 293; 717–722. 27. Boehmer, F. 1865. Zur pathologischen Anatomie 13. Aristotle 1995. History of animals. In J. Barnes der Meningitis cerebromedullaris epidemica. (ed.). The Complete Works of Aristotle, the Ärztliches Intelligenzblatt für Bayern 12:539– Revised Oxford Translation. Princeton Univer- 550. sity Press: Chichester, West Sussex, UK, pp. 28. Bolte, A.L., M. Voss, E. Vielitz, and E.F. Kaleta. 774–993. 2001. Response of domestic geese to lentogenic 14. Bähr, J. 1997. Bevölkerungsverteilung und Bev- and velogenic strains of Newcastle disease virus. ölkerungsstruktur. In J. Bähr (ed.). Bevölkerungs- Deutsche Tierärztliche Wochenschrift 108:155– geographie. 3rd ed. Eugen Ulmer Verlag: 159. Stuttgart, pp. 29–172. 29. Boughton, I.B., and E.A. Tunnicliff. 1925. 15. Baldamus, A.C.E. 1876. Cholera—chicken European fowl pest in Illinois. The Journal of the cholera. In A.C.E. Baldamus (ed.). Illustriertes American Veterinary Medical Association 67: Handbuch der Federviehzucht. Band 1: Die 183–185. 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 179

30. Brieg, A. 1919. Übersicht über die wichtigsten 43. Cernaianu, C., and I. Popovici. 1944. Beiträge Krankheiten des Federviehs mit besonderer zum Studium des Gefl ügelpestvirus und der von Berücksichtigung der pathologischen Anatomie. diesem Virus in Rumänien in den Jahren 1941 Deutsche Tierärztliche Wochenschrift 27:309– bis 1944 bedingten Krankheit [The fowl plague 310. (Newcastle disease) outbreak in Rumania 1941– 31. Brühann, W. 1983. Geschichtlicher Überblick. 1944]. Archiv für die Gesamte Virusforschung In: W. Brühann (ed.). Das öffentliche Vete- 3:231–249. rinärwesen. Verlag Paul Parey: Berlin, pp. 1– 44. Chu, C.M. 1948. Agglutination of red blood cells 54. of different animal species by infl uenza and 32. Brusaferro, S. 1901. Un epizoozia dei polli nelle Newcastle disease viruses. Journal of Hygiene province di Parma e di Reggio. La Clinica Vete- 46:239–246. rinaria, Milano 24:49–54; 61–66; 77–81. 45. Collier, W.A. 1932. Übertragung des Gefl ügel- 33. Burnet, F.M., and J.D. Ferry. 1934. The differen- pestvirus auf Mäusegehirn und Rattenhoden. tiation of fowl plague and Newcastle disease: Zeitschrift für Hygiene und Infektionsk- experiments using the technique of chorio-allan- rankheiten, medizinische Mikrobiologie, Immu- toic membrane inoculation of the developing egg. nologie und Virologie 113:751–753. British Journal of Experimental Pathology 15:56– 46. Columella, L.I.M. 1982. Achtes Buch. Über die 64. Haltung von Haushühnern und die Einrichtung 34. Burnet, F.M. 1936a. Infl uenza on the developing der HÜhnerställe. In W. Richter (translator and egg. I. Changes associated with the development editor). Zwölf Bücher über Landwirtschaft. of egg-passage strain of virus. British Journal of Artemis: Munich and Zurich, pp. 229–307. Experimental Pathology 17:282–293. 47. Comberg, G. 1984. Die deutsche Tierzucht im 35. Burnet, F.M. 1936b. Infl uenza on the developing 19. und 20. Jahrhundert. Eugen Ulmer Verlag: egg. II. Titration of egg passage virus by the Stuttgart. pock-counting method. Australian Journal of 48. Cominotti, L. 1916. Peste aviaria nelle anitre. La Experimental Biology and Medical Sciences Clinica Veterinaria, Milano 39:129–135. 14:241–246. 49. Cooper, H. 1931. Ranikhet disease: a new disease 36. Burnet, F.M. 1937. Infl uenza virus on developing of fowls in India due to a fi lter-passing virus. egg. IV. The pathogenicity and immunising Indian Journal of Veterinary Science and Animal power of egg virus for ferrets and mice. British Husbandry 1:107–123. Journal of Experimental Pathology 18:37–43. 50. Cornil, A.-V. 1882. Observations histologiques 37. Calamida, D. 1904. Beitrag zum Studium der sur les lésions des muscles déterminanées par Natur der Hühnerseuchen. Zentralblatt für l’injection du microbe du choléra des poules. Bakteriologie, Parasitenkunde und Infektionsk- Archives de Physiologie 10:615–643. rankheiten. I. Abteilung, Originale 35:37–42. 51. Crawford, R.D. 1990. Origin and history of 38. Caporale, G. 1943. Gegenüberstellung der histo- poultry species. In R.D. Crawford (ed.). Poultry pathologischenVeränderungen am Zentralner- breeding and genetics. Elsevier: Amsterdam, 1– vensystem bei Gefl ügelpest und “Hühnerseuche 41. 1940.” Berliner und Münchener Tierärztliche 52. Daubney, R., W. Mansi and G. Zahran. 1949. Wochenschrift 59:390–393. Vaccination against fowl plague. The Journal of 39. Ceconi, A. 1901. Sinopsie. Giornale della Reale Comparative Pathology and Therapeutics 59:1– Accademia di Medicina di Torino 7:15–16. 18. 40. Centanni, E., and E. Savonuzzi. 1901. La peste 53. Daubney, R., and G. Ishak. 1953. Mutations in aviaria. La Clinica Veterinaria, Milano 24:292– pathogenicity of some recently isolated strains of 295; 305–307; 314–317; 323–326. fowl-plague virus. Journal of Comparative Pathol- 41. Centanni, E. 1902. Die Vogelpest. Beitrag zu dem ogy and Therapeutics 63:255–282. durch Kerzen fi ltrierbaren Virus. Zentralblatt für 54. Dawson, J.M., and W.J. Elford. 1949. The inves- Bakteriologie, Parasitenkunde und Infektionsk- tigation of infl uenza and related viruses in the rankheiten. I. Abteilung, Originale 31:145–152; electron microscope by a new technique. Journal 182–201. of General Microbiology 3:298–311. 42. Centanni, E., and I. Prampolini. 1901. Sopra una 55. De Blieck, L. 1925. Untersuchung der singolare localizzazione del virus della peste Gefl ügelkrankheiten in den Niederlanden. aviaria nei piccioni. Gazzetta degli Ospedalie Deutsche Tierärztliche Wochenschrift 33:908– delle Cliniche 87:912. 910. 180 Avian Influenza

56. Demnitz, A., and B. Schneider. 1947. Die Dif- Comparative Pathology and Therapeutics 40:144– ferentialdiagnose zwischen der klassischen und 169. atypischen Gefl ügelpest mit Hilfe der Hämagglu- 70. Dubois, A. 1902. Une maladie infectieuse des tinations-Hemmung. Monatshefte für Tierhe- poules à microbes invisibles. Comptes Rendus ilkunde 2:205–208. des Séances de la Société de Biologie et de ses 57. Depperich, C. 1907. Beiträge zur Kenntnis der Filiales 71:1162–1163. “neuen Hühnerseuche” (Hühnerpest). Fortschritte 71. Eckert, J. 1957. Epizootiologie und Seuchenkarte der Veterinär-Hygiene 4:217–226; 244–250. der Gefl ügelpest in Europa. Veterinary Disserta- 58. Dinter, Z. 1944. Vergleichende Untersuchungen tion, Tierärztliche Hochschule Hannover. über die atypische und klassische Gefl ügelpest. 72. Eggebrecht, M. 1909. Über ein seuchenartiges Archiv für die Gesamte Virusforschung 3:207– Hühnersterben. Zeitschrift für Infektionsk- 219. rankheiten der Haustiere 5:453–458. 59. Dinter, Z. 1949a. Eine Variante des Virus der 73. Elford, W.J., and C. Todd. 1933. The size of the Gefl ügelpest in Bayern? Tierärztliche Umschau virus of fowl-plague estimated by the method of 4:185–186. ultrafi ltration analysis. British Journal of Experi- 60. Dinter, Z. 1949b. Weitere Untersuchungen über mental Pathology 14:240–246. das Virus N. Tierärztliche Umschau 4:229–231. 74. Elford, W. J., C.M. Chu, I.M. Dawson, J.A. 61. Dinter, Z., and K. Bakos. 1950. Über die Bezie- Dudgeson, F. Fulton, and J. Smiles. 1948. Physi- hungen des Virus N zu dem Virus der klassischen cal properties of the viruses of Newcastle disease, Gefl ügelpest. Berliner und Münchener Tierärztli- fowl plague and mumps. British Journal of che Wochenschrift 63:101–105. Experimental Pathology 29:590–599. 62. Dinter, Z. 1952. Über einige Virusarten der Infl u- 75. Enders, P. 1902. Beiträge zur Kenntnis einer enzagruppe. Berliner und Münchener Tierärztli- neuen Infektionskrankheit—Phasianidenseuche, che Wochenschrift 65:241–243. Phasianidenseptikämie, Darmseuche, Intesti- 63. Doerr, R., and A. Pick. 1915. Untersuchungen nalmycose—der echten Hühner (Phasianiden). über das Virus der Hühnerpest. Zentralblatt für Berliner Thierärztliche Wochenschrift 14:339– Bakteriologie, Parasitenkunden und Infektionsk- 342; 362–366; 374–379; 389–390. rankheiten. I. Abteilung, Originale 76:476–494. 76. Ercolani, G.B. 1861. Epizoozie tifi che. In: Delle 64. Doerr, R., and E. Zdansky. 1924. Untersuchun- Mallattie Degli Uccelli Domestici. Giornale di gen über das Virus der Hühnerpest. III. Medicina Veterinaria, pp. 92–106. Mitteilung. Zeitschrift für Hygiene und Infek- 77. Erdmann, R. 1916. Attenuation of the living tionskrankheiten, Medizinische Mikrobiologie, agents of cyanolophia. Proceedings of the Society Immunologie und Virologie 101:125–139. for Experimental Biology and Medicine 13:189– 65. Doerr, R., S. Seidenberg, and L. Whitman. 1931. 139. Untersuchungen über das Virus der Hühnerpest. 78. Erdmann, R. 1920. Immunisierung gegen Hüh- IV. Mitteilung. Zeitschrift für Hygiene und nerpest. Archiv für Protistenkunde 41:190–241 Infektionskrankheiten, Medizinische Mikrobiolo- +8 drawings. gie, Immunologie und Virologie 112:732–753. 79. Farinas, E.C. 1930. Avian pest, a disease of birds 66. Doerr, R., and E. Gold. 1932. Untersuchungen hitherto unknown in the Philippine Islands. Phil- über das Virus der Hühnerpest. V. Mitteilung. ippine Journal of Agriculture 1:311–375. Zeitschrift für Hygiene und Infektionskrank- 80. Findlay, G.M., and R.D. Mackenzie. 1937. The heiten, Medizinische Mikrobiologie, Immunolo- histopathology of fowl pest. British Journal of gie und Virologie 113:645–670. Experimental Pathology 18:146–160. 67. Doerr, R., and S. Seidenberg. 1932. Untersuc- 81. Fiorentini, A. 1896. Hämorrhagische Septikämie hungen über das Virus der Hühnerpest. VI. der Schwäne. Zentralblatt für Bakteriologie, Mitteilung. Zeitschrift für Hygiene und Infek- Parasitenkunde und Infektionskrankkheiten. I. tionskrankheiten, Medizinische Mikrobiologie, Abteilung, Originale 19:932–936. Immunologie und Virologie 113:671–681. 82. Flückiger, G. 1950. Bericht. Internationale 68. Doll, P. 1981. Das 20. Jahrhundert beginnt. In P. Bekämpfung der Gefl ügelpest. Schweizer Archiv Doll (ed.). Chronik des Bundes Deutscher für Tierheilkunde 92:657–661. Rassegefl üügelzÜchter e. V. Oertel und Spoerer: 83. Foà, P., and A. Cesaris-Demel. 1899. Sulla Reutlingen, pp. 16–31. recente epizoozia dei polli in vari paesi del 69. Doyle, T.M. 1927. A hitherto unrecorded disease Piemonte. Giornale della Reale Accademia di of fowls due to a fi lter-passing virus. Journal of Medicina di Torino 62:253–256. 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 181

84. Freese, R. 1908. Über Hühnerpest mit besonderer und Infektionskrankheiten. I. Abteilung, Origi- Berücksichtigung der pathologischen Anatomie. nale 37:308–311. Deutsche Tierärztliche Wochenschrift 16:173– 96. Giemsa, G., and S. Prowazek. 1908. Weitere 177. Untersuchungen über sogenannte ultramikros- 85. Frei, W. 1921. Die Zuverlässigkeit der patholo- kopische Infektionserreger. Zur Filtration des gisch-anatomischen Diagnose bei Seuchen. Sch- Hühnerpestvirus. Münchener Medizinische weizer Archiv für Tierheilkunde 63:391–419. Wochenschrift 55:1524–1525. 86. Fröhner, E. 1901. Veterinärpolizeiliche Überwa- 97. Gink, van, C.S.Th. 1992. Poultry Paintings 1890– chung von Gefl ügelausstellungen. Deutsche Thi- 1968. Stichting Fonds vor Pluimveebelangen, erärztliche Wochenschrift 9:186–187. NHDB, the Netherlands. 87. Fukushima, T. 1933a. Zur Frage der Nekrosebil- 98. Glisson, J.R., C.L. Hofacre, and J.P. Christensen. dung bei Hühnerpest-Enzephalitis. Deutsche 2003. Fowl cholera. In: M. Hofstad, B.W. Calnek, Tierärztliche Wochenschrift 41:177–179. C.F. Helmboldt, W.M. Reid, and H.W. Yoder, Jr. 88. Fukushima, T. 1933b. Zur Kenntnis der Hühner- (eds.). Diseases of Poultry, 11th ed. Iowa State pest-Pathologie (Zugleich ein Aufschluss über University Press: Ames, IA, pp. 658–676. die in Korea auftretenden Hühnerseuchen) 99. Gram, H.C. 1884. Über die isolirte Färbung der (German with Japanese summary). Journal of the Schizomyceten in Schnitt- und Trockenpräparaten. Japanese Society of Veterinary Sciences 12:147– In: Fortschritte der Medicin. 2:185–189. 165. 100. Gratzl, E., and H. Köhler. 1967. Gefl ügelpest. In: 89. Fukushima, T., and K. Shimomura. 1933. Beiträge E. Gratzl and H. Köhler (eds.). Spezielle Patholo- zur pathologisch-anatomischen Kenntnis der gie und Therapie der Gefl ügelkrankheiten. Ferdi- Farinasschen avian pest, insbesondere zur Ver- nand Enke Verlag: Stuttgart, pp. 1–2. gleichung mit solchen wie der in Korea auftreten- 101. Grausgruber, W. 1958. Untersuchungen zum den Hühnerseuche. (German with Japanese biologischen und serologischen Nachweis des summary). Journal of the Japanese Society of Virus der Newcastle-Krankheit und der klas- Veterinary Sciences 12:1–6. sischen Gefl ügelpest. Wiener Tierärztliche 90. Fukushima, T., K. Shimomura, and S. Oyama. Wochenschrift 45:76–114. 1933. Pathologische Anatomie der durch ein fi l- 102. Greve, L. 1901. Beobachtungen über eine von der trierbares Virus verursachten Hühnerseuche in Braunschweiger Gefl ügelausstellung in die Stadt Korea (German with Japanese summary). Journal und das Amt Oldenburg eingeschleppte Hühner- of the Japanese Society of Veterinary Sciences seuche. Deutsche Thierärztliche Wochenschrift 12:302–316. 9:373–376. 91. Gerlach, A.C. 1853. Der Thierarzt in socialer und 103. Gross, W.B. 1978. Spirochetosis. In M.S. Hofstad, gewerblicher Beziehung. In: A.C. Gerlach (ed.). B.W. Calnek, C.F. Helmboldt, W.M. Reid, and Lehrbuch der allgemeinen Therapie für Thi- H.W. Yoder Jr. (eds.). Diseases of Poultry, 7th erärzte, August Hirschfeld Verlag: Berlin, pp. ed. Iowa State University Press: Ames, IA, pp. 7–62. 330–334. 92. Gerlach, F. 1927. Gefl ügelpest. In: W. Kolle, R. 104. Haber, P. 1937. Affi nité du virus de la peste Kraus, and P. Uhlenhuth (eds.). Handbuch der aviaire (souche daptée a souris) pour l’embryon Pathogenen Mikroorganismen, Vol. 9. Gustav de poulet. Comptes Rendus des Séances de la Fischer Verlag: Jena, pp. 165–187. Societé de Biologie et de ses Filiales 126:885– 93. Gerlach, F., and J. Michalka. 1926. Über die im 886. Jahre 1925 in Österreich beobachtete Gefl ügel- 105. Habicht, M.R., and Struwe. 2006. Zur Haltung pest. Deutsche Tierärztliche Wochenschrift 34: und Nutzung von Gefl ügel von der Antike bis 897–902. zur Neuzeit. Tierärztliche Umschau 61:207– 94. Giemsa, G. 1902. Färbemethoden für Malariapar- 216. asiten. Zentralblatt für Bakteriologie, Parasiten- 106. Halász, F. 1911. Adatok a tyúkpestes ismere- kunde und Infektionskrankheiten. I. Abteilung, téhez. (Beiträge zur Kenntnis der Gefl ügelpest) Originale 32:307–313. [Data of the knowledge of hen pest] (Hungarian 95. Giemsa, G. 1904. Eine Vereinfachung und with German summary). Veterinary Dissertation, Vervollkommnung meiner Methylenazur- University of Budapest, Hungary. Methylenblau-Eosin-Färbemethode zur Erzielung 107. Hallauer, C. 1931. Über das Verhalten von Hüh- der Romanowsky-Nochtschen Chromatinfärbung. nerpestvirus in der Gewebekultur. Zeitschrift für Zentralblatt für Bakteriologie, Parasitenkunde Hygiene und Infektionskrankheiten, Medizinische 182 Avian Influenza

Mikrobiologie, Immunologie und Virologie 120. Hertel, M. 1904. Über Gefl ügelcholera und Hüh- 113:61–74. nerpest. Arbeiten aus dem Kaiserlichen Gesund- 108. Hallauer, C. 1934. Immunitätsstudien bei Hüh- heitsamte 20:453–511. nerpest. I. Mitteilung. Zeitschrift für Hygiene und 121. Hindle, E. 1912. Attempts to transmit “fowl pest” Infektionskrankheiten, Medizinische Mikrobiolo- by Argas persicus. Bulletin de la Societé de gie, Immunologie und Virologie 116:456–479. Pathologie Exotique 26:165–167. 109. Hallauer, C. 1935. Immunitätsstudien bei Hüh- 122. Hirst, G.K. 1941. The agglutination of red cells nerpest. II. Mitteilung. Über das Schicksal von by allantoic fl uid of chick embryos infected with Hühnerpestvirus im immunisierten Tierkörper. infl uenza virus. Science 94:22–23. Zeitschrift für Hygiene und Infektionsk- 123. Hoare, E.W. 1913. Avian plague. In: E.W. Hoare rankheiten, Medizinische Mikrobiologie, Immu- (ed.). A system of veterinary medicine. Vol. 1. nologie und Virologie 117:451–469. Microbial diseases. Bailliére, Tindall and Cox: 110. Hallauer, C. 1936. Immunitätsstudien bei Hüh- London, pp. 484–493. nerpest. III. Mitteilung. Über aktive Immunisier- 124. Hotz, G., and W. Schäfer. 1955. Ultrahistolo- ung mit formalinisiertem Virus. Zeitschrift für gische Studie über die Vermehrung des Virus der Hygiene und Infektionskrankheiten, Medizinische klassischen Gefl ügelpest. Zeitschrift für Natur- Mikrobiologie, Immunologie und Virologie 117: forschung 10b:1–5. 711–721. 125. Hutyra, F., and J. Marek. 1920. Gefl ügelpest. In: 111. Hallauer, C., and S. Seidenberg. 1937. Beitrag zur F. Hutyra and J. Marek (eds.). Spezielle Patholo- Epidemiologie der Hühnerpest. Zeitschrift für gie und Therapie der Haustiere, Vol. 1, 5th ed. Hygiene und Infektionskrankheiten, Medizinische Gustav Fischer Verlag: Jena, pp. 306–311. Mikrobiologie, Immunologie und Virologie 126. Ilieff, T. 1944. Sur la nature d’une maladie à 120:110–120. virus, sévissant dans divers pays d’Europe et en 112. Hallauer, C. 1939. Studien über die Variabilität Bulgarie, semblable à la peste aviaire. Annuaire des Hühnerpestvirus im Gewebeexplantat. Archiv de l’Université de Sofi a 20:1944–1945; 76, ref. in für die Gesamte Virusforschung 1:70–84. Bulletin de l’Offi ce International des Epizooties 113. Hallauer, C. 1947. Beitrag zur Klassifi kation der 27:320–322 (1947). Gefl üügelpeststämme. Archiv für die Gesamte 127. Jansen, J., and O. Nieschulz. 1933. Over het Virusforschung 3:356–374. infecteeren van muizen met hoenderpestvirus. 114. Hallauer, C., and G. Kronauer. 1960. Immunisier- Tijdschrift voor Diergeneeskunde 60:245–248. ungsversuche mit experimentell induzierten 128. Jansen, J., and O. Nieschulz. 1934. Over de Varianten des klassischen und atypischen gevoeligheid van ratten voor het hoenderpes- Gefl ügelpestvirus. Archiv für die Gesamte Virus- tvirus. Tijdschrift voor Diergeneeskunde 61:15– forschung 10:46–71. 17. 115. Hanson, R.P. 1978. Newcastle disease. In: M.S. 129. Jess, P. 1901. Die Braunschweiger Hühnerseuche. Hofstad, B.W. Calnek, F. Helmboldt, W.M. Reid, Zentralblatt für Bakteriologie, Parasitenkunde and H.W. Yoder, Jr. (eds.). Diseases of Poultry, und Infektionskrankheiten, I. Abteilung, Origi- 7th ed. Iowa State University Press: Ames, IA, nale 29:755–757. pp. 513–535. 130. Joest, E. 1902. Beitrag zur Kenntnis der Bak- 116. Hecker, C. 1901. Gefl ügelseuche. Deutsche Thi- terienfl ora des Hühnerdarmes nebst einigen erärztliche Wochenschrift 9:110. Bemerkungen über eine neue Hühnerseuche. Ber- 117. Heelsbergen, van, T. 1927. Ervaring opgedaan bij liner Thierärztliche Wochenschrift 18:241–243. het onderzoek van pluimveeziekten in Nederland. 131. Johnson, S.R. 1925. European fowl pest in Mich- Vogelpest. Tijdschrift voor Diergeneeskunde 54: igan. Journal of the American Veterinary Medical 1–4. Association 67:195–202. 118. Heelsbergen, van, T. 1929. Gefl ügelpest. In: 132. Jouan, C., and A. Staub. 1920. üÈtude sur la peste T. van Heelsbergen (ed.). Handbuch der aviaire. Annales de I’Institute Pasteur 34:343– Gefl ügelkrankheiten und der Gefl ügelzucht. 357. Ferdinand Enke Verlag: Stuttgart, pp. 279–295. 133. Jungherr, E.L., E.E. Tyzzer, C.A. Brandly, and 119. Henle, J.F.G. 1840. Von den Miasmen und Con- H.E. Moses. 1946. The comparative pathology of tagien und von den miasmatisch-contagiösen fowl plague and Newcastle disease. American Krankheiten. In: J.F.G. Henle (ed.). Pathologische Journal of Veterinary Research 7:250–288. Untersuchungen. August Hirschwald: Berlin, pp. 134. Kaleta, E.F., and C. Baldauf. 1988. Newcastle 1–45. disease in free-living and pet birds. In: D.J. Alex- 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 183

ander (ed.). Newcastle disease. Kluwer Academic brandinfection durch Fütterung. Arbeiten aus Publishers: Doordrecht, pp. 197–227. dem Kaiserlichen Gesundheitsamte 2:147–161. 135. Kaleta, E.F. 1995. From roots to fruits of contem- 148. Koch, R., and Rabinowitsch, M. 1907. Die Tuber- porary avian medicine. Jahrbuch für Papageien- kulose der Vögel und ihre Beziehungen zur kunde 1:163–184. Säugetiertuberkulose. Virchows Archiv 190, 136. Kaleta, E.F., and A. Hönicke, A. 2004. Review Beiheft 246. of the literature on avian infl uenza A viruses in 149. Komarov, A. 1934. Study on “cell inclusion pigeons and experimental studies on the suscep- disease” in fowls. 1. On the identity of acute cell- tibility of domestic pigeons to infl uenza A viruses inclusion disease and fowl plague. Journal of of the haemagglutinin subtype H7. Deutsche Comparative Pathology and Therapeutics 47:282– Tierärztliche Wochenschrift 111:467–472. 295. 137. Kaleta, E.F., G. Hergarten, and A. Yilmaz. 2005. 150. Konno, T., Y. Ochi, and K. Hashimoto. 1929. Avian infl uenza A viruses in birds—an ecologi- Neue Gefl ügelseuche in Korea. Deutsche Tierärz- cal, ornithological and virological view. Deutsche tliche Wochenschrift 37:515–517. Tierärztliche Wochenschrift 112:448–456. 151. Kraneveld, F.C. 1926. Korte medeelingen uit het 138. Kaleta, E.F., and A. Hönicke. 2005. A retrospec- laboratorium voor veearssennijkundig ondersoek tive description of a highly pathogenic avian to Buitenzorg. Nederlandsch-Indische Bladen infl uenza A virus (H7N1/Carduelis/Germany/72) voor Diergeneeskunde 38:448–451. in a free-living siskin (Carduelis spinus Linnaeus, 152. Kraus, R., and O. Loewy. 1905. Ueber Hühner- 1758) and its accidental transmission to yellow pest. 1. Mitteilung. Zentralblatt für Bakteriologie, canaries (Serinus canaria Linnaeus, 1758). Parasitenkunde und Infektionskrankheiten. I. Deutsche Tierärztliche Wochenschrift 112:17– Abteilung, Originale 38:540–••. 19. 153. Kraus, R., and O. Loewy. 1907. Ueber Hühner- 139. Kaupp, B.F. 1917. Chicken cholera—fowl pest. 2. Mitteilung. Zentralblatt für Bakteriologie, cholera. In: B.F. Kaupp (ed.). Poultry Diseases, Parasitenkunde und Infektionskrankheiten. I. 2nd ed. American Veterinary Publishing: Chicago, Abteilung, Originale 43:343–348. pp. 106–111. 154. Kraus, R., and J. Schiffmann. 1907. Studie über 140. Kiernan, J.A. 1999. Histological and Histo- Immunisierung gegen das Virus der Hühnerpest. chemical Methods, Theory and Practice, 3rd ed. 1. Die aktive Immunisierung der Gans. Zentralb- Arnold: London. latt für Bakteriologie, Parasitenkunde und Infek- 141. Kitt, T. 1886. Werth und Unwerth der Schutzimp- tionskrankheiten. I. Abteilung, Originale 43: fungen gegen Thierseuchen. Berlin, p. 64. 824–832. 142. Kitt, T. 1894. Zur Kenntnis der Immunitätsver- 155. Kraus, R., and R. Doerr. 1908. Ueber das Ver- hältnisse bei der Gefl ügelpest. Monatshefte für halten des Hühnerpestvirus im Zentralnerven- praktische Thierheilkunde 5:198–200. system empfänglicher, natürlich und künstlich 143. Kitt, T. 1908. Lombardische Hühnerpest. In: T. unempfänglicher Tiere. Zentralblatt für Bakteri- Kitt (ed.). Bakterienkunde und pathologische ologie, Parasitenkunde und Infektionskrank- Mikroskopie, 5th ed. Verlag Moritz Perles: Wien, heiten. I. Abteilung, Originale 46:709–715. pp. 245–246. 156. Kraus, R., V. Eisler., and P. Fukuhara. 1909. 144. Klee, R. 1910. Die Lombardische Hühnerpest. In: Ueber Adsorption des fi ltrierbaren Virus. R. Klee (ed.). Die hauptsächlichsten Gefl ügel- Zeitschrift für Immunitätsforschung 1:307–315. Krankheiten, 4th ed. Expedition der Gefl ügel- 157. Kraus, R., and O. Loewy. 1915. Ueber Hühner- Börse: Radebeul, pp. 14–16. pest. 3. Mitteilung: Ueber die Variabilität des 145. Kleine, F.K. 1905. Neue Beobachtungen zur Hühnerpestvirus. Zentralblatt für Bakteriologie, Hühnerpest. Zeitschrift für Hygiene und Infek- Parasitenkunde und Infektionskrankheiten. I. tionskrankheiten, Medizinische Mikrobiologie, Abteilung, Originale 76:343–348. Immunologie und Virologie 51:177–182. 158. Krausz, A. 1901. Ueber eine bisher nicht bes- 146. Kleine, F.K., and B. Moellers. 1905. Über Hüh- chriebene Hühnerepizootie. Zentralblatt für nerpest bei Gänsen. Zentralblatt für Bakteriolo- Bakteriologie, Parasitenkunde und Infektion- gie, Parasitenkunde und Infektionskrankheiten. I. skrankheiten. I. Abteilung, Originale 29:980– Abteilung, Originale 39:545–549. 982. 147. Koch, R., G. Gaffky, and F.A.J. Loeffl er. 1882. 159. Künnemann, O. 1902. Beobachtungen über die Experimentelle Studien Über die künstliche Vogelpest. Deutsche Tierärztliche Wochenschrift Abschwächung der Milzbrandbacillen und Milz- 10:413–415; 421–424. 184 Avian Influenza

160. Kuiken, T., G. Rimmelzwaan, D. van Riel, G. van Hebdomadaires des séances de l’Académie des Amerongen, M. Baars, R. Fouchier, and A. Oster- Sciences 202:2018–2020. haus. 2004. Avian H5N1 infl uenza in cats. Science 173. Lipschütz, B. 1909. Ueber mikroskopisch sicht- 306:241. bare, fi ltrierbare Virusarten (Ueber Strongylo- 161. Kujumgiev, J. 1948. La peste aviaire en Bulgarie plasmen). Zentralblatt für Bakteriologie, (1943–1945). Bulletin de l’Offi ce International Parasitenkunde und Infektionskrankheiten. I. des Epizooties 29:143. Abteilung, Originale 48:77–90. 162. Lagrange, E. 1929. Une nouvelle maladie des 174. Lisi, G. 1895. Di una setticema virulenta nei polli poules à virus fi ltrable observée en Egypte. Bul- della provincia di Massa e Carrara. La Clinica letin de la Société de Pathologie Exotique 22:64– Veterinaria, Milano 18:177–184. 68. 174. Lode, A., and J. Gruber. 1901. Bakteriologische 163. Lagrange, E. 1932. études sur la peste aviaire Studien über die ätiologie einer epidemischen d’Egypte. Annales de l’Institut Pasteur, Paris Erkrankung der Hühner in Tirol. Zentralblatt für 48:208–267. Bakteriologie, Parasitenkunde und Infektion- 164. Landsteiner, K. 1906. Beobachtungen über das skrankheiten. I. Abteilung, Originale 30:593– Virus der Hühnerpest. Zentralblatt für Bakteriolo- 604. gie, Parasitenkunde und Infektionskrankheiten. I. 176. Lode, A. 1902a. Notizen zur Biologie des Erre- Abteilung, Originale 38:540–542. gers der Kyanolophie der Hühner. Zentralblatt für 165. Landsteiner, K., and M. Berliner. 1912. über die Bakteriologie, Parasitenkunde und Infektionsk- Kultivierung des Virus der Hühnerpest. Zentral- rankheiten. I. Abteilung, Originale 31:447–451. blatt für Bakteriologie, Parasitenkunde und 177. Lode, A. 1902b. Eine ätiologisch interessante Infektionskrankheiten. I. Abteilung, Originale Hühnerepizootie. Beilage zur Hygienischen 67:165–168. Rundschau 5:262. 166. Lanni, F., D.G. Sharp, E.A. Eckert, E.S. Dillon, 178. Lode, A. 1907. Zur Biologie des Erregers der D. Beard, and J.W. Beard. 1949. The egg white Hühnerpest (Kyanolophia gallinarum). Zentralb- inhibitor of infl uenza virus haemagglutination. I. latt für Bakteriologie, Parasitenkunde und Infek- Preparation and properties of semipurifi ed inhib- tionskrankheiten. I. Abteilung, Originale 43: itor. Journal of Biological Chemistry 179:1275– 355–359. 1287. 179. Loeffl er, F., and P. Frosch. 1898. Bericht der 167. Leclainche, M.E. 1904. La peste aviaire. Revue Kommission zur Erforschung der Maul- und Générale de Médicine Vétérinaire 3:49–54. Klauenseuche bei dem Institut für Infektionsk- 168. Lépine, P., and P. Haber. 1935. Inclusiones leu- rankheiten in Berlin. Zentralblatt für Bakteriolo- cocytaires dans la peste aviaire. Démonstration de gie, Parasitenkunde und Infektionskrankheiten. leur non spécifi té par l’electropyrexie. Comptes I. Abteilung, Originale 23:371–391. Rendus des Séances de la Société de Biologie et 180. Longo, T. 1880. Osservationi ed esperimenti de ses Filiales 119:1083–1086. intorno a due forme cliniche differenti di tifo da 169. Lépine, P., J. Basset and M. Macheboeruf. 1936. cui sono colpiti i gallinacei ed i tacchini. Giornale Action des ultrapressures sur de la peste aviaire. di Medicina Veterinaria 42:241–270. Pouvir antigènie du virus ultrapressé. Comptes 181. Lucam, F. 1949. Existance de foyers de peste Rendus des Séances de la Société de Biologie et aviaire dans le Sud-Est. Bulletin de la Academie de ses Filiales 121:202–203. de France 22:67–70. 170. Lépine, P., and V. Sautter. 1936. Existence de 182. Lüpke, J.C.G. 1901. Die neue Gefl ügelseuche. lésions nucléaires spécifi ques dans la peste Berliner Thierärztliche Wochenschrift 17:628– aviaire. Comptes Rendus des Séances de la 629. Société de Biologie et de ses Filiales 121:511– 183. Lush, D. 1943. The chick red cell agglutination 512. test with the viruses of Newcastle disease and 171. Levaditi, C., and P. Haber. 1936a. Evolution de fowl plague. Journal of Comparative Pathology la virus de peste aviaire dans les cellules hépati- and Therapeutics 53:157–160. ques de la souris. Comptes Rendus Hebdomadai- 184. Mackenzie, R.D., and G.M. Findlay. 1937. Vari- res des Séances de l’Académie des Sciences ations in fowl pest virus. British Journal of Exper- 202:1214–1217. imental Pathology 18:138–145. 172. Levaditi, C., and P. Haber. 1936b. Affi nité du 185. Maggiora, A., and G.L. Valenti. 1903. über eine virus de la peste aviaire pour les cellules néoplas- Seuche von exsudativem Typhus bei Hühnern. 1. tics (èpithelioma) de la Souris. Comptes Rendus Mittheilung. Zeitschrift für Hygiene und Infek- 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 185

tionskrankheiten, Medizinische Mikrobiologie, 198. Maue, o. V. 1904. Immunisierungsversuche bei Immunologie und Virologie 42:185–243. Hühnerpest. Arbeiten aus dem Kaiserlichen 186. Maggiora, A., and G.L. Valenti. 1904. über das Gesundheitsamte 21:537–552. Virus des exsudativen Typhus bei Hühnern. 199. Mazza, C. 1899. Bakteriologische Untersuchun- II. Mittheilung. Zeitschrift für Hygiene und gen über eine neuerdings aufgetretene Hühner- Infektionskrankheiten, Medizinische Mikrobio- epizootie. Zentralblatt für Bakteriologie, logie, Immunologie und Virologie 48:280– Parasitenkunde und Infektionskrankheiten. I. 327. Abteilung, Originale 26:181–185. 187. Maggiora, A., and O.V. Garofani. 1909. Di un 200. Mazzuoli, S. 1916. Osservazioni sulla peste notevole reperto microscopico riscontrado nel aviaria. La Clinica Veterinaria, Milano 39:1–6. tifo essudativo. Atti della Societá Italiana di 201. Miessner, H., and R. Berge. 1926. Die Gefl ügelpest Pathologia VI. Riunione, Modena, September bei Gänsen. Deutsche Tierärztliche Wochen- 1909, p. 143 (cited in Ref. 213). schrift 34:385–393. 188. Magnus von, P. 1951a. Propagation of the PR8 202. Miyaji S. 1914. Beiträge zur Kenntnis des strain of infl uenza virus in chick embryos. I. The Hühnerpestvirus. Zentralblatt für Bakteriologie, infl uence of various expermental conditions on Parasitenkunde und Infektionskrankheiten. I. virus multiplication. Acta Pathologica et Micro- Abteilung, Originale 74:540–547. biologica Scandinavia 28:250–277. 203. Mrowka, 1912. Das Virus der Hühnerpest ein 189. Magnus von, P. 1951b. Propagation of the PR8 Globulin. Zentralblatt für Bakteriologie, Parasi- strain of infl uenza virus in chick embryos. IV. tenkunde und Infektionskrankheiten. I. Abteilung, Studies on the factors involved in the formation Originale 67:249–268. of incomplete virus upon serial passage of undi- 204. Nakamura, N., and Y. Kawamura. 1926. An luted virus. Pathologica et Microbiologica Scan- experimental study on the virus of fowl-pest. On dinavia 30:311–335. the susceptibility of the pigeon. Journal of the 190. Mann, G. 1894. Über die Behandlung der Ner- Japanese Society of Veterinary Sciences 5:281– venzellen für experimentell-histologische Unter- 283. suchungen. Zeitschrift für wissenschaftliche 205. Nakamura, N., S. Oyama, and N. Tomonaga. Mikrobiologie 11:479–494. 1932. Über die Virulenz des Virus der sog. 191. Manninger, R. 1932. Über die Beziehungen der “Korea-Hühnerseuche.” (Japanese with German Newcastle-Krankheit zur Gefl ügelpest. Zugleich summary). Journal of the Japanese Society of ein Beitrag zur Frage der Pluralität der fi ltrier- Veterinary Sciences 11:285–301. baren Krankheitserreger. Archiv für Wissenscha- 206. Nakamura, N., S. Oyama, and S. Wagatsuma. ftliche und Praktische Tierheilkunde 65:256– 1937. Vaccination of fowls against Chosen 265. disease (Newcastle disease) and fowl plague. 192. Manninger, R. 1936. Fowl plague and Newcastle Journal of the Japanese Society of Veterinary Sci- disease. Journal of Comparative Pathology and ences 16:55–58. Therapeutics 49:279–283. 207. Nakamura, N., and T. Iwasa. 1942. On the fowl- 193. Manninger, R. 1949. Zur Geschichte der pest infection in cat (Japanese with English trans- Gefl ügelpest. Acta Veterinaria Hungarica 1:98– lation) Japanese Journal of Veterinary Science 100. 4:511–523. 194. Marchoux, E. 1908. Cultures in vitro du virus de 208. Negri, A. 1903. Beitrag zum Studium der Aeti- la peste aviaire. Comptes Rendus Hebdomadaires ologie der Tollwuth. Zeitschrift für Hygiene und des Séances de l’Academie des Sciences 147:357– Infektionskrankheiten, Medizinische Mikrobiolo- 359. gie, Immunologie und Virologie 43:507–528 195. Marchoux, E. 1910. La pest aviaire n’est pas une 209. Nitzschke, E. 1953. Zur Diagnostik der atypischen maladie contagieuse. Comptes Rendus des Gefl ügelpest mittels der Hämagglutinationshem- Séances de la Société de Biologie et de ses Filia- mungsprobe und der Eikultur. Berliner und les 68:346–347. Münchener Tierärztliche Wochenschrift 66:301– 196. Marcone, G. 1904. La peste aviaire. La peste 304; 321–325; 338–340. aviaire des faisans. Revue Générale de Médicine 210. Nitzschke, E. 1956. Untersuchungen über die Vétérinaire 3:409–423; 465–481. Komplementbindungs- und Komplementsbind- 197. Mattel, R. 1926. Über histologisch-pathologische ungs-Hemmungs-Reaktion mit Hühner-Immun- Befunde bei der Hühnerpest. Veterinary Disser- serum gegen die Viren der atypischen und der tation, University of Vienna, Austria. klassischen Gefl ügelpest sowie der Schweine- 186 Avian Influenza

Infl uenza. Zentralblatt für Veterinärmedizin B 223. Pearson, J. E. 2004. Sampling methods. In: OIE 3:75–87. (ed.). Manual of Diagnostic Tests and Vaccines 211. Nosotti, I. 1880. L’attuale epizoozia dominante for Terrestrial Animals, Vol. I, 5th ed., pp. 3– nei polli. Milano (cited in Ref. 249). 13. 212. Oesterlen, F. 1873. Begriff epidemischer 224. Perroncito, E. 1878. Epizoozia tifoide nei gallina- Krankheiten oder Seuchen und deren Erklärungs- cei. Annali della Reale Accademia d’Agricoltura versuche durch Ansteckung oder Infektion. In: F. di Torino 21:87–126. Oesterlen (ed.). Die Seuchen ihre Ursachen, 225. Perroncito, E. 1879. Ueber das epizootische Gesetze und Bekämpfung, Verlag H. Laupp: Typhoid der Hühner. Archiv für Wissenschaftli- Tübingen, pp. 1–153. che und Praktische Thierheikunde 5:22–51. 213. Ostertag von, R. 1912. Hühnerpest. In: W. Kolle 226. Perroncito, E. 1894. Intorno ad una “epizoozia and A. von Wassermann (eds.). Handbuch der tifoide” del pollame che non è il cholera dei gal- pathogenen Mikroorganismen. Gustav Fischer linacei, palmipedi e colombi. Giornale della Reale Verlag: Jena, pp. 280–297. Accademia di Medicina di Torino 42:245–246. 214. Ostertag von, R., and K. Wolffhügel. 1902. 227. Perroncito, E. 1899. Sulla malatti dominante nei Untersuchungen über die Hühnerpest, die neue polli. Giornale della Reale Accademia di Medi- Gefl ügelseuche. Monatshefte für Praktische Thi- cina di Torino 62:182–189. erheilkunde 14:49–70. 228. Petényi, J. S. 1833. Erfahrungsbericht. Tagung 215. Ostertag von, R., and G. Bugge. 1906. Weitere der Naturforscher und Ärzte in Wien. Isis 454– Untersuchungen über die Hühnerpest. Zeitschrift 456. für Infektionskrankheiten, Parasitäre Krankheiten 229. Pfeiffer, R. 1893. Die Aetiologie der Infl uenza. und Hygiene der Haustiere 2:1–9. Zeitschrift für Hygiene und Infektionskrankheiten, 216. Otte, W. 1928. Gefl ügelpest. Pestis avium. In: W. Medizinische Mikrobiologie Immunologie und Otte (ed.). Die Krankheiten des Gefl ügels mit Virologie 13:357–386. besonderer Berücksichtigung der Anatomie und 230. Pfenninger, W. 1929. Immunisierungsversuche der Hygiene. Richard Schoetz: Berlin, pp. 196– bei Hühnerpest. Zentralblatt für Bakteriologie, 200. Parasitenkunde und Infektionskrankheiten. I. 217. Ottolenghi, D. 1913. Über einen besonderen Abteilung, Originale 111:448–456. Befund bei der Gefl ügelpest. Zentralblatt für 231. Pfenninger, W., and E. Metzger. 1926. Studien Bakteriologie, Parasitenkunde und Infektionsk- über Hühnerpest. I. Mitteilung: Die natürliche rankheiten. I. Abteilung, Originale 67:510– und die experimentelle Infektion. Schweizer 519. Archiv für Tierheilkunde 68:2–21. 218. Pasteur, M.L. 1870. études sur la Maladie des 232. Pfenninger, W., and Z. Finik. 1926. Studien über vers à soie, Moyen Practique Assuré de la Com- Hühnerpest. II. Mitteilung: Beiträge zur Kenntnis battre et den Prévenir le Retour. Gauthier-Villars: der histologischen Veränderungen im Zentra- Paris. lnervensystem. Zentralblatt für Bakteriologie, 219. Pasteur, L. 1880a. Sur les maladies virulentes, et Parasitenkunde und Infektionskrankheiten. I. en particular sur la maladie appellée vulgairment Abteilung, Originale 99:145–153. choléra des poules. Comptes Rendus Hebdoma- 233. Piana, o. V. 1876. Ricerce sopra una epizoozia daires des Séances de l’Académie des Sciences dei gallinacei osservata nella provincia di 90:239–248. Bologna. Estrato della Gazzetta Medico-Veteri- 220. Pasteur, L. 1880b. Sur le choléra des poules; naria N. III e IV, p. 146 (cited in Ref. 249). etudes des conditions de la non-récidive de la 234. Picard, W.K. 1928. Pseudovogelpest. Veeartse- maladie et de quelques autres de ses caractéres. nijkundige Medeeling 65:1–46. Nederlands- Comptes Rendus Hebdomadaires des Séances de Indische Blaaden for Diergeneeskunde 40:1–10. l’Académie des Sciences 90:952–958; 1030– 235. Plinius Gaius Secundus the Elder 1986. 1033. Vogelkrankheiten und Brüten. In: R. König and 221. Pasteur, L. 1880c. De l’atténuation du virus du G. Winkler (translators and editors). Naturalis choléra des poules. Comptes Rendus Hebdoma- historiae, liber X. Artemis: Munich and Zurich, daires des Séances de l’Académie des Sciences pp. 109–114. 91:673–680. 236. Pop, A., N. Muntiu, and A. Turburi. 1943. Unter- 222. Pavlov, G. 1935. La service vétérinaire en Bulga- suchungen über eine bösartige Kükenseuche: rie de 1929 à 1933. Bulletin de l’Offi ce Interna- “Morbus fi laret.” Deutsche Tierärztliche Wochen- tional des Epizooties 11:2–52. schrift 51:247–250. 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 187

237. Prowazek, S. 1908. Zur Ätiologie der Hühner- 252. Röhrer, H. 1947. Die Histopathologie der experi- pest. Münchener Medizinische Wochenschrift mentellen klassischen Gefl ügelpest beim Huhn. 55:165–166. Monatshefte für Veterinärmedizin 2:33–38. 238. Purchase, H.S. 1930. Active immunisation of 253. Rosenthal, W. 1906. über Beziehungen zwischen fowls against fowl plague. Journal of Compara- Hühnerpest und Lyssa. Zentralblatt für Bakteri- tive Pathology and Therapeutics 43:151–157. ologie, Parasitenkunde und Infektionskrankheiten. 239. Purchase, H.S. 1931a. An atypical fowl-plague I. Abteilung, Originale 40:204–206. virus from Egypt. Journal of Comparative Pathol- 254. Rott, R., and W. Schäfer. 1960. Physikalisch- ogy and Therapeutics 44:71–83. chemische und biologische Eigenschaften des 240. Purchase, H.S. 1931b. Experiments on the viabil- Virus N und seine Beziehungen zur Infl uenza ity of the virus of fowl-plague under trade condi- A-Untergruppe der Myxoviren. Zentralblatt für tions. The Veterinary Record 11:644–648. Veterinärmedizin B 7:237–248. 241. Purchase, H.S. 1931c. A study of cross-immunity 255. Rülke, C.P.A. 2007. Veterinärhistorische Studie with viruses of fowl-plague and observations on über die Klassische Gefl ügelpest der Vögel: Ent- the duration of immunity. British Journal of wicklung von der ersten Beschreibung bis zum Experimental Pathology 12:199–201. heutigen Kenntnisstand und volkswirtschaftliche 242. Pyl, G. 1938. Die Haltbarkeit von Virus-Arten in Bedeutung. Veterinary Dissertation, University Abhängigkeit von der Wasserstoffi onenkonzen- of Giessen. tration. Behringwerk-Mitteilungen 9:6–19. 256. Ruska, E. 1935. The electron microscope as ultra- 243. Rasch, K. 1942. über Gefl ügelpest. Tierärztliche microscope. Research and Progress 1:18–19. Rundschau 48:133–136; 171. 257. Russ, V.K. 1907. Beobachtungen über das Virus 244. Reinacher, M. E., and E. Weiss. 1975. Electron der Hühnerpest. Archiv für Hygiene 59:286–312. microscopical study of initial and fi nal stages of 258. Sangiorni, G. 1912. Versuche mit dem fi ltrier- fowl plague virus replication in chick embryo baren Virus der “Meerschweinchenpest.” Zentral- cells. Archives of Virology 49:187–197. blatt für Bakteriologie, Parasitenkunde und 245. Reinhardt, R. 1925. Hühnerpest. Pestis avium. Infektionskrankheiten. I. Abteilung, Originale In: R. Reinhardt (ed.). Lehrbuch der Gefl ügel- 72:70–73. krankheiten, 2nd ed. M. & H. Schaper: Hannover, 259. Sassenhoff, I. 1939. Vergiftungen bei Hühnern germany, pp. 127–134. und Wildgefl ügel mit Metallphosphidgetreide. 246. Reinhardt, R. 1950. Hühnerpest. Pestis avium. Archiv für Tierheilkunde 74:513–517. In: R. Reinhardt (ed.). Lehrbuch der Gefl ügel- 260. Schäfer, W., and G. Schramm. 1950. Über die krankheiten, 4th ed. M. & H. Schaper: Hannover, Isolierung und Charakterisierung des Virus der Germany, pp. 102–108. klassischen Gefl ügelpest. Zeitschrift für Natur- 247. Rice, J.P. 1924. Some contagious diseases of forschung 5b:91–102. poultry. The Veterinary Record 4:207–214. 261. Schäfer, W. 1952. Sammelreferat: Gefl ügelpest. 248. Rimmelzwaan, G.F., D. van Riel, M. Baars, T.M. Deutsche Tierärztliche Wochenschrift 59:25–27. Bestebroer, G. van Amerongen, R.A.M. Fouch- 262. Schäfer, W., K. Munk, and O. Armbruster. 1952. ier, A.D.M.E. Osterhaus, and T. Kuiken. 2006. Eigenschaften tierischer Virusarten, untersucht Infl uenza A virus (H5N1) infection in cats causes an den Gefl ügelpestviren als Modell. III. systemic disease with potential novel routes of Mitteilung: Weitere Untersuchungen über die virus spread within and between hosts. American physikochemischen und morphologischen Eigen- Journal of Pathology 168:176–183. schaften der Gefl ügelpestviren. Zeitschrift für 249. Rivolta, S., and D. Delprato. 1880. Delle lesioni Naturforschung 7b:29–33. patologiche del sangue. Tifo essudativo. In: S. 263. Schäfer, W. 1955. Vergleichende sero-immunol- Rivolta and D. Delprato (eds.). L’ornitoiatria o la ogische Untersuchungen über die Viren der Infl u- Medicina degli Uccelli Domestici e Semidome- enza und der klassischen Gefl ügelpest. Zeitschrift stici. Uebelhart: Pisa, pp. 460–462. für Naturforschung 10b:81–91. 250. Robertson, A., J.G. Campbell, and D.N. Graves. 264. Scheurlen, W., and K. Buhl. 1901. Zur Kenntnis 1945. Experimental zinc phosphide poisoning in der seuchenhaften Bauchfellentzündung der fowls. Journal of Comparative Pathology and Haushühner. Berliner Thierärztliche Wochen- Therapeutics 55:290–300. schrift 17:369–370. 251. Rodier, E.A. 1928. Philippine fowl disease. Pro- 265. Schiffmann, J. 1906. Zur Histologie der Hühner- ceedings of the Society for Experimental Biology pest. Wiener Klinische Wochenschrift 19:1347– and Medicine 25:781–783. 1348. 188 Avian Influenza

266. Schiffmann, J. 1908. Zur Histologie der Hühner- bocytes in vitro infected with fowl plague virus pest. Zentralblatt für Bakteriologie, Parasiten- (FPV). Medical Microbiology and Immunology kunde und Infektionskrankheiten. I. Abteilung, 159:151–160. Originale 45:393–403. 279. Straub, o.V. 1877. Gefl ügelseuchen. Mitteilungen 267. Schmidt, H. 1985. Die wilden Stammformen. aus den Jahresberichten der Oberamtsthierärzte In H. Schmidt (ed.). Handbuch der Nutz- und für das Jahr 1876. Repertorium der Thierhe- Rassehühner. Neumann-Neudamm: Melsungen, ilkunde 38:252–254. pp. 13–20. 280. Strebel, U., and B. Reicherter. 1885. Typhoid des 268. Schmidt, U. 1968. Die Gefl ügelpest. In: H. Röhrer Gefl ügels, Hühnercholera oder Hühnerpest. In U. (ed.). Handbuch der Virusinfektionen bei Tieren, Strebel and B. Reicherter (eds.). Neues, illustri- Band III/1, Spezieller Teil 2. Gustav Fischer ertes Haustierarzneibuch. Eine ausführliche Bes- Verlag: Jena, pp. 283–437. chreibung der Zucht und Haltung sämtlicher 269. Schürmann, E. 1943. Zur Diagnose der Haustiere, der inneren und äußeren Krankheiten, Gefl ügelpest. Berliner und Münchener Tierärztli- und deren Behandlung, der Tierseuchen, der che Wochenschrift und Wiener Tierärztliche Hauptmängel einschließlich der bezüglichen Monatsschrift 27/28:195–218. Gesetze, sowie der Geburtshilfe, und des Hufbe- 270. Schweizer, P. 1922. Untersuchungen über die schlags. Süddeutsche Verlagsbuchhandlung: Natur der fi ltrierbaren Vira und die Resistenz des Stuttgart, pp. 734–737. Hühnerpestvirus gegen zwellschädigende Ein- 281. Stubbs, E.L. 1943. Fowl pest. In: H.E. Biester and fl üsse (Gerbstoffe, Oligodynamie). Archiv für L. Devries (eds.). Diseases of Poultry, 3rd ed. Hygiene 90:155–174. Iowa State College Press: Ames, IA, pp. 493– 271. Seifried, O. 1938. Einfl uss von Virulenz und 502. Infektionsart auf den anatomischen Charakter der 282. Swayne, D.E. 2003. Avian intestinal spirocheto- sog. Laryngotracheitis der Hühner. Zeitschrift für sis. In Y.M. Saif, H.J. Barnes, J.R. Glisson, A.M. Infektionskrankheiten, parasitäre Krankheiten Fadly, L.R. McDougald, and D.E. Swayne (eds.). und Hygiene der Haustiere 52:108–123. Diseases of Poultry. 11th ed. Iowa State Univer- 272. Smith, W., C.H. Andrews, and P.P. Laidlaw. sity Press: Ames, IA, pp. 826–836. 1935. Infl uenza: experiments on the immuniza- 283. Swayne, D.E., and D.A. Halvorson. 2003. Infl u- tion of ferrets and mice. British Journal of Exper- enza. In Y.M. Saif, H.J. Barnes, J.R. Glisson, imental Pathology 16:291–302. A.M. Fadly, L.R. McDougald, and D.E. Swayne 273. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- (eds.). Diseases of Poultry. 11th ed. Iowa State Heng, N. Meemak, N. Pariyothorn, S. Payung- University Press: Ames, IA, pp. 135–160. porn, A. Theamboonlers, and Y. Poovorawan. 284. Todd, C. 1928. Experiments on the virus of fowl 2006. Avian infl uenza H5N1 in naturally infected plague. British Journal of Experimental Pathol- domestic cat. Emerging Infectious Diseases ogy 9:19–27; 101–106. 12:681–683. 285. Todd, C., and J.P. Rice. 1930. Fowl plague. In: 274. Spears, H.N. 1946. Immunisation against fowl C. Todd, (ed.). A System of Bacteriology in Rela- plague with crystal-violet vaccine. Journal of tion to Medicine, 7th ed. Medical Research Comparative Pathology and Therapeutics 56: Council, His Majesty’s Stationary Offi ce: London, 160–169. pp. 119–131. 275. Spinola, W.T.J. 1858. Katarrhalische Leiden des 286. Trétrop, E. 1900. La maladie des cygnes cosco- Hausgefl ügels und Pips der Hühner. In: W.T.J. roba. Annales de l’Institut Pasteur 14:224–231. Spinola (ed.). Handbuch der speciellen Patholo- 287. Tumova, B. 1986. Avian infl uenza and para- gie und Therapie für Thierärzte, Vol. 1. August myxoviruses in central and eastern Europe. A Hirschfeld: Berlin, pp. 402–403. review. In: B.C. Easterday and C.W. Beard (eds.). 276. Staub, A. 1926. Vaccination contre la peste Proceedings of the Second International Sympo- aviaire. Comptes Rendus des Séances de la sium on Avian Infl uenza, Georgia Center for Société de Biologie et de ses Filiales 95:1193– Continuing Education, the University of Georgia, 1194. Athens, Georgia, USA, September 3–5, 1986. 277. Stazzi, P. 1906. Il tifo essudativo o peste aviaria Symposium on Avian Infl uenza, US Animal nei psittacidi. La Clinica Veterinaria, Milano Health Association: Richmond, VA, pp. 84–89. 29:337–344; 337–344. 288. Ullner, W. 1951. Die Bewegung der Tierseuchen 278. Sterz, I., and E. Weiss. 1974. Electron micro- in Hessen in den Jahren 1938–1950 mit der Dar- scopical and virological studies of chicken throm- legung der Ursachen und Auswirkungen dieser 7 / The Beginning and Spread of Fowl Plague Across Europe and Asia 189

Bewegung. Veterinary Dissertation, University of for Terrestrial Animals (Mammals, Birds, Bees), Giessen. 5th ed. OIE: Paris, pp. 3–13. 289. Varro, M.T. 2002. Das Vogelhaus. In: C. Flach 298. Wiemann, J., and G. Francke. 1928. Der deutsche (translator and editor). Gespräche über die Viehbestand und die Tierseuchen in Zahlen, Landwirtschaft, Buch 3. Wissenschaftliche Karten und Tafeln, 5th ed., J. Wiemann and G. Buchgesellschaft: Darmstadt, pp. 143–180. Francke (eds.). Richard Schoetz: Berlin, pp. 129– 290. Vaida, M. 1925. Miscarea epizootilor in anul 132. ′ 1924. (Tierseuchenbewegung im Jahre 1924) (in 299. Woernle, H. 1955. Ein Beitrag zum Infektionsab- Romanian). Buletinul Direcsiei Generale Zoo- lauf der atypischen und klassischen Gefl ügelpest. ′ tehnice si Sanitare Veterinare 6:396–356. Tierärztliche Umschau 10:324–328. ′ 291. Vianello, G. 1940. Sulla diffusione in Lombardia 300. Woernle, H., and O. Siegmann. 1952. Blutunter- del cholera aviario, della tifosi aviaria e della suchung zur Diagnosestellung der Gefl ügelpest peste aviaria (über die Verbreitung der am toten Tier. Deutsche Tierärztliche Wochen- Gefl ügelcholera, des Gefl ügeltyphus und der schrift 59:373–375. Gefl ügelpest in der Lombardei). La Clinica Vet- 301. Wolstein, J.G. 1778. Von den Thieren, ihren erinaria, Milano 63:297–300. Seuchen und Aerzten. In: J.G. Wolstein (ed.). 292. Virchow, R. 1858. Die Cellularpathologie in ihrer Anmerkungen über Viehseuchen in österreich Begründung auf Physiologische und Patholo- nebst einer Abhandlung gegen das Umbringen gische Gewebelehre. August Hirschfeld: Berlin. der Thiere in Seuchen. Joseph Camesina & 293. Wagener, K. 1941. Die Hühnerpest (Gefl ügelpest) Company: Vienna, pp. 1–12. als Kriegstierseuche. Berliner und Münchener 302. Zeiss, C. 1872. Production of microscopes Tierärztliche Wochenschrift 57:537–538. equiped with condensor. Available at http://www. 294. Weidenmüller, H. 1951. Vergleichende Desin- zeiss.de. Carl Zeiss AG, Jena, Germany. Accessed fektionsversuche mit dem Virus N und dem August 2006. Virus der atypischen Gefl ügelpest. Tierärztliche 303. Zschokke, E. 1912. Beobachtungen über Hühner- Umschau 6:407–408. pest. Schweizer Archiv für Tierheilkunde 54:282– 295. Weineck, E. 1938. Ueber das Verhalten des 287. fi ltrierbaren Virus (Hühnerpest, Maul-und- 304. Zureck, F. 1954. über die in den Jahren 1950 Klauenseuche) im Gewebe von Huhnembryonen. bis 1953 im Landesveterinäruntersuchungsamt Zentralblatt für Bakteriologie, Parasitenkunde Berlin festgestellten Gefl ügelkrankheiten unter und Infektionskrankheiten I. Abteilung, Originale besonderer Berücksichtigung der Gefl ügelpest. 141:14–21. Monatshefte für Tierheilkunde 6:89–98. 296. West, B., and B.-X. Zhou. 1989. Did the chicken 305. Zürn, F.A. 1882. Entophyten. Die im Inneren go north? New evidence for domestication. des Hausgefl ügels wohnenden Schmarotzer- World’s Poultry Science Journal 45:205–218. pfl anzen. In: F.A. Zürn (ed.). Krankheiten 297. Wiegers, A. 2004. Sampling methods. In OIE des Hausgefl ügels. Bernhard Friedrich Voigt: (ed.). Manual of Diagnostic Tests and Vaccines Weimar. 8 High Pathogenicity Avian Influenza in the Americas

David E. Swayne

INTRODUCTION 1966, H5N2 HPAI in the United States during Highly pathogenic avian infl uenza (HPAI), termed 1983–1984, H5N2 HPAI in Mexico during 1994– fowl plague or fowl pest until 1981 (6), was fi rst 1995, H7N3 HPAI in Chile during 2002, H7N3 reported in the Americas during the 1920s (23). The HPAI in Canada during 2004, H5N2 HPAI in the early cases were often called European fowl pest United States during 2004, and H7N3 HPAI in because the fi rst reports of the disease were from Canada during 2007 (86 21a). For this chapter, the Europe and all diagnostic information, published occurrence of HPAI on a farm will be referred to as literature, and training originated from Europe. a “case,” while the collection of cases will be termed However, many of the early outbreaks in the Amer- the “outbreak” or “epidemic.” icas were poorly documented and others may have gone unnoticed because of inadequate diagnostics FOWL PLAGUE IN NORTHEASTERN AND and insuffi cient training or knowledge. For example, UPPER MIDWESTERN UNITED STATES Mohler in 1926 reported fowl plague outbreaks in DURING 1924–1925 both Argentina and Brazil but provided minimal In the fall of 1924, fowl plague appeared in chickens information and an untraceable citation to two Latin in live poultry markets of New York City, causing American authors (50). Some have questioned the high morbidity and mortality (36, 49). During this diagnosis of fowl plague in Argentina and suggested outbreak, the disease was commonly referred to as an alternative diagnosis of Newcastle disease (20). European fowl pest. The disease was subsequently The limited available information indicated the diagnosed in Pennsylvania, New Jersey, Connecti- Argentine disease was a high mortality nervous dis- cut, Indiana, Michigan, West Virginia, Missouri, order of chickens that had clinical presentation of and Illinois before being eradicated in spring of ataxia and movement of the head in a circle (50). 1925 (36). The diagnostic confusion between cases of fowl plague and Newcastle disease were acknowledged Timeline in the 1930s and 1940s in various texts on poultry The outbreak began with reports of high mortality diseases (8, 78). As a result, Newcastle disease was in chickens among several large live poultry markets often referred to as “pseudo-fowl pest” (78). in New York City during the latter part of August This chapter reviews the nine well-documented 1924 (41). An undiagnosed high-mortality disease and distinct epidemics that have been extensively was noted as early as June in some of the markets investigated and had suffi cient information in the (11). These losses were local and limited until mid- scientifi c literature or in government documents to September, when they became generalized and support a diagnosis of HPAI. These epidemics spread to all parts of New York City (41). Losses include fowl plague in the United States during were severe by September 13, 1924 (5). The disease 1924–1925 and 1929, H5N9 HPAI in Canada during was most prevalent at Thanksgiving and Christmas

Avian Influenza Edited by David E. Swayne 191 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 192 Avian Influenza time (77), presumably from increased transport and Detroit outbreaks, shipping from the city was pro- mixing of poultry to supply the family holiday meals. hibited. The last Detroit cases were identifi ed on In 1924, the live poultry market (LPM) system of February 20, 1925. During February 1925, infection New York City was composed of 307 establish- of poultry was identifi ed on one premises in St. ments and 25 commission merchants, with the large Louis, Missouri (50). wholesale markets handling 20,000 birds a week and The fi rst cases of fowl plague in Indiana occurred smaller retail markets selling 1000 birds per week on February 1, 1925 (39). On February 6, the legis- (41). The markets were supplied from private fl ocks lature appropriated emergency funds for control and in New York City which averaged 15 to 175 birds eradication. A survey of 2.5 million poultry on all per fl ock, but 75% of the birds were shipped in by farms was made by practicing veterinarians from railroad from outside of New York City with farms February 9 through 18 and identifi ed fi ve affected located in New England and northeastern and mid- farms and three affected feeding stations. In total, western United States. For 1924, poultry sales in 1197 chickens were destroyed and all loading and New York City were 49 million birds with 99% assembly premises were cleaned and disinfected being chickens and the remaining 1% being a (C&D). In West Virginia, one diseased fl ock was mixture of turkeys, ducks, and geese. With the out- found on February 10, 1925 (50). On February 25, break of fowl plague, economic losses were great 1925, a single fl ock was diagnosed in northeastern and a conservative estimate of losses in New York Illinois, in a Chicago suburb (13). This fl ock was City alone was 600,000 birds (41). Total poultry adjacent to a railroad line from which the owner production in the United States for 1924 was 678 had picked up dead chickens in the railroad million chickens (5). right-of-way. From New York City, fowl plague spread to New Most of the infected poultry were identifi ed in the Jersey with the fi rst case reported on October 3, markets of New York City, Philadelphia, Jersey 1924, in Ocean County with later spread to 12 addi- City, and three or four cities in Connecticut (50). tional counties before ending with a fi nal case on Eradication was complete in Connecticut and New February 4, 1925 (5, 11). The fi rst case in Pennsyl- Jersey by late January 1925 (50). The eradication vania was identifi ed on October 22, 1924, among was completed on May 1, 1925, although an infected poultry owned by a dealer in Philadelphia (5, 75, fl ock was identifi ed in Philadelphia on May 1, 1925, 76). Additional cases were diagnosed in Pennsylva- and one additional small affected fl ock of chickens nia fl ocks after Thanksgiving and cases were reported was identifi ed on Long Island, New York; both were until January 17, 2005 (76). Most cases had a history immediately eliminated (36, 50). of purchase of holiday chickens from a Philadelphia poultry market from which the birds were intro- Signalment and Lesions duced into home fl ocks. The disease was seen primarily in chickens, guinea The fowl plague spread to Long Island by October fowl, and a few turkeys but not in pigeons, ducks, 28, 1924, with initial cases on three farms involving or geese (16, 41). All breeds and ages of chickens 627 chickens over 21 days (18). The cases continued were susceptible, although some reports suggested through November and December with reports from the heavy breeds were more severely affected than upstate New York (17, 52). The cases were traced the light breeds, such as Leghorns. Most birds died back to the LPM in New York City. On December without any clinical signs, or if signs were present, 23, 1924, a carload of poultry from New York City they were mild and birds died within 1 to 2 hours transiting through Delphos, Ohio, arrived with 3000 (11, 41). Clinical signs when present included ces- dead chickens and guinea fowl affected with fowl sation of egg lay, fever, listlessness, loss of appetite, plague, an additional 300 sick chickens, and a few dull attitude, ruffl ed feathers, somnolence, droopy geese and ducks that were unaffected (5, 16). The wings and tail, and prostration (41, 50) (Figs. 8.1 carload was condemned and destroyed. No fowl and 8.2). Some investigators reported various respi- plague was found in native Ohio poultry. ratory signs, including labored breathing, rales and On January 15 1925, cases of fowl plague were wheezing, open mouth breathing, and mucus nasal identifi ed in larger markets of Detroit but no cases discharge, but the concurrent appearance of infec- were traced to farms in Michigan (5, 37). With the tious bronchitis raises doubt that the respiratory 8 / High Pathogenicity Avian Influenza in the Americas 193

Figure 8.1. Lethargic condition with periorbital edema in a chicken 36 hours after subcutaneous injection of fi ltered blood containing the fowl plague virus. (Used with permission of Journal of the American Veterinary Medical Association [37].) Figure 8.2. Severe edema of wattles, earlobes, and eyelids in a chicken 40 hours after subcutaneous injection of fi ltered blood containing the fowl plague virus. (Used with permission of Journal of the American Veterinary Medical Association [37].) Figure 8.3. Petechiae on the external surface and mucosa of proventriculus and ventriculus (gizzard) in a chicken after subcutaneous injection of fi ltered blood containing the fowl plague virus. (Used with permission of Journal of the American Veterinary Medical Association [37].)

Table 8.1. The frequency of gross lesions in groove and mucosa of proventriculus and ventricu- chickens during the 1924–1925 fowl plague lus (11, 41, 50) (Fig. 8.3). The latter commonly had outbreak in the United States based on 106 ulceration under the cuticle (50). Additional and less necropsies performed in Pennsylvania on frequent lesions seen included cyanotic wattles; natural cases (81). edema of eyelids, head, submandibular tissue, ear Lesion Frequency (%) lobes, and occasionally down to the breast (Figs. 8.1 and 8.2); serous ocular discharge, hemorrhage Cyanosis of head 75 throughout respiratory and alimentary tracts; and Swollen wattles 29 petechial hemorrhages in pancreas, ovary, and ret- Swollen ear lobes 22 roperitoneal fat. Some birds developed nervous Swollen head 19 signs or greenish-yellow diarrhea (50, 73). Experi- mentally, most inoculated birds died without signs Hemorrhages in proventriculus 91 or lesions, while others had lesions typical of fowl Hemorrhages in ventriculus 56 plague cases (41). Mortality ranged from 50% to (gizzard) 100%. Hemorrhages in abdominal fat 55 Natural exposure produced very virulent disease pad in chickens and to lesser extent in turkeys but no Hemorrhages on the heart 26 disease in domestic ducks (50). Others noted that no Hemorrhages in the intestines 10 cases were seen in domestic ducks or geese, even on Hemorrhages on inner surface of 10 farms with high mortality in chickens (77). breast bone Hemorrhages on oral mucosa 10 Experimental Studies Multiple veterinarians and researchers within the signs were from fowl plague (50). The appearance U.S. Department of Agriculture (USDA), various of individual clinical signs varied with individual state government laboratories, and universities cases (Table 8.1). studied the fowl plague virus of 1924–1925. Exper- The most consistent lesions were cyanotic comb imentally, chickens, turkeys, and guinea fowl were and hemorrhages in epicardial fat in the coronary very susceptible to the virus by either feeding of 194 Avian Influenza infected liver or intramuscular or subcutaneous Epidemiology injection of small volumes of blood from affected The virus was most often introduced into the markets chickens (16, 42, 50). However, infection by feeding or private fl ocks by purchase of diseased or exposed required more virus than by injection to produce the chickens, which were the most important means of lethal infection (42). The virus could also be trans- disseminating the virus (11, 41). Usually, the fl ock mitted by scratching the skin and applying either died in a few days, but some took up to a week. In infected blood or oral secretions from infected birds. establishments without the introduction of diseased Dropping infected blood into conjunctival sacs of birds, the virus was introduced on the clothing or chickens could transmit the virus. There were no shoes of persons or by wild birds. Occasionally, differences in susceptibility of different chicken scavenging chickens picked it up from vacant lots breeds (42). Passage of the virus in chickens resulted where dead chickens or viscera were dumped. in increased virulence evident as shortening of time However, the movement of diseased chickens was from inoculation to death (16, 42). the primary means of dissemination, and improper Similar experiments in domestic geese, ducks, disposal of infected carcasses was a serious problem and pigeons failed to produce illness (50), although in instituting control (50). Feeding of offal was a one investigator indicated that ducks and geese common practice, and to prevent transmission of the could be infected and killed by subcutaneous injec- virus, heating the offal to 60º C for 30 minutes was tion of large doses, and smaller amounts of virus effective at killing the virus (17). Current cooking 11 caused infection and death in pigeons and guinea standards indicate 10 EID50 of HPAI virus per fowl (42). Various mammals including mice, rabbits, gram of meat would be inactivated in 13.3 min guinea pigs, and macaque monkeys did not become (Chapter 22, p. 499). ill or die when injected with the virus, suggesting Once the virus entered a market establishment, that the virus had low potential to affect humans (16, the continual addition of susceptible chickens sup- 42, 50, 77). Pigeons and sparrows were noted to be ported a continuous cycle of reinfection. Further- less susceptible than chickens (16, 76, 77)—in one more, management practices within the markets study, 16 pigeons were injected with the virus but perpetuated the disease through refi lling of contam- only 2 died, and they lacked signs and had few inated coops with healthy birds, reusing leftover lesions. feed, and reattaching drinking troughs between Experimental infections were not always easy to coops. The virus was spread to supply farms princi- produce or reproduce, even in chickens through pally by returning contaminated crates from the contact exposure. Some studies failed to produce markets and restocking with birds purchased in infection and death in chickens when birds were the markets. Also, rodents may have served as placed in the same unclean caging of chickens that mechanical carriers of the virus (50). The cool tem- died of fowl plague (42, 77). However, direct contact peratures in New England and Midwest in fall and with diseased birds was usually effi cient at transmit- winter prolonged the survival of the virus in the ting the virus. environment and most likely contributed to spread. The most effi cient means of experimental trans- In one study, virus in blood from an infected chick- mission was injection of infected blood, either sub- ens, when kept in an ice box, remained viable for cutaneously or intramuscularly. The oral mucus had 30 days (17). a higher quantity of virus than did the feces; in The incubation period in chickens following some experiments, injection of fecal extracts did not natural exposure was 5 to 7 days, while experimen- produce death as quickly as oral secretions suggest- tal inoculation produced an incubation period of 1 ing lesser quantity of virus in the fecal materials to 3 days (50). Experimentally, chickens placed in (42). Most chicken died within 26 to 72 hours after contaminated caging died within 4 to 6 days (16). inoculation or from a few hours to 24 hours after the appearance of clinical signs (77). In some studies, Diagnosis clinical signs appeared less than 24 hours after inoc- Clinically, fowl plague and the septicemic form of ulation (73). Clinical signs and lesions in experi- fowl cholera are similar (50). The diagnosis of fowl mental studies were similar to those in natural plague was made by identifying specifi c lesions cases. including edema and cyanosis of head, hemorrhage 8 / High Pathogenicity Avian Influenza in the Americas 195 in proventriculus and under the cuticle of ventricu- ally eradicating the disease (37). However, success lus, and acute swelling of kidneys, along with exper- of the eradication was attributed to establishing imental reproduction of the disease by inoculation closer and more cordial relationships and formulat- of blood into healthy chickens with mortality in 2 ing uniform recommendations in the eradication days (11). Such inoculated chickens died without between the Bureau of Animal Health (USDA), state lesions or had lesions typical of fowl plague. Some governments, and the commodity association (National veterinarians suggested the inoculum either must be Poultry, Butter and Egg Association) (50). fi ltered before inoculation to eliminate any septicemia producing bacteria, such as the “Pasteurella Movement Controls and Quarantines aviseptica” (Pasteurella multocida) that caused fowl The losses in New York City continued to mount in cholera, or should be cultured and found to be neg- late October with a peak in cases during early ative for specifi c bacterium before fowl plague could November that lasted until after the Christmas be confi rmed (17, 18, 50). holiday (41, 77). The city authorities, as did author- With fowl cholera, the disease has similarities to ities in other eastern states, theorized that diseased fowl plague—the high mortality rate, rapid onset, birds were arriving into New York City via the rail- prostration, cyanosis, and diarrhea in chickens (78). road transport system and effective December 11, However, fowl cholera bacterium produces whitish- 1924, New York City embargoed all shipments of yellow pinpoint foci in the liver, which are not diseased birds from eight Midwest states including present in cases of fowl plague. The hemorrhages North and South Dakota, Nebraska, Kansas, Mis- are more generalized with fowl plague while more souri, Iowa, Illinois, and Indiana (41, 50). However, localized to heart and intestines with fowl cholera. by some authorities this was interpreted as an Furthermore, the fowl cholera bacterium, when embargo on all shipments of poultry from the inoculated into rabbits, domestic ducks, and geese, Midwest. On December 22, 1924, the USDA issued will cause illness and death, while fowl plague virus a prohibition on the interstate transport of live chick- does not (50, 77). Other diseases that must be dif- ens, turkeys, and geese affected with fowl plague or ferentiated from fowl plague include (1) other sep- that had been exposed to sick poultry (i.e., danger- ticemic diseases such as fowl typhoid (Salmonella ous contacts) from 27 states (49, 50, 93). These gallinarium) and apoplectiform septicemia (Strepto- shipping restrictions decreased the number of birds coccus zooepidemicus), both of which can be ruled for sale in New York City by 75% and had a great out by bacterial cultures; and (2) phosphorus poison- impact on the drop in fowl plague cases following ing, which produces proventricular hemorrhages the Christmas holiday. In addition, all farms with but, unlike fowl plague, causes severe ulceration of infected birds were placed under quarantine (49). the mouth and esophagus, and the carcass has a All sick birds were destroyed and the carcasses of distinct odor of phosphorus (50). all birds, that died or were destroyed, were disposed Confusing the diagnostic situation in 1924–1925 by burning or burial deep in the ground (49). was a concurrent problem with infectious bronchitis A negative aspect of the shipping restrictions by in the Midwest that was misdiagnosed as fowl New England states was the placement of embar- plague (50). This confusion, and at times hysteria, goes on all poultry from Midwest states up to 1500 was responsible for many of the rumors related to miles away, resulting in severe economic hardship fowl plague coming to New York from Midwestern on the farmers (93). Careful reexamination of the farms. However, in retrospect, the outbreak began embargoed states found very few fowl plague cases, in New York City and was moved to other locations, and most of these cases resulted from transport of including a few Midwestern states, via the railway the virus from the east to the Midwest via the rail- system. road, not vice versa (50, 77). Complicating the decision for embargoes was the confusion created by Control Measures reports of mortality in chickens in the Midwest from The most critical aspect of the eradication program another disease, infectious bronchitis, which was was “immediate action” (36). Movement controls, misdiagnosed as fowl plague (77). Although the quarantines, and sanitation were the most important restrictions in transport were meant to prevent the principle measures used in controlling and eventu- introduction of fowl plague diseased birds from 196 Avian Influenza the Midwest into Northeastern cities, in retrospect, for use in processing plants and premises, and some the source and epicenter of the fowl plague outbreak have a total ban because of negative environmental was New York City and the implemented movement and human health impact. controls effectively stopped the dissemination of disease from the large urban centers of Northeast Financial Aspects United States to the Midwest states, thus minimizing On December 17, 1924, Congress provided an emer- the number of outbreaks in the Midwest and averting gency $100,000 ($1.14 million, 2006 US$) appro- a national animal health disaster. priation to pay for federal fi eld control measures Human nature has a tendency to look outwardly such as federal inspectors and C&D costs (36, 50). for the source of problems instead of looking inward. In a unique move by one state, Indiana appropriated Today, some highly pathogenic notifi able avian $50,000 ($572,000, 2006 US$) for a proactive sur- infl uenza (HPNAI)-infected countries have placed veillance program to identify infected farms and embargoes on poultry and poultry products from paid farmers with infected poultry indemnities HPNAI-free countries because of political pressure totaling $840.11 ($9612, 2006 US$). However, the and poor understanding of scientifi c information. federal government did not participate in any indem- This is counterproductive to disease control. nity programs (50). Estimates for losses incurred in the outbreak was $1 million ($11.4 million, 2006 Cleaning and Disinfection US$), but this most likely did not take into consid- The second measure taken in controlling fowl plague eration the closure of multiple large processing was the C&D of farms, premises, transportation plants with loss of income or the thousands of facilities, and equipment, especially equipment used employees without work (50). in the shipping of the poultry to markets (49). Since the distribution of rail cars and coops that had been Source in the markets of New York City were not traceable, Investigations by USDA suggested the virus was all railcars and coops were part of the C&D program introduced in the summer of 1923 from Pasteur (50). All C&D was done under offi cial supervision Institute in France by a “well-meaning scientist of the USDA and used 140 federal inspection working on fi lterable viruses” at a prestigious eastern stations and 500 employees (49). However, the university and this scientist distributed the virus to state authorities accomplished the largest share of two other investigators in spring of 1924 (93). To the C&D (50). Dilapidated coops and crates were maintain the virus at the original institution, an burned. Disinfection was done with coal-tar prepara- assistant to the importing scientist took the virus to tions and carbolic acid, or live steam and boiling his father’s farm and propagated the virus in chick- water. In total, the C&D program completed the ens during June, July, August, and September 1924. task on 2718 feeding and assembling premises, 8245 Finally, a fi rm of New York poultry dealers regu- railroad cars, 354,358 poultry transport cages, and larly purchased poultry from one of the institutions 125,975 pieces of miscellaneous equipment (49). carrying on the experimentation. Most of the cases After completion of C&D, farms were not allowed were found in or traced to states in or near where to repopulate for 14 to 30 days (50). Prior to full this experimentation took place and provided the repopulation, test birds (i.e., sentinel chickens) were basis for reasonable suspicion by USDA as to the introduced to confi rm the success of C&D and that source. In a 1925 editorial in the Journal of the virus was not lingering in unidentifi ed places the American Veterinary Medical Association, the (49). If the test birds remained healthy, full restock- proposed “well-meaning scientist” theory was given ing was allowed but on a gradual basis (49, 50). high credibility and the individual was harshly crit- A complication of the eradication efforts was the icized, where the evidence “strongly indicates that negative impact of some disinfectants. The USDA this criminal carelessness was the origin” (34). The received complaints of large quantities of poultry editor goes on to state “it is deeply regretted that contaminated with the odor of coal-tar disinfectants such utter carelessness, inexcusable ignorance and and, as a result, USDA recommended the alternative open disregard for the safety and welfare of one of use of other disinfectants in the markets such as hot our most important industries. . . . Let us hope that lye solution or 4% formaldehyde (50). Today, we a lesson has been taught that will stand for all time.” know that none of these disinfectants are acceptable However, another historical review article has cast 8 / High Pathogenicity Avian Influenza in the Americas 197 doubt to this “well-meaning scientist” theory (2). FOWL PLAGUE IN NEW JERSEY, UNITED The determination of the source for the fowl plague STATES, DURING 1929 outbreak was not defi nitive in 1925 and other pos- A limited, small outbreak of fowl plague was sibilities were acknowledged in the offi cial report reported in chickens on four infected and four (93). One prominent poultry virologist suggested exposed small fl ocks in Morris County, New Jersey, that wild birds might have been the source (11). beginning with a case on May 15, 1929, and with Today, other possibilities must be considered the fi nal case on June 29, 1929 (12, 51). Eradication such as the fowl plague virus could have arisen was declared complete by the USDA on August 15, directly from mutation of a circulating H5 or H7 1929 (94). The principal measures used in disease LPAI virus as has been documented with 1983– eradication were destruction of diseased chickens 1984, 1994–1995, 2002, and 2004 HPAI outbreaks and C&D of premises (35). The diagnosis was made in the Americas. Frozen materials from the 1924– using established criteria recognized by USDA and 1925 outbreak had been saved and was maintained state agricultural experiment stations (Table 8.2). at Cook College, Rutgers University, by Dr. F. R. The clinical signs and lesions were similar to Beaudette, but these materials were lost in a freezer those reported from fowl plague 1924–1925 out- failure in the early 1990s (David Tudor, personal break except these chickens had a prominent severe communication, April 11, 1997). Because none of hemorrhagic tracheitis with peritracheal edema and the original materials exist, the determination of H prominent vesicle production on the comb (12). In and N subtype and the virus lineage (i.e., North experimental work, the virus was determined to be American verses Eurasian) may never be defi ni- a fi lterable virus with inoculum from a 14-lb Mandler tively known. The circulating fowl plague viruses in fi lter reproducing fowl plague, while inoculum from Europe and Asia before 1959 have retrospectively a 9-lb Mandler or Seitz fi lters did not reproduce the been identifi ed as H7N7 and H7N1 HPAI viruses, disease when given by subcutaneous or intramuscu- but this is not known for the 1924–1925 fowl plague lar injections (12). A few chickens that were injected virus. became ill and recovered and their blood con- In 1925, it was recognized, and USDA had legal tained virus-neutralizing substance, which today we authority to prohibit importation of animals from know to be neutralizing antibodies against the hem- countries with exotic diseases as a measure to agglutinin protein (12). Because of this virus- prevent introduction of such diseases and protect neutralizing substance in recovered birds, attempts domestic livestock health (93). Dr. Mohler, the chief at artifi cial immunization of chicken using Todd’s veterinary offi cer, also recognized that “the place to study dangerous foreign pests and diseases is in the countries where they exist and not in laboratories Table 8.2. Criteria for diagnosis of fowl here where there is a potential danger that they may plague in 1920s (51). escape to do great damage to our animal industry” (93). However, with modern primary containment Criteria facilities and secondary safety equipment, and 1. Acute, high mortality in chickens understanding and training in biosecurity and biosafety, such exotic diseases can be studied in a safe 2. Specifi c lesions predominantly petechial to environment with negligible risk to the animal and ecchymotic hemorrhages in the proventriculus poultry commodities within the country (7). The and ventriculus, and on the epicardial surface; knowledge gained has had a tremendous impact on cyanosis of comb and wattles; edema of head, prevention and control of exotic diseases within comb, wattles, and earlobes such investing countries. However, great care must 3. Inoculum derived from tissues after passage be practiced among veterinary and biomedical through Mandler (14 lb) fi lter reproduced fowl researchers when working with exotic, dangerous plague in chickens and other gallinaceous and economically signifi cant agents such as H5N1 poultry HPAI virus of today. Any escape of a virus due to 4. Pigeons, ducks, and geese resistant to fowl inadequate biocontainment can be catastrophic and plague using inoculum above research with exotic viruses must be closely regu- 5. Mammals not susceptible to fowl plague using lated by federal governments. inoculum above 198 Avian Influenza phenol-glycerin vaccine were tried but failed to chickens had lesions typical of HPAI and included produce protection (12, 90). Transmission of fowl edema of the head and neck and petechiae in the plague virus by mosquito bites between diseased and mucosa of the proventriculus and ventriculus naïve chickens failed (12). (gizzard) (43). The virus was also highly lethal in The exact source of the virus is unknown, but the turkeys (54, 69). Histologically, in these species, the owner of the initial cases had purchased and intro- infections produced multifocal necrosis in the brain duced new birds from an itinerate dealer on April and viscera, accompanied by exudative processes in 15, 1929. This introduction was proposed as a pos- the subcutis of the head in chickens and minimal sible initial source, but the 1-month lag from intro- hemorrhagic lesions in both turkeys and chickens duction to mortality is too long (12). This conclusion (54). Turkeys had severe depression, torticollis, and was supported by experimental studies that could diarrhea (69). not reproduce the disease if materials were taken In contrast to chickens and turkeys, other gallina- from survivors at 14 or more days after initial inoc- ceous poultry varied in the severity of experimental ulation with fowl plague materials (12). Other pos- disease. In intranasally inoculated Japanese quail sible sources of the fowl plague virus were proposed (Coturnix coturnix japonica), 15% became ill and as (1) introduction by importation of 1200 Hungar- died, but all the survivors had virological or sero- ian partridges from Czechoslovakia into New Jersey logical evidence of infection (69). Intranasal inocu- on April 1, 1925, of which over 150 died by July 17, lation of pheasants and domestic ducks failed to but fi ltrates from two dead birds did not reproduce produce clinical signs or death (69). The pheasants fowl plague, and (2) undiagnosed cases that contin- developed anti-nucleoprotein antibodies (AGID ued from 1924–1925 fowl plague outbreak, but with test) but not the ducks (69). Intranasally inoculated the high chicken susceptibility and chickens being pigeons were resistant to infection, with 2 of 19 the major poultry population in the United States, it becoming infected and 1 died (69). is unlikely that high mortality events would not have The HPAI virus had the ability to hemagglutinate occurred and gone unrecognized from 1925–1929 a variety of erythrocytes of birds and mammals (12). The source remains unknown. including those from chicken, turkey, goose, pigeon, sea gull, tern, owl, horse, calf, pig, sheep, goat, dog, H5N9 HIGH PATHOGENICITY AVIAN cat, rabbit, guinea pig, hamster, mouse, and humans INFLUENZA IN ONTARIO, CANADA, (43). During the laboratory investigation, this HPAI DURING 1966 virus was determined to not be related serologically A limited outbreak of H5N9 HPAI occurred in a to the classic fowl plague viruses (i.e., H7N7) but turkey breeder fl ock in Ontario, Canada, in a single was related to the viruses reported in chickens from company on two farms during 1966 (43). The out- Scotland in 1959 and in terns from South Africa in break began on March 24 with sudden illness evident 1961 (i.e., H5 HPAI viruses) (43). At the time, these as listlessness, drooping wings, and severe drop in latter outbreaks were termed “fowl plague–like” feed consumption and egg production. The outbreak because the viruses were infl uenza A and the clinical in the second fl ock occurred 3 weeks later, and the and pathological features were similar to fowl two farms did not share attendants but did share an plague, but on HI tests, the viruses were distinct insemination crew. Mortality over 5 weeks resulted from classic fowl plague viruses (43). Based on in loss of 10% of the 8100 birds in the two fl ocks with today’s knowledge, these three viruses were the fi rst similar percentage of losses between hens and toms. of a new hemagglutinin subtype of HPAI virus, the The turkeys did not have outdoor access. After 5 H5 subtype, while all the classic fowl plague viruses months, the remaining birds were slaughtered. Sixty were H7N7 or H7N1 (see Table 6.1). blood samples from slaughter were positive for H5 The low mortality in the infected fl ocks and the hemagglutination inhibition (HI) antibodies (43). high number of surviving birds with hemagglutinat- Although the virus infection in the fi eld was asso- ing antibodies suggest the fl ock had a mixed infec- ciated with low mortality, experimental studies in tion of H5 LP and HPAI viruses. An H5 LPAI virus chickens produced 82% mortality at 3 and 10 days was isolated from turkey breeders in Ontario 3 after intranasal inoculation of 6-week-old chickens months prior to the HPAI outbreak, and this H5 indicating the virus was HP (43). The resulting LPAI virus was antigenically similar to the 1966 8 / High Pathogenicity Avian Influenza in the Americas 199

H5N9 HPAI outbreak virus (43, 44). The subtle respiratory disease and circulation of this virus in clinical and laboratory features suggest the index commercial poultry for 6 months followed by muta- fl ock could have been initially infected with an tion of the LPAI virus to an HPAI strain. H5N9 LPAI virus that mutated to an HPAI virus as it spread through the fl ock. Future sequencing of The Beginning as an LPAI Outbreak these H5 AI viruses will be able to ascertain if this In April 1983, an acute respiratory disease with H5 LPAI virus was the progenitor to the H5N9 increasing mortality and declining egg production HPAI outbreak virus. was diagnosed in an egg laying fl ock in Pennsylvania (24). An H5N2 LPAI virus was isolated from the H5N2 HIGH PATHOGENICITY AVIAN fl ock and a similar diagnosis was made in 24 addi- INFLUENZA IN NORTHEASTERN UNITED tional fl ocks between April and October 1983 (24), STATES DURING 1983 AND 1984 but the HPAI Eradication Task Force estimated 100 The largest epidemic of HPAI in the United States fl ocks became infected between April and October occurred in the states of Pennsylvania, Maryland, 1983 (25). In total, 700,000 broilers in 12 fl ocks, Virginia, and New Jersey during 1983–1984. over 1 million layers in 10 fl ocks, 17,000 broiler However, Pennsylvania had the majority of cases breeders in 2 fl ocks, and 65,000 layer pullets in a being the origin site and central geographic focus of single fl ock were affected (24). A serological survey infection. This is reminiscent of the 1924–1925 conducted in late August to early September on 105 HPAI epidemic where Pennsylvania held a central layer, 16 pullet, 33 broiler, 7 layer breeder, 6 broiler role in the outbreak and two generations of veteri- breeder, 7 backyard, 1 quail, 1 duck, and 7 turkey farms nary diagnosticians, Drs. Evan Stubbs and Robert failed to identify any additional infected farms, indi- Eckroade, played key roles in diagnosis and control cating the LPAI infection was limited in scope (24). of the outbreaks (Fig. 8.4). The unique feature of the In layers, the clinical presentation varied (24). 1983–1984 HPAI epidemic was the initial appear- Typically, the birds had mild to moderate respiratory ance of an H5N2 LPAI virus in chickens with distress, 4% to 43% decrease in egg production, and

Figure 8.4. Drs. Evan Stubbs (right) and Robert Eckroade, prominent veterinary diagnosticians at the University of Pennsylvania, who were key personnel in the control of the 1924–1925 and 1983–1984 HPAI outbreaks in Pennsylvania, United States, respectively. (Source: R. Eckroade.) Figure 8.5. Massive mortality in broiler chickens during the 1983–1984 HPAI outbreak in the northeastern United States (Source: U.S. Department of Agriculture.). 200 Avian Influenza

0% to 2.7% mortality (24). Typically, egg produc- from vesicles to severe edema to cyanosis with some tion returned to normal levels within 2 to 4 weeks having petechiae and ischemic necrosis (Figs. 8.6 (24). Post mortem lesions identifi ed included caseous and 8.7) (1, 24), sometimes with edema and subcu- tracheal exudate, which occluded airways in some taneous hemorrhage of the feet and leg shanks (1, birds, tracheal edema, and petechiae and catarrhal 24). Petechiae hemorrhages occurred on and in infl ammation of respiratory tract (24). In some many visceral organs such as the pancreas but were broiler fl ocks, the infection was asymptomatic with most consistent on the mucosa of proventriculus diagnosis being made on serology at slaughter (24). (24). Lesions varied between broilers and layers (1). However, in other broiler fl ocks, the infection pro- Broilers had few lesions except diffuse mild pete- duced respiratory disease with up to 15% mortality chiae on sternum and abdominal fat and dehydration (24). In broiler breeders and pullets, the mortality (1). Microscopically, chickens had nonsuppurative was less than 5% and typical presentation was respi- encephalitis, necrotizing pancreatitis, and necrotiz- ratory disease (24). ing myositis as the most consistent lesions (1).

The Change to an HPAI Virus Diagnostics and Surveillance Between October 1983 and April 1985, 174,114 Signalment samples from 41,471 submissions were tested for AI In early October 1983, a layer fl ock presented with virus by virus isolation in embryonating chicken signifi cant declines in feed and water consumption, eggs (61). Of these samples 675 resulted in virus and cessation of egg production in a few days (24). isolates and 325 were pathotyped in chickens by in Over the 10-day observation period, mortality vivo chicken testing (61). Of these, 96% of the increased from 50% to 89% (24) (Fig. 8.5). Chick- HPAI isolates were made on the fi rst egg passage ens exhibited listlessness, and a few had nervous and tracheal swabs were positive more frequently signs such as tremors, but respiratory signs were not than cloacal swabs (61). Also, HPAI virus was prominent and many times were absent (24). isolated from housefl ies as well as eggs and egg handling equipment (61). Lesions The lesions in layers included dehydration, edema- Epidemiology tous comb and wattles, and periorbital edema (1, This H5N2 epidemic represented the fi rst docu- 24). The type and severity of comb lesions varied mented change of an LPAI virus to an HPAI virus

Figure 8.6. Edema, necrosis, and ruptured vesicles and bulla in the comb of a layer naturally infected with H5N2 HPAI virus in Pennsylvania 1983–1984. (Used with permission of Veterinary Pathology [1].) Figure 8.7. Subepidermal vesicle and acute infl ammation of dermis in the comb of a layer naturally infected with H5N2 HPAI virus in Pennsylvania 1983–1984. (Used with permission of Veterinary Pathology [1].) 8 / High Pathogenicity Avian Influenza in the Americas 201 during a fi eld outbreak. This was a shocking and pen-reared chukar partridge and one hunter-killed new fi nding, as stated by Dr. Robert Eckroade: “We ring-necked pheasant (55, 56). This low infection had literally watched this H5N2 AI [virus] change rate indicates wild birds and rodents were not from a non-pathogenic agent into a highly patho- involved with spread of HPAI virus between farms genic one” (24). and had negligible involvement in the outbreak. Of particular concern was the inconsistent clinical features and mortality rates in infected chickens Control Measures resulting from mixed infections with H5N2 LPAI The federal eradication effort began in early Novem- and HPAI viruses (24). As a result, the decision was ber 1983 (24). Initially, the federal program focused made in latter stages of the eradication program to on eradication of H5N2 HPAI, but the states were destroy both LP- and HP-infected fl ocks because concerned with all pathotypes of AI regardless of distinction was not practical, and eventually sero- pathogenicity (26). As the eradication program pro- positive fl ocks, which most likely represented gressed, the use of the in vivo laboratory lethality resolved infections by LPAI viruses were eliminated test as an instrument to identify HPAI infected fl ocks (27). The HP phenotype was demonstrated in two for stamping out was determined to not be success- thirds of virus-positive Pennsylvania fl ocks, but ful because some fl ocks with high mortality had LPAI virus was demonstrated in all fl ocks from Vir- isolates that produced low mortality in the labora- ginia and the remaining third of Pennsylvania fl ocks tory. As a result, the eradication program was mod- (27). The detection of antibodies using the AGID ifi ed to include all H5N2 AI–infected fl ocks in the test with either egg yolk or sera was helpful in iden- quarantine area for depopulation (26). Soon after tifying infected fl ocks for elimination. the 1983–1984 epidemic, criteria other than in vivo The introduction of AI virus from Pennsylvania testing in chickens were noted as a need for identify- into Virginia and Maryland occurred via a contami- ing AI viruses for future eradication (3). These new nated transport truck and coops used to haul turkeys, criteria were focused on quick laboratory methods while the New Jersey incident was started by a con- that could predict virulence or potential virulence taminated feed truck (19). However, the spread of in poultry such as cleaved hemagglutinin protein, the LPAI virus between farms was attributed to growth in tissue culture without exogenous trypsin, movement of live and dead birds; contaminated and amino acid sequence at hemagglutinin protein eggs, feed, and water; insect vectors; and contami- compatible with other known HPAI viruses. This nated people (clothes and shoes) and equipment, evolved into specifi c national and international cri- with specifi c risks identifi ed as feed delivery, vac- teria for virulence (59, 92). Furthermore, because cination crews, service personnel, egg processors, some H5 and H7 LPAI viruses have been shown to mechanical equipment repairmen, and relatives who mutate to HPAI viruses, regulations in the United worked on other farms (24, 27). In eggs collected States and World Organization of Animal Health from an infected chicken breeder farms, the HPAI (Offi ce Internationale des Epizooties [OIE]) have virus was not isolated from dead embryos or eggs in categorized H5 and H7 LPAI as LP notifi able AI which the embryos did not develop (61). The hatched (LPNAI) with eradication requirements because of chicks were serologically negative and no virus was the potential for these viruses to mutate and become isolated (61). However, it was unknown if they were HPAI viruses (59, 92). collected during a viremic phase (61). In another A total of 17 million birds from 448 fl ocks in series of fi eld cases from broiler breeders and layers, Pennsylvania, Virginia, and New Jersey were HPAI virus was isolated from internal contents and destroyed (27). Seventy-three of these fl ocks were on shell surface of eggs from chickens 1 to 18 days in Shenandoah Valley of Virginia and 1 layer fl ock after the appearance of clinical signs (21). In an was in New Jersey (66). Most cases were in com- experimental study, HPAI virus was present in 85% mercial chickens and turkeys, but a few small non- to 100% of eggs laid by hens on days 3 and 4 post- commercial fl ocks were infected and eliminated inoculation (10). (27). During the epidemic, $41 million was dis- There were 4466 wild birds and rodents tested in pensed in indemnities and the total cost to the federal the quarantined areas of Pennsylvania, 313 in Vir- government was $63 million (27). The primary ginia, and 1511 in the nonquarantined area of eradication procedures included strict quarantine, Maryland with isolation of HPAI virus from one immediate destruction of all fl ocks with diagnoses 202 Avian Influenza of AI, surveillance to identify free and infected LPM system of New York, which sells 50,000 birds fl ocks, environmental decontamination through per week. A survey of the LPM system resulted in C&D, and education to enhance biosecurity (27). isolation of LPAI viruses from poultry within 26 of HPAI virus–infected fl ocks were eliminated primar- 44 and 12 of 26 markets in New York and New ily by on-site burial and less frequently by landfi ll Jersey, respectively (29). Several H5N2 LPAI burial (27) (see Chapter 15, Methods for Disposal viruses were also isolated from poultry in the Miami, of Poultry Carcasses). Incineration proved expen- Florida, LPM system (29). The LPM markets contain sive and slow, which prevented signifi cant use, and and sell a mixture of poultry species including rendering was not attempted on actively infected chickens, turkeys, guinea fowl (Numida meleagris), fl ocks because the industry was concerned with pheasants (Phasianus colchicus), domestic ducks spreading the virus and the inability to sell the fi nal (Anas platyrhynchos), and chukar partridges (Alec- rendered product (27). Some have suggested that toris chukar) (29). These H5N2 LPAI viruses were recovered LPAI-infected fl ocks could have been closely related to the LPAI and HPAI viruses associ- marketed under controlled conditions without sig- ated with the 1983–1984 outbreak and were the last nifi cant risk of disease spread (27). In Virginia, two recorded H5 viruses in this genetic lineage (29). fl ocks were slaughtered and three seropositive fl ocks were rendered (66). The most diffi cult problems Source faced were depopulation of large numbers of birds The initial focus of the 1983–1984 H5N2 LPAI virus and the disposal of manure from infected farms (27). source was wild birds, but surveys of wild birds in The last seropositive fl ock was depopulated Septem- the outbreak area failed to identify wild birds as the ber 12, 1984, and the quarantine was lifted October introduction source (55, 56, 98). Later, as protein 4, 1984, in Pennsylvania (19, 25). antigenic and molecular analytic tools emerged, the Another challenge in the eradication was the cool source focused on LPM. The H5N2 LPAI viruses environmental conditions during the fall and winter isolated from poultry in LPM of northeast United in the northeastern United States. For example, the States during 1985 and 1986 were closely related virus was shown to survive in wet manure up to 105 antigenically and genetically to the HPAI viruses days after depopulation if stored in the barn when from the 1983–1984 outbreak (Fig. 8.8) (9, 97). The the weather was very cool and under wet conditions data suggest that LPM may have been the introduc- (27). tion source of the H5N2 LPAI viruses resulting in Repopulation was only acceptable after a 30-day outbreaks among Pennsylvania commercial poultry minimum down-time following C&D and testing to in April 1983 and again in December 1985 (97). demonstrate the absence of viable AI virus (27). Before repopulation, the immediate area must be Virus negative for active AI cases and all replacement The H5N2 HPAI virus isolated from the index farm fl ocks were serologically monitored after placement during October 1983 was derived from predecessor (27). The conveyance of the birds had to be accom- H5N2 LPAI virus through a single mutation at posi- plished in a biosecure manner (27). Indemnities tion 11 of the hemagglutinin resulting in the loss of were paid by the federal government at 100% of the a shielding glycosylation site from the proteolytic appraised fair-market value of the fl ocks (19). cleavage site (40). However, the hemagglutinin proteolytic cleavage site of the H5N2 LP viruses already Related H5N2 LPAI Outbreak during 1985–1986 had important amino acid substitutions, compared On January 8, 1986, a broiler fl ock in Pennsylvania with the consensus sequences of North American was identifi ed with H5N2 LPAI infection, which LPAI viruses, resulting in four basic amino acids subsequently spread to farms in New York, New being present at the cleavage site for a sequence of Jersey, Massachusetts, and Ohio (29). The outbreak PQKKKR/GLF (68). These two molecular features involved 21 fl ocks with 134,059 broilers and roast- endowed the H5N2 AI virus from October with the ers, 42,217 turkeys, 190,082 layers, and 3065 other ability to be cleaved by ubiquitous proteases con- poultry. The outbreak was eliminated by stamping- tained in multiple cell types throughout the body, out strategies (29). The infected farms were all con- thus expressing the HP phenotype in both chickens nected through poultry transport crates from the and turkeys. Analysis of the H5 viruses from North H5 HA1

CK/Scotland/59 (HP) Tern/South Africa/61(HP) TK/Ireland/1378/83 (HP) Eurasian CK/Italy/1485/97 (HP) Hong Kong/156/97 (HP) Vietnam/1196/04 (HP) TK/Ontario/7732/66 (HP) TK/WI/68 Mallard/WI/34/75 CK/PA/1/83 CK/PA/1370/83 (HP) TK/VA/6962/83 TK/VA/21833/84 CK/VA/40018/84 CK/PA/10210/86 PA/83 CK/NJ/12508/86 lineage CK/FL/27716-2/86 CK/FL/22780-2/88 CK/FL/2507/89 CK/NY/12004-3/87 CK/MA/11801/86 CK/OH/22911-10/86 Mallard/WI/428/75 Mallard/WI/169/75 Mallard/Ohio/345/88 DK/Michigan/80 TK/MN/3689-1551/81 Mallard/WI/944/82 TK/TX/14802/82 Ruddy Turnstone/DE/244/91 North CK/FL/25717/93 American Emu/TX/39442/93 LBM/93 CK/NJ/17169/93 CK/PA/13609/93 TK/MN/10734/95 Ruddy Turnstone/NJ/2242/00 UN/NY/118547-11/01 DK/NJ/117228-7/01 UN/NY/9899-6/01 DK/ME/151895-7A/02 TK/CA/D0208651-C/02 Chukkar/MN/14951-7/98 Environment/NY/5626-1/98 Environment/NY/5626-2/98 Unknown/NY/101250-18/01 Recent DK/NY/191255-79/02 LBM DK/NY/185502/02 DK/NY/191255-59/02 DK/NY/186875/02 Environment/NY/5626-17/98 Pheasant/NJ/1355/98 Avian/NY/31588-2/00 CK/NY/51375/00 Avian/NY/31588-3/00 DK/NY/44018-1/00 DK/NY/44018-2/00 CK/TX/167280-4/02 CK/TX/298313/04 (HP) CK/Puebla/8624-604/94 (HP) CK/Queretaro/14588-19/95 (HP) 10 changes

Figure 8.8. Phylogenetic tree based on nucleotide sequences of the H5 HA gene. The tree was generated by the maximum parsimony method with the PAUP*4.0b10 program with bootstrap replication (100 bootstraps) and a heuristic search method. The tree is rooted with A/chicken/ Scotland/59. Previously identiﬁ ed highly pathogenic viruses are indicated as HP in parentheses. *, Ruddy Turnstone/NJ/2242/00 virus belongs to the recent live-bird market phylogenetic lineage, but the virus was not recovered from live-bird market (LBM). Abbreviations: CK, chicken; DK, duck; TK, turkey. Standard two-letter abbreviations are used for states in the United States. (Used with permission of Journal of Virology [45].)

203 204 Avian Influenza

Table 8.3. Results of experimental studies in birds inoculated in the caudal thoracic air sac, intratracheally/orally, or intranasally with the HPAI strain, A/chicken/Pennsylvania/1370/1983 (H5N2), obtained from the 1983–1984 HPAI outbreak (4, 61, 98). Last day of virus Seroconversion Species Mortality Virus isolation shedding in survivors

Chicken 100% (10/10) NRa 4NSa Turkey 100% (10/10) NR 4 NS Guinea fowl 50% (15/30) 100% (30/30) 10 100% (15/15) Pheasants 38% (6/16) 100% (16/16) 7 100% (10/10) 0% (0/18) 100% (18/18) 15 100% (18/18) Partridge 19% (5/27) 89% (24/27) 7 100% (23/23) Japanese quail 10% (1/10) NR 4 78% (7/9) Domestic ducks 0% (0/32) 0% (0/32) NAa 13% (4/32) 0% (0/10) 0% (0/10) 0% (0/10) 0% (0/12) 8% (1/12) 100% (12/12) a NR = not reported, NS = no survivors, NA = not applicable.

America identifi ed a distinct lineage of H5 AI broiler chickens in central Mexico (22, 96). In May viruses with a close relationship between the 1983 1994, an H5N2 LPAI virus was identifi ed in samples LP and HPAI outbreak viruses (PA/83 lineage) (45) from 6-week-old broilers experiencing respiratory (Fig. 8.8). Also, this PA/83 lineage contained LPM disease and increased mortality rates that varied viruses from 1985–1986 New England H5N2 LPAI from 12% to 18%. By the end of 1994, LPAI was viruses, with the last recorded virus in the PA/83 diagnosed in commercial poultry from 11 Mexican lineage being from a bird in the LPM of Miami, states, most experiencing respiratory disease, but Florida, obtained during 1989 (45). layers and broiler breeders also exhibited decreased In experimental studies, gallinaceous birds were egg production. In experimental studies, intravenous easily infected and shed the virus, but mortality or intranasal inoculated chickens had interstitial varied with host species including 100% mortality pneumonia; lymphoid depletion and necrosis in the in chickens and turkeys but lower rates in other gal- spleen, cloacal bursa, and thymus; and necrosis and linaceous species (Table 8.3) (4, 61, 98). Also, infl ammation in the kidneys (81). The viruses had unlike the classic fowl plague viruses, this H5 HPAI hemagglutinin proteolytic cleavage site sequence of virus took longer to kill chickens than the H7N7 PQRETR/GLF, consistent with an LPAI virus. A HPAI viruses. Domestic ducks were resistant to survey in May 1994 covering 80% of commercial H5N2 HPAI experimental infection and did not broiler and broiler breeders in Queretaro identifi ed become ill or die, and only a few showed serological an 85% infection rate, but samples in December evidence of infection (4, 61), but in one study with 1994 covering 80% of commercial poultry popula- a high intratracheal challenge dose, all ducks became tion identifi ed only a 5% infection rate (67). infected as evident by seroconversion (98). The LPAI viruses were shed for up to 30 days after The Change to HPAI inoculation in chickens (61). Two separate events of conversion of H5N2 LPAI to HPAI viruses were reported (63). First, in the last H5N2 HIGH PATHOGENICITY AVIAN week of November 1994, 25- to 90-week-old layers INFLUENZA IN MEXICO DURING 1994–1995 on a 1.25-million bird farm in Puebla, Mexico, developed clinical signs of depression, with some The Beginning as an LPAI Outbreak prostration, more rapid and greater drops in egg In the fall of 1993 and into early 1994, problems of production, and a few birds exhibiting nervous signs undiagnosed respiratory disease were reported in such as torticollis (22, 67, 96). Compared with the 8 / High Pathogenicity Avian Influenza in the Americas 205 previous reports of LPAI virus infections, respira- and the latter was derived from the LPAI viruses. tory signs were milder and inconsistent in the Puebla These LPAI and HPAI viruses occupied a separate cases. Chickens on other layer farms in the area were lineage among the North American H5 AI viruses affected and had the presentation of generalized (Fig. 8.8) (46, 80). Most likely, the lineage repre- depression, and severe reduction in feed and water sents a single introduction, but since introduced in consumption, while some birds had pneumonia, air- 1993 to poultry, these H5N2 viruses have undergone sacculitis, peritonitis, petechiae in epicardial and changes with further development of several distinct abdominal fat, and hemorrhages in the ventriculus sublineages of H5 in different geographic regions and proventriculus (67). However, the mortality such as in Guatemala and El Salvador (80). pattern was 10% to 20% per house and egg production drops of only 20% to 25% (67). The mortality Control may have been low because of preexisting immunity The concurrent widespread infection with LPAI in chickens from H5N2 LPAI virus infections (67). virus brought new challenges in diagnosis and The isolated virus when intravenously inoculated control of HPAI. The variable presence of antibod- into chickens did not cause high mortality. However, ies to H5N2 LPAI virus in poultry of 11 Mexican chickens that died had lesions consistent with HPAI states in June-December 1994 produced partial pro- and the virus had an amino acid sequence at the tection from HPAI virus, thus limiting the morbidity hemagglutinin cleavage site of PQRKRKTR/GLF and mortality rates, and lesions following HPAI that was compatible with HPAI virus (63, 81, 85). virus exposure (67). In addition, the wide distribu- Passage of this original fi eld isolate in the 14-day tion of LPAI throughout central Mexico and the embryonating-chicken-egg (ECE) model resulted in abrupt appearance of HPAI made a stamping-out derivatives that were highly lethal for chickens and policy beyond the immediate resources of the produced lesions consistent with HPAI (81). In addi- country. The government devised and implemented tion, the derivatives produced more severe necrotic a control program using immediate stamping-out lesions in the heart and pancreas than the original of HPAI-infected fl ocks, disinfection of affected fi eld isolates (81). premises, quarantine measures in the region, strict Second, in early January 1995, a broiler breeder movement control of poultry and poultry products, fl ock in Villa del Marques, Queretaro, experienced increased biosecurity, implementation of serologic a complete decline in egg production over the 6-day surveillance, use of inactivated H5N2 vaccine, and clinical period, and additional cases in breeders and controlled marketing of LPAI virus–infected chick- broilers were noted in the state of Queretaro (67, ens (95, 96). The last HPAI virus was isolated in 96). Typically, the mortality was 10% to 15% in June 1995, but H5N2 LPAI continues to circulate breeders, while in broilers, the mortality was 50% poultry in central Mexico and has spread to Guate- to 70% and typically seen in birds 6 to 6.5 weeks of mala and El Salvador in 2000 and 2001, respectively age, but cases as young as 6 days of age were (80, 95). Vaccination began in January 1995 with an reported (67). In all cases, the lesions were typical inactivated AI vaccine, and in 1998, a recombinant for HPAI (67). Based on the IVPI, all isolates for fowlpox vaccine containing an H5 AI virus gene next 2 months in Queretaro were highly lethal for insert was also licensed and used (95). By mid-2005, chickens and had hemagglutinin proteolytic cleav- over 1.5 billion doses of inactivated AI and 1.6 age of PQRKRKTR/GLF (28, 33, 64). However, an billion doses of recombinant fowlpox vaccine have isolate from June 1995 had double duplication with been used in Mexico, Guatemala, and El Salvador insertion of four basic amino acids at the cleavage (82). site for a sequence of PQRKRKRKTR/GLF, but having the same IVPI and lesions as the earlier H7N3 HIGH PATHOGENICITY AVIAN H5N2 HPAI viruses (64, 81). HPAI was also diag- INFLUENZA IN CHILE DURING 2002 nosed in the state of Jalisco (67). Beginning with the end of April and continuing into fi rst week of May 2002, broiler breeders on a large Source farm in San Antonio, central Chile, developed a Based on hemagglutinin sequence, the Mexican syndrome characterized by a slight decline in egg H5N2 LPAI and HPAI viruses were closely related production (10%), egg peritonitis and low mortality, 206 Avian Influenza which was diagnosed as H7N3 LPAI virus (48, 65). Virus On May 23, 2002, the disease made an abrupt The virus from samples collected on May 9, 2002, change, causing high mortality and cessation in egg from the index farm was an H7N3 LPAI virus with production in chickens in some barns on the same intravenous pathogenicity index of 0.0 (48). Samples farm (48, 65). The overall mortality was 26.7% collected on June 1–8, 2002, during the depopula- before the depopulation occurred (48). tion of the index farm, were H7N3 HPAI viruses On the fi rst of June 2002, a clinical disease with with IVPI ranging from 2.43 to 3.00 (79). The initial upper respiratory signs, followed by explosive H7N3 LPAI virus had a hemagglutinin proteolytic increase in deaths, was noted in two barns of turkey cleavage site of PEKPKTR/GLF typical of other breeder poults that were located 4 km from the index LPAI viruses and did not produce cytopathic effect farm and functionally connected to the index farm in chicken embryo fi broblast cultures without the (65). addition of trypsin (79). In the HPAI viruses, the hemagglutinin proteolytic cleavage site had inser- Lesions tions of 10 amino acids through nonhomologous The chickens with H7N3 HPAI virus infection had recombination mechanism with the inserts being cyanotic combs and wattles, petechiae in skeletal derived from 30 base pairs obtained from the nucleo- muscle and heart, subcutaneous petechiae of the protein gene (nucleotides 1268–1297) of the same legs, petechiae in the pancreas and other organs, and virus (79). Among the H7N3 HPAI viruses, two subcutaneous edema (48). In experimental studies, cleavage site sequences were identifi ed: PEKPKTC- the H7N3 LPAI virus produced infection in only SPLSRCRETR/GLF and PEKPKTCSPLSRCRETR/ 25% of specifi c-pathogen–free (SPF) chickens that GLF (79). The HPAI viruses plaqued in chicken received a high intranasal challenge dose (106 mean embryo fi broblast cultures with or without the addi- embryo infectious doses [EID50]), indicating the tion of trypsin (79). The mutation from LP to HP virus was poorly adapted to chickens and most likely was presumed to have occurred in the fi rst barn on a recent introduction (38). Intravenous inoculation the index farm (48). using the standard pathogenicity test produced 25% The Chilean H7N3 AI viruses were not closely mortality and infected birds sampled on day 3 after related to North American H7 AI viruses; that is, inoculation had severe nephrosis with lymphocytic they were genetically distinct from North American infl ammation, mild histiocytic interstitial pneumo- Avian, Eurasian Avian, and Australian Avian and nia, and mild necrotizing pancreatitis with AI viral Equine type 1 lineages of H7 infl uenza A viruses antigen in necrotic kidney tubules (38). These (79). Initially, the closest related virus was A/turkey/ changes were similar to lesions produced by other Oregon/1971, which had only 82.1% hemagglutinin LPAI viruses intravenously inoculated into chickens similarity to Chilean H7 viruses, quite distant in (87–89). However, the H7N3 HPAI virus produced genetic relationship (79). Subsequently, an H7N3 a lethal infection in 100% of chickens when given LPAI virus was isolated from a cloacal swab obtained by either intravenous or intranasal routes (38). Spe- from a cinnamon teal (Anas cyanoptera) on Lake cifi c lesions seen with HPAI infection included focal Titicaca, Bolivia, on October 27, 2001 (70). This hemorrhages on the comb, petechial hemorrhages at cinnamon teal LPAI virus had 96.6% sequence sim- the esophageal–proventricular junction, proventric- ilarity for the hemagglutinin to Chilean LPAI virus ular mucosa, edema, and congestion of the lung, and represents the closest relative to the Chilean and petechiation of the spleen (38). Histopathologi- outbreak H7N3 AI viruses (70). However, the teal cal changes seen included severe necrosis, hemor- virus was not the immediate source for the Chilean rhage, and/or infl ammation in multiple visceral outbreak viruses because the neuraminidase, matrix, organs with infl uenza virus localized predominantly and nonstructural gene segments were more closely in endothelium of blood vessels throughout most related to other AI viruses, indicating multiple reas- tissues, and less frequently in histiocytes and cel- sortment events had occurred (70). lular debris of lymphoid tissues (38). Even less consistently, cardiac myocytes, hepatocytes, Kupffer’s Control Measures cells, glandular epithelial cells, microglial cells, and During the initial phase of the eradication program, neurons became infected (38). the primary diagnostic and surveillance tests used 8 / High Pathogenicity Avian Influenza in the Americas 207 were virus isolation in embryonating chicken eggs needed to create the Chilean H7N3 LPAI virus, and antibody detection by ELISA, agar gel immu- whether in wild birds or agricultural systems. nodiffusion (AGID), and hemagglutination inhibition tests (48). At the end of the outbreak, the H7N3 HIGH PATHOGENICITY AVIAN real-time reverse transcriptase–polymerase chain INFLUENZA IN BRITISH COLUMBIA, reaction (RRT-PCR) test was the offi cial diagnostic CANADA, DURING 2004 test for virus detection (48). Quarantine was put in place on the infected farms The Beginning as an LPAI Outbreak (48). Actions taken included movement restrictions, On February 5, 2004, 52-week-old chickens in a continuous offi cial supervision, and diagnostic and 9000-bird broiler breeder fl ock in Frazier Valley, surveillance sample collection and testing (48). The British Columbia, experienced a sudden decrease in program used a 3-km infected zone and 10-km sur- feed consumption, a 20% drop in egg production, veillance zone for each of the two outbreak farms and an 0.5% increase in mortality over a 72-hour (48). In total, 150,500 broiler breeders died, and period (15). On February 15, 2004, 24-week-old 465,000 broiler breeders, 18,500 turkey breeders, chickens in an 8000-bird broiler breeder fl ock in an and 116,000 hatching eggs were destroyed (48, 65). adjacent barn on the same farm experienced 25% The depopulation of broiler breeders on the fi rst mortality in 48 hours (15). Lesions in the younger outbreak farm occurred June 1–8, 2002, and depop- birds were more pronounced than in the older birds ulation of turkeys in two of eight barns on the second and included mild generalized fi brinous pneumonia outbreak farm occurred on June 15–19, 2002 (48). in the hilus, which was nonspecifi c (15). Histologi- National serological and virological surveys were cally, there was renal tubule necrosis, lymphocytic conducted in July and August 2002, and workers did tracheitis with epithelial necrosis, moderate necrosis not fi nd evidence of additional H7N3 AI cases (48). of pulmonary epithelium with interstitial edema, and However, complicating the diagnosis and eradica- associated mixed infl ammatory cells (15). tion efforts was the detection of antibodies to H5N2 On March 4, 2004, a second farm, 3 km west, had AI virus in broilers and broiler breeders in another a chicken fl ock with sudden increased mortality, and region of the country (65). There was no evidence H7N3 HPAI virus was isolated (60). Additional of clinical disease, and the antibodies were attrib- infected farms in Frazier Valley were identifi ed uted to an inclusion body hepatitis vaccine that was based on screening cloacal and oropharyngeal swabs contaminated with inactivated H5N2 AI viral antigen using RRT-PCR assay with a described assay (72). (65). An H5 AI viral hemagglutinin gene was detected in the hepatitis vaccine by RRT-PCR, and Lesions gene sequencing and phylogenetic analysis revealed Gross lesions in experimentally inoculated SPF 4- to that this virus was related to the H5 Mexican lineage 6-week-old chickens and commercial mature layers (E. Spackman, unpublished data, February 20, were compatible with HPAI and included edema and 2007). Repeated vaccination of a group of SPF hemorrhage in nonfeathered skin and multiple inter- chickens with the contaminated vaccine produced nal organs (60). Histological lesions were more antibodies to type A infl uenza virus (D. Swayne, severe in oronasally inoculated layers than the intra- unpublished data, February 20, 2007). venously inoculated SPF 4- to 6-week-old chickens, probably because of the rapid death induced by Source intravenous route of exposure (60). Histologically, The immediate source of the H7N3 LPAI virus was the lesions were usually hemorrhage, edema, and unknown. However, the low infectivity of the H7N3 necrosis in respiratory and digestive tracts, skin, LPAI virus in intranasally inoculated chickens sug- kidneys, liver, pancreas, and lymphoid organs (60). gests a poorly adapted AI virus for chickens and Lesions that were unique in these cases compared most likely a recent introduction from a non- with prior HPAI virus infections in chickens included galliforme host, such as a wild bird (38). The H7N3 mild lesions in the brain of intravenous inoculated LPAI virus obtained in fall 2001 from a cinnamon chickens, presence of necrosis in lamina propria of teal was the closest relative but not the immediate the esophagus and esophageal glands, and severe source (70). Multiple gene reassortments were necrosis of hepatocytes (60). 208 Avian Influenza

Virus during 2000, but there was no direct epidemiologic An H7N3 virus was isolated from the 52-week-old connection and the relatedness was not high (15). chickens on the index farm had an IVPI of 0.0 Retrospective serological evaluation on the index and hemagglutinin proteolytic cleavage site of farm taken 3 weeks before the outbreak failed to PENPKTR/GLF (15, 60). The lesions in chickens on identify infl uenza A antibodies (15). the index farm were not consistent with a specifi c etiology but did resemble lesions seen in natural and Control Measures experimental cases of LPAI in chickens (83, 88–89), The outbreak lasted 91 days (14). On the index farm, and an LPAI virus was confi rmed by sequence and mass euthanasia was done by carbon dioxide gas on pathotyping data (15). Although the H7N3 AI virus February 21, 2004 (15). The chickens were moved obtained from the second fl ock of the index farm had from the barn to an outside composting facility 400 an identical hemagglutinin cleavage site sequence m away (15). On March 11, 2004, a control area was (PENPKTR/GLF) to the H7N3 LPAI virus from the designated with restrictions on movement of any fi rst fl ock, it produced cytopathic effect in QT-35 captive birds including day-old chicks and hatching cell cultures in the absence of exogenous trypsin eggs, poultry products, and any equipment or sup- (60), a trait more consistent with HPAI virus. Fur- plies exposed to birds (58). A stamping-out program thermore, the in vivo pathotyping test yielded an was implemented on all farms with infected birds. IVPI of 2.96, and chickens that died had severe However, on April 5, 2004, to stop spread and to depression, labored breathing, periorbital edema, accelerate the eradication, strategic depopulation cyanosis of comb and wattles, and hemorrhages of was begun on noninfected premises within the the feet and legs (60). A virus reisolated from tissue control area (i.e., within 3 km of infected premises) of a dead chicken had a cleavage site sequence (14, 57). Poultry from these 410 non-infected of PENPKQAYRKRMTR/GLF (60). These latter premises were slaughtered, rendered, or centrally characteristics suggested an HPAI virus for the composted and represented 90% of the poultry in the second barn on the index premise, and the initial Frazier Valley (14). Also, 553 backyard fl ocks rep- characterization of an LPAI virus cleavage site may resenting more than 18,000 birds were depopulated have been the result of amplifi cation and sequencing because of proximity to infected premises (14). an LPAI virus from a sample composed of a mixture On May 20, 2004, the birds on the last infected of LPAI and HPAI viruses (60). The insertion of premises were destroyed (58). In total, 1,204,564 seven amino acids resulted from nonhomologous birds from 42 commercial farms and 11 backyard recombination with insertion of nucleotides 737– fl ocks were diagnosed as infected and were stamped- 757 of the matrix gene into the hemagglutinin pro- out (58). In addition, approximately 16 million com- teolytic cleavage site (60). mercial poultry were eliminated in the strategic A total of 40 isolations of H7N3 AI virus were depopulation program (58). On June 18, 2004, the made from the affected farms, but the hemagglutinin last affected premise underwent C&D, and the out- cleavage site sequences had slight variations with break declared eradicated on July 9 (58). The poultry some additional amino acid substitutions within the industry endured a 51-day downtime from the last seven-amino-acid matrix gene insert (60). Thirty- detection of HPAI virus until repopulation with seven of the isolates had the insert and included birds began (14). Individually infected premises had sequences of PENPKQAYQKRMTR/GLF (n = 24), to remain empty of birds for 21 days after C&D was PENPKQAYKKRMTR/GLF (n = 4), PENPKQAY- complete on the last infected premises in the control HKRMTR/GLF (n = 3), PENPKQAHQKRMTR/ area. GLF (n = 1), PENPRQAYRKRMTR/GLF (n = 4), Key components in the successful eradication and PENPKQACQKRMTR/GLF (n = 1) (60). The program included zoning; animal identifi cation and remaining H7N3 AI viruses had the LPAI hemag- traceability; demographics and mapping; biosecu- glutinin proteolytic cleavage site of PENPKTR/GLF rity and biocontainment, including movement (60). restrictions and disinfection checkpoints; preventing The source of the virus on the index farm was unauthorized access to infected properties; targeted unknown (15). The virus was most closely related depopulation with rapid isolation, containment, and to an H7 virus isolated from turkeys in Ontario elimination of infected birds; and composting as the 8 / High Pathogenicity Avian Influenza in the Americas 209 means of infected carcass disposal (57). The farm- wearing glasses but had feather exposure to the to-farm spread of the virus most likely occurred conjunctiva (91). Neither were on prophylactic through movement of people, contaminated equip- anti-infl uenza drugs (oseltamivir), nor were they ment, and infected birds, but airborne transmission vaccinated against human infl uenza virus (91). Clin- in the densely populated poultry area through dust ical signs of the infections included conjunctivitis, and feathers from depopulation activities of nearby headache, and coryza 1 to 3 days after exposure to infected fl ocks may have contributed (57). There- infected poultry (30). Both patients subsequently fore, appropriate containment must be used to avoid received oseltamivir and had an uneventful recovery airborne and wind-blown dispersion of virus from (91). The two human H7N3 isolates had hemagglu- aerosols generated by open air carcass grinding, tinin proteolytic cleavage sites of PENPKQAY- wind-blown feathers and dust from litter removal, QKQMTR/GLF and PENPKQAYQKRMTR/GLF or transport of affected birds for off-site disposal, (30). The latter was HP based on in vivo pathotyping especially through densely populated poultry areas in chickens, and the sequence of the former matched (15). that of chicken isolates that were HPAI viruses (30). Financial Aspects The economic cost for the outbreak were great: H5N2 HIGH PATHOGENICITY AVIAN $CDN 360 million in gross economic losses, $CDN INFLUENZA IN TEXAS DURING 2004 63.3 million in costs to farmers and farm organiza- A case of H5N2 HPAI was diagnosed in a small tions, $CDN 63.7 million in federal indemnity com- fl ock of broilers in Gonzalez, Texas, on February pensation, and $CDN156 million in uncompensated 16, 2004 (46, 62) (Fig. 8.9). The owner had noted downstream losses to poultry allied service industry increased mortality (3%) and respiratory signs (14). Approximately 1700 people lost jobs or had (moist rales) and submitted chickens for diagnostic reduced employment during the outbreak. investigation (46). These birds had fi brinous airsacculitis and focal pulmonary edema (46). Histologi- Human Infections cally, the lesions were fi brinoheterophilic pneumonia Two workers on the depopulation crews became with edema and moderate severe lymphocytic tra- infected and developed clinical signs (30, 91). One cheitis with epithelial cell necrosis (46). Experi- was not wearing eye protection, and the other was mentally, intranasally inoculated virus in chickens

Figure 8.9. Location of 2004 H5N2 HPAI outbreak in Gonzales, Texas, United States. (Used with permission of Journal of the American Veterinary Medical Association [62].) Figure 8.10. Map of 2004 H5N2 HPAI outbreak surveillance in Texas, United States. (Used with permission of Journal of the American Veterinary Medical Association [62].) 210 Avian Influenza produced tracheitis and bronchitis with the infl uenza called a “pseudo-HPAI virus” because in vivo virus replication limited to respiratory epithelium pathotyping test in chickens failed to produce illness (46). In intravenously inoculated chickens, the or death and passage of the virus in an ECE model lesions were predominately necrosis in the kidneys failed to increase virulence as has been shown in the and pancreas (46), similar to lesions produced with past with other H5 LPAI viruses (i.e., the virus failed intravenously inoculated LPAI viruses (87–89). The to be classifi ed as HPAI based on in vivo testing in Texas/04 HPAI virus was pathobiologically similar chickens) (31, 32, 46, 84). However, the H5N2 AI to LPAI virus. virus had a hemagglutinin proteolytic cleavage site The affected farm had 6608 chickens and was a of PQRKKR/GPL, which was identical to the H5N1 supplier to retail markets in the LPM system of HPAI virus A/chicken/Scotland/1959 and thus was Houston, Texas (62). The chickens on the index classifi ed as HPAI virus based on molecular criteria farm were depopulated on February 21, 2004 (46). (59, 92). Finally, the presence of AI viral antigen in In addition, birds in two LPM retail establishments some cardiac myocytes after passage in ECE model were confi rmed to have HPAI infections, and an suggests an early stage in transition for LPAI to additional three markets were identifi ed as danger- HPAI virus (46). ous contacts and were depopulated (46). The initial The Texas H5N2 2004 HPAI virus was most introduction was determined to be between February closely related to a previous H5N3 LPAI virus iso- 2 and 11, 2004 (62). Epidemiological investigation lated from chickens in Texas LPM system during suggested the virus was introduced to the farm from 2002 (46). These two H5 AI viruses were most birds taken and then returned to the farm from closely related to the LPM H5 LPAI virus lineage, Houston LPMs (62). Following fi ve rounds of sur- which is distinctly different from Pennsylvania/83, veillance in commercial and noncommercial poultry Mexican/94, and Eurasian lineages of H5 HPAI for antibodies (2928 samples) and virus (3595 viruses (46) (Fig. 8.8). However, the Texas/04 H5N2 swabs), all with negative results, the outbreak was HPAI virus was a reassortant, only having similar declared over on April 1, 2004 (46). sequences with hemagglutinin, nonstructural, matrix, The initiation of diagnostic investigation was not and nucleoprotein genes of Texas/02 H5N3 LPAI made based on a high mortality event that triggered virus (46). The polymerase complex and neuramin- a diagnostic investigation, but the diagnosis was idase genes were phylogenetically more closely made based on routine diagnostic samples submitted related to other AI viruses (46). The N2 neuramini- for determination of the cause of nonspecifi c respira- dase gene of the Texas/04 H5N2 HPAI virus had a tory diseases (62). Success for quick eradication was stalk deletion, which is a marker for poultry adapta- attributed to diagnosis of the index farm, depopulation and suggests the virus had been circulating in tion and disposal within 24 hours of diagnosis, poultry for some time, probably in the LPM system existence of experienced veterinary laboratory diag- (46, 47). nostic infrastructure and testing for rapid surveil- The Texas/04 HPAI virus was well adapted to lance support, and cooperation from commercial and chickens as evident by replication to high titers noncommercial poultry operations and their employ- in respiratory system of intranasally inoculated ees (62). This was the fi rst detection of AI through chickens (46). use of the RRT-PCR on routine diagnostic samples. However, molecular testing had been used during Control the H7N3 HPAI epidemics in Chile (2002) and In the control plan, movement restrictions were H7N7 HPAI outbreak in the Netherlands (2003) (48, placed on all poultry fl ocks within 8 km (Infected 74). The fi rst use of RRT-PCR as an AI diagnostic Zone), and surveillance testing was conducted on tool was in April 8 through 10, 2002, in the H7N2 all fl ocks within 16 km (Surveillance Zone) (62) LPAI eradication efforts in the New York and New (Fig. 8.10). Commercial fl ocks within 50 km (Buffer Jersey LPMs (53, 71). Zone) were also surveyed. In addition, movement restrictions and surveillance were conducted on all Virus fl ocks linked epidemiologically to the infected fl ock The isolated H5N2 AI virus presented with some (62). Surveillance samples were analyzed for anti- unique biological properties. This virus has been nucleoprotein antibodies by AGID test and virus 8 / High Pathogenicity Avian Influenza in the Americas 211 detection by RRT-PCR on 39 premises in the Canada during 2007. There were many common Infected Zone, 167 premises in the Surveillance features of these outbreaks, including recognition of Zone and 162 premises in the Buffer Zone, which most frequent gross lesions of edema and cyanosis involved 3608 AGID tests and 3685 RRT-PCR tests of comb and wattles, petechiae in mucosa of proven- of pooled tracheal or cloacal swab samples (62). triculus and ventriculus (gizzard), and petechiae in This surveillance identifi ed two infected LPM retail fat of heart and viscera. In most outbreaks, control establishments in Houston. However, as a voluntary was accomplished by identifi cation of infected and action, all fi ve retail markets in Houston were dangerous contact premises; quarantine of these depopulated and completed a C&D program. All premises, enhancements to biosecurity; elimination other samples were negative for AI virus or infection of infected poultry; movement restrictions on people, by AI virus. equipment, and birds; surveillance for virus and antibody evidence of infections in poultry, and C&D H7N3 HIGH PATHOGENICITY AVIAN of equipment and facilities. INFLUENZA IN SASKATCHEWAN, Several new features were recognized during CANADA DURING 2007 these outbreaks. First, the 1983–1984 epidemic was An outbreak of H7N3 HPAI was reported in a mul- the fi rst documented cases of an LPAI virus mutat- tiage commercial broiler breeder fl ock and hatchery ing and becoming an HPAI virus. Second, from this in Regina Beach, Saskatchewan, Canada; an area of outbreak, came the development of national and sparsely populated commercial and non-commercial international molecular criteria for the classifi cation poultry (21a, 59a). The outbreak began with of AI viruses as HP, which is now used in addition increased mortality in one barn of 24 week-old to in vivo chicken pathotyping for declaration of an spiker males on September 22, which over the next HPAI virus. Third, the development of the rapid 5 days experienced over 95% mortality for a total RRT-PCR diagnostic test during the 2002 H7N2 farm mortality of 1.1%. A poultry veterinarian iden- LPAI LPM closure program in the United States and tifi ed lesions compatible with HPAI on September its initial deployment for use in the Chilean H7N3 23 and farm quarantine was implemented on Sep- HPAI outbreak was a success. tember 23. An H7N3 HPAI virus was detected, isolated and sequenced between September 23 and 26. ACKNOWLEDGMENTS The 48,560 remaining chickens were euthanized by Richard Slemons, Elizabeth Buckles, and Susan whole house CO2 gas on September 30 and on farm Wilzer are thanked for assistance in identifying and burial was completed by October 4. Surveillance by obtaining publications from the 1920s on fowl October 3 was negative in the 3 km zone in six plague in the United States. backyard fl ocks and the 10 kg surveillance zone in 1 commercial and 8 backyard fl ocks. Serological REFERENCES evidence suggested the 32,000 breeder hens in two 1. Acland, H.M., L.A. Silverman Bachin, and R.J. barns had been previously infected by H7 LPAI Eckroade. 1984. Lesions in broiler and layer virus which protected the hens from disease and chickens in an outbreak of highly pathogenic avian mortality when the HPAI virus infected spiker males infl uenza virus infection. Veterinary Pathology were introduced into the barns. The source of the 21:564–569. virus is unknown but wild birds are suspected. 2. Alexander, D.J. 1987. Avian infl uenza—historical aspects. In: B.C. Easterday and C.W. Beard (eds.). CONCLUSIONS Proceedings of the Second International Sympo- From 1924 to 2004, there were nine HPAI epidem- sium on Avian Infl uenza, Georgia Center for Continuing Education, the University of Georgia, ics in the Americas: fowl plague in the United States Athens, Georgia, USA, September 3–5, 1986. during 1924–1925 and 1929, H5N9 HPAI in Canada Symposium on Avian Infl uenza, US Animal Health during 1966, H5N2 HPAI in the United States during Association: Richmond, VA, pp. 4–13. 1983–1984, H5N2 HPAI in Mexico during 1994– 3. Alexander, D.J. 1987. Criteria for the defi nition of 1995, H7N3 HPAI in Chile during 2002, H7N3 pathogenicity of avian infl uenza viruses. In: B.C. HPAI in Canada during 2004, H5N2 HPAI in the Easterday and C.W. Beard (eds.). Proceedings of United States during 2004, and H7N3 HPAI in the Second International Symposium on Avian 212 Avian Influenza

Infl uenza, Georgia Center for Continuing Educa- 13. Boughton, I.B., and E.A. Tunnicliff. 1925. Euro- tion, the University of Georgia, Athens, Georgia, pean Fowl pest in Illinois. Journal of the American USA, September 3–5, 1986. Symposium on Avian Veterinary Medical Association 20:183–185. Infl uenza, US Animal Health Association: Rich- 14. Bowes, V.A. 2007. After the outbreak: how the mond, VA, pp. 228–245. British Columbia commercial poultry industry 4. Alexander, D.J., G. Parsons, and R.J. Manvell. recovered after H7N3 HPAI. Avian Diseases 1986. Experimental assessment of the pathogenic- 51(Suppl.):313–316. ity of eight infl uenza A viruses of H5 subtype for 15. Bowes, V.A., S.J. Ritchie, S. Byrne, K. Sojonky, chickens, turkeys, ducks and quail. Avian Pathol- J.J. Bidulka, and J.H. Robinson. 2004. Virus char- ogy 15:647–662. acterization, clinical presentation, and pathology 5. Anonymous. 1925. History of fowl pest outbreak. associated with H7N3 avian infl uenza in British Journal of the American Veterinary Medical Columbia broiler breeder chickens in 2004. Avian Association 20(2):273. Diseases 48(4):928–934. 6. Bankowski, R.A. 1981. Introduction and objec- 16. Broerman, A. 1925. A disease of fowls caused by tives of the symposium. In: R.W. Bankowski (ed.). a fi lterable virus. The Veterinary Alumni Quarterly Proceedings of the First International Symposium (Ohio State University, College of Veterinary on Avian Infl uenza, April 1981, Beltsville, Medicine) 12(1):107–109. Maryland. Symposium on Avian Infl uenza, US 17. Brunett, E.L. 1925. A fi lterable virus disease of Animal Health Association: Richmond, VA, pp. chickens (fowl plague?). Cornell Veterinarian vii–xiv. 15(1):4–8. 7. Barbeito, M.S., G. Abraham, M. Best, P. Cairns, 18. Brunett, E.L. 1925. The occurrence of a disease of P. Langevin, W.G. Sterritt, D. Barr, W. Meulepas, chickens in New York State, caused by a fi ltrable J.M. Sanchez-Vizcaino, M. Saraza, E. Requena, virus. Journal of the American Veterinary Medical M. Collado, P. mani, R. Breeze, H. Brunner, C.A. Association 19:497–498. Mebus, R.L. Morgan, S. Rusk, L.M. Siegfried, and 19. Buisch, W.W., A.E. Hall, and H.A. McDaniel. L.H. Thompson. 1995. Recommended biocontain- 1984. 1983–1984 lethal avian infl uenza outbreak. ment features for research and diagnostic facilities Proceedings of the United States Animal Health where animal pathogens are used. Revue Scienti- Association 88:430–446. fi que et Technique—Offi ce International des 20. Buscaglia, C., C. Espinosa, M.V. Terrera, and R. Epizooties 14(3):873–887. De Benedetti. 2007. Avian infl uenza surveillance 8. Barger, E.H., and L.E. Card. 1935. Fowl plague. in backyard poultry of Argentina. Avian Diseases In: Diseases and Parasites of Poultry. Lea & 51(Suppl.):467–469. Febiger: Philadelphia, pp. 166–170. 21. Cappucci, D.T., Jr., D.C. Johnson, M. Brugh, T.M. 9. Bean, W.J., Y. Kawaoka, and R.G. Webster. 1986. Smith, C.F. Jackson, J.E. Pearson, and D.A. Senne. Genetic characterization of H5N2 infl uenza viruses 1985. Isolation of avian infl uenza virus (subtype isolated from poultry in 1986. In: B.C. Easterday H5N2) from chicken eggs during a natural out- and C.W. Beard (eds.). Proceedings of the Second break. Avian Diseases 29:1195–1200. International Symposium on Avian Infl uenza, 21a. Castallan, D.M., C. Argue, and S. Stephens. 2007. Georgia Center for Continuing Education, the Uni- Detection of HPNAI H7N3 subtype in Saskatche- versity of Georgia, Athens, Georgia, USA, Sep- wan, 2007. Presented at 111th annual meeting of tember 3–5, 1986. Symposium on Avian Infl uenza, the U.S. Animal Health Association October 18– US Animal Health Association: Richmond, VA, 24, 2007, Reno, Nevada. pp. 207–214. 22. Chacon, M.F., and P.P. Ramirez. 1995. Aislamien- 10. Beard, C.W., M. Brugh, and D.C. Johnson. 1984. tos en Mexico del virus de infl uenza aviar. Procee- Laboratory studies with the Pennsylvania avian dings of the National Convention of Asociacion infl uenza viruses (H5N2). Proceedings of the Nacional de Expecialistas en Ciencias Avicolas United States Animal Health Association 88:462– 20:97–99. 473. 23. Easterday, B.C., and B. Tumova. 1972. Avian 11. Beaudette, F.R. 1925. Observations upon fowl infl uenza. In: M.S. Hofstad, B.W. Calnek, C.F. plague in New Jersey. Journal of the American Helmbolt, W.M. Reid, and H.W. Yoder, Jr. (eds.). Veterinary Medical Association 20:186–194. Diseases of Poultry, 6th ed. Iowa State University 12. Beaudette, F.R., C.B. Hudson, and A.H. Saxe. Press: Ames, IA, pp. 670–700. 1932. An outbreak of fowl plague in New Jersey in 24. Eckroade, R.J., and L.A. Silverman-Bachin. 1986. 1929. Journal of Agriculture Research 49:83–92. Avian infl uenza in Pennsylvania. The beginning. 8 / High Pathogenicity Avian Influenza in the Americas 213

In: B.C. Easterday and C.W. Beard (eds.). Pro- Kobasa, T. Booth, and M. Paetzel. 2004. Novel ceedings of the Second International Symposium avian infl uenza H7N3 strain outbreak, British on Avian Infl uenza, Georgia Center for Continuing Columbia. Emerging Infectious Diseases Education, the University of Georgia, Athens, 10(12):2192–2195. Georgia, USA, September 3–5, 1986. Symposium 31. Horimoto, T., and Y. Kawaoka. 1995. Molecular on Avian Infl uenza, US Animal Health Associa- changes in virulent mutants arising from avirulent tion: Richmond, VA, pp. 22–32. avian infl uenza viruses during replication in 14- 25. Fichtner, G.J. 1984. Problems associated with day-old embryonated eggs. Virology 206:755– lethal avian infl uenza eradication. In: B.C. Easter- 759. day and C.W. Beard (eds.). Proceedings of the 32. Horimoto, T., and Y. Kawaoka. 1998. A possible Second International Symposium on Avian Infl u- mechanism for selection of virulent avian infl u- enza, Georgia Center for Continuing Education, enza A viruses in 14-day-old embryonated eggs. the University of Georgia, Athens, Georgia, USA, Journal of Veterinary Medical Science 60(2):273– September 3–5, 1986. Symposium on Avian Infl u- 275. enza, US Animal Health Association: Richmond, 33. Horimoto, T., E. Rivera, J. Pearson, D. Senne, S. VA, pp. 415–420. Krauss, Y. Kawaoka, and R.G. Webster. 1995. 26. Fichtner, G.J. 1987. Population surveillance for Origin and molecular changes associated with Avian infl uenza (H5N2)-Pennsylvania-1983–1984. emergence of a highly pathogenic H5N2 infl uenza In: B.C. Easterday and C.W. Beard (eds.). Pro- virus in Mexico. Virology 213:223–230. ceedings of the Second International Symposium 34. Hoskins, H.P. 1925. Poultry diseases. Journal of on Avian Infl uenza, Georgia Center for Continuing the American Veterinary Medical Association Education, the University of Georgia, Athens, 20(2):137–140. Georgia, USA, September 3–5, 1986. Symposium 35. Hyde, A.M. 1929. Report of the Secretary of on Avian Infl uenza, US Animal Health Associa- Agriculture. p. 77. tion: Richmond, VA, pp. 150–154. 36. Jardine, W.M. 1925. Outbreak and suppression of 27. Fichtner, G.J. 1987. The Pennsylvania/Virginia European fowl pest. Report of the Secretary of experience in eradication of avian infl uenza Agriculture November 14:66. (H5N2). In: B.C. Easterday and C.W. Beard (eds.). 37. Johnson, S.R. 1925. European fowl pest in Michi- Proceedings of the Second International Sympo- gan. Journal of the American Veterinary Medical sium on Avian Infl uenza, Georgia Center for Association 20:194–202. Continuing Education, the University of Georgia, 38. Jones, Y.L., and D.E. Swayne. 2004. Comparative Athens, Georgia, USA, September 3–5, 1986. pathobiology of low and high pathogenicity H7N3 Symposium on Avian Infl uenza, US Animal Health Chilean avian infl uenza viruses in chickens. Avian Association: Richmond, VA, pp. 33–38. Diseases 48(1):119–128. 28. Garcia, M., J.M. Crawford, J.W. Latimer, M.V.Z.E. 39. Julien, R.C. 1925. Fowl pest in Indiana. Journal of Rivera-Cruz, and M.L. Perdue. 1996. Heterogene- the American Veterinary Medical Association ity in the hemagglutinin gene and emergence of the 20:178–179. highly pathogenic phenotype among recent H5N2 40. Kawaoka, Y., and R.G. Webster. 1989. Interplay avian infl uenza viruses from Mexico. Journal of between carbohydrate in the stalk and the length General Virology 77:1493–1504. of the connecting peptide determines the cleav- 29. Garnett, W.H. 1987. Status of avian infl uenza in ability of infl uenza virus hemagglutinin. Journal of poultry: 1981–86. In: B.C. Easterday and C.W. Virology 63:3296–3300. Beard (eds.). Proceedings of the Second Interna- 41. Krohn, L.D. 1925. A study on the recent outbreak tional Symposium on Avian Infl uenza, Georgia of a fowl disease in New York City. Journal of the Center for Continuing Education, the University of American Veterinary Medical Association 20:146– Georgia, Athens, Georgia, USA, September 3–5, 170. 1986. Symposium on Avian Infl uenza, US Animal 42. Krumwiede, C., H. Gerber, and D.J. Provost. 1925. Health Association: Richmond, VA, pp. 61–66. Laboratory observations on the virus of the malig- 30. Hirst, M., C.R. Astell, M. Griffi th, S.M. Coughlin, nant disease recently epizootic among poultry. M. Moksa, T. Zeng, D.E. Smailus, R.A. Holt, S. Journal of the American Veterinary Medical Jones, M.A. Marra, M. Petric, M. Krajden, D. Association 20(2):171. Lawrence, A. Mak, R. Chow, D.M. Skowronski, 43. Lang, G., O. Narayan, B.T. Rouse, A.E. Ferguson, Tweed S Aleina, S. Goh, R.C. Brunham, J. Robin- and M.C. Connell. 1968. A new infl uenza A virus son, V. Bowes, K. Sojonky, S.K. Byrne, Y. Li, D. infection in turkeys II. A highly pathogenic variant, 214 Avian Influenza

A/turkey/Ontario/7732/66. Canadian Veterinary Animal Health. Offi ce Internationale des Epizo- Journal 9:151–160. oties: Paris, pp. 81–89. 44. Lang, G., B.T. Rouse, O. Narayan, A.E. Ferguson, 58. Offi ce Internationale des Epizooties. 2004. Highly and M.C. Connell. 1968. A new infl uenza virus pathogenic avian infl uenza in Canada. Follow-up infection in turkeys. I. Isolation and characteriza- report No. 7 (fi nal report). Accessed on May 17, tion of virus 6213. Canadian Veterinary Journal 2005. 9(1):22–29. 59. Offi ce Internationale des Epizooties. 2006. Avian 45. Lee, C.W., D.A. Senne, J.A. Linares, P.R. Wool- infl uenza. Offi ce Internationale des Epizooties. cock, D.E. Stallknecht, E. Spackman, D.E. Swayne, Accessed on September 8, 2006. and D.L. Suarez. 2004. Characterization of recent 59a. Offi ce Internationale des Epizoofi es. 2007. Highly H5 subtype avian infl uenza viruses from US pathogenic avian infl uenza, Canada. Immediate poultry. Avian Pathology 33(3):288–297. notifi cation (09/28/2007), and follow-up reports 46. Lee, C.W., D.E. Swayne, J.A. Linares, D.A. Senne, No. 1 (10/5/2007) and 2 (10/18/2007). Accessed and D.L. Suarez. 2005. H5N2 avian infl uenza out- on October 27, 2007. break in Texas in 2004: the fi rst highly pathogenic 60. Pasick, J., K. Handel, J. Robinson, J. Copps, D. strain in the United States in 20 years? Journal of Ridd, K. Hills, H. Kehler, C. Cottam-Birt, J. Virology 79(6):3692–3702. Neufeld, Y. Berhane, and S. Czub. 2005. Interseg- 47. Matrosovich, M., N. Zhou, Y. Kawaoka, and R. mental recombination between the haemagglutinin Webster. 1999. The surface glycoproteins of H5 and matrix genes was responsible for the emer- infl uenza viruses isolated from humans, chickens, gence of a highly pathogenic H7N3 avian infl uenza and wild aquatic birds have distinguishable proper- virus in British Columbia. Journal of General ties. Journal of Virology 73(2):1146–1155. Virology 86(Pt 3):727–731. 48. Max, V.K., J.R. Herrera, R.Z. Moreira, and 61. Pearson, J.E., D.A. Senne, E.A. Carbrey, J.G. Gus- H.O. Rojas. 2007. Avian infl uenza in Chile: a suc- tafson, J.G. Landgraf, D.R. Cassidy, and G.A. cessful experience. Avian Diseases 51(Suppl.):363– Erickson. 1987. Laboratory support for the Penn- 365. sylvania/Virginia avian infl uenza outbreak. In: 49. Mohler, J.R. 1925. Eradication of European fowl B.C. Easterday and C.W. Beard (eds.). Proceed- pest. Report of the Chief of the Bureau of Animal ings of the Second International Symposium on Industry September 29:18–19. Avian Infl uenza, Georgia Center for Continuing 50. Mohler, J.R. 1926. Fowl pest in the United States. Education, the University of Georgia, Athens, Journal of the American Veterinary Medical Georgia, USA, September 3–5, 1986. Symposium Association 21:549–559. on Avian Infl uenza, US Animal Health Associa- 51. Mohler, J.R. 1929. Report of the Chief of the tion: Richmond, VA, pp. 39–50. Bureau of Animal Industry. August 24, 1929:1. 62. Pelzel, A.M., B.J. McCluskey, and A.E. Scott. 52. Moore, V.A. 1925. Fowl plague, a new chicken 2006. Review of the highly pathogenic avian disease, in America. The Cornell Veterinarian infl uenza outbreak in Texas, 2004. Journal of the 15(1):1–3. American Veterinary Medical Association 228(12): 53. Mullaney, R. 2003. Live-bird market closure activ- 1869–1875. ities in the Northeastern United States. Avian Dis- 63. Perdue, M.L., M. Garcia, J.R. Beck, M. Brugh, eases 47(Suppl):1096–1098. and D.E. Swayne. 1996. An Arg-Lys insertion 54. Narayan, O., G. Lang, and B.T. Rouse. 1969. A at the hemagglutinin cleavage site of an H5N2 new infl uenza A virus infection in turkeys. V. avian influenza isolate. Virus Genes 12:77– Pathology of the experimental disease by strain 84. turkey-Ontario 7732–66. Arch Gesamte Virus- 64. Perdue, M.L., M. Garcia, D. Senne, and M. Fraire. forsch 26:166–182. 1997. Virulence-associated sequence duplication 55. Nettles, V.F., J. Wood, and R.G. Webster. 1984. at the hemagglutinin cleavage site of avian infl u- Wildlife studies on avian infl uenza. Foreign Animal enza viruses. Virus Research 49:173–186. Disease Report 12:2–4. 65. Rojas, H., R. Moreira, P. Avalos, I. Capua, and S. 56. Nettles, V.F., J.M. Wood, and R.G. Webster. 1985. Marangon. 2002. Avian infl uenza in poultry in Wildlife surveillance associated with an outbreak Chile. Veterinary Record 151(6):188. of lethal H5N2 avian infl uenza in domestic poultry. 66. Roth, A.J. 1984. Avian infl uenza task force depop- Avian Diseases 29:733–741. ulation concern in Virginia. Proceedings of the 57. Offi ce Internationale des Epizooties. 2004. Canada. United States Animal Health Association 88:421– New activities of the veterinary services. In: World 429. 8 / High Pathogenicity Avian Influenza in the Americas 215

67. Salem, M. 1995. Avian infl uenza in Mexico. In: 78. Stubbs, E.L. 1943. Fowl pest. In: H.E. Biester and Proceedings of the 30th National Meeting on L. Devries (eds.). Diseases of Poultry. Iowa State Poultry Health and Processing, 30th ed. Ocean College Press: Ames, IA, pp. 493–502. City, Maryland, pp. 99–105. 79. Suarez, D.L., D.A. Senne, J. Banks, I.H. Brown, 68. Senne, D.A., B. Panigrahy, Y. Kawaoka, J.E. S.C. Essen, C.W. Lee, R.J. Manvell, C. Mathieu- Pearson, J. Suss, M. Lipkind, H. Kida, and R.G. Benson, V. Moreno, J.C. Pedersen, B. Panigrahy, Webster. 1996. Survey of the hemagglutinin (HA) H. Rojas, E. Spackman, and D.J. Alexander. 2004. cleavage site sequence of H5 and H7 avian infl u- Recombination resulting in virulence shift in avian enza viruses: amino acid sequence at the HA cleav- infl uenza outbreak, Chile. Emerging Infectious age site as a marker of pathogenicity potential. Diseases 10(4):693–699. Avian Diseases 40(2):425–437. 80. Suarez, D.L., E. Spackman, and D.A. Senne. 2003. 69. Slemons, R.D., and B.C. Easterday. 1972. Host Update on molecular epidemiology of H1, H5, and response differences among fi ve avian species H7 infl uenza virus infections in poultry in North to an infl uenza virus A/turkey/Ontario/7732/66 America. Avian Diseases 47:888–897. (Hav5N?). Bulletin of the World Health Organiza- 81. Swayne, D.E. 1997. Pathobiology of H5N2 tion 47:521–525. Mexican avian infl uenza virus infection of 70. Spackman, E., K.G. McCracken, K. Winker, and chickens. Veterinary Pathology 34:557–567. D.E. Swayne. 2006. H7N3 avian infl uenza virus 82. Swayne, D.E. 2006. Principles for vaccine protec- found in a South American wild duck is related to tion in chickens and domestic waterfowl against the Chilean 2002 poultry outbreak, contains genes avian infl uenza: Emphasis on Asian H5N1 high from equine and North American wild bird lin- pathogenicity avian infl uenza. Annals of the New eages, and is adapted to domestic turkeys. Journal York Academy of Sciences 1081:174–181. of Virology 80(15):7760–7764. 83. Swayne, D.E., and D.J. Alexander. 1994. Confi r- 71. Spackman, E., D.A. Senne, L.L. Bulaga, T.J. mation of nephrotropism and nephropathogenicity Myers, M.L. Perdue, L.P. Garber, K. Lohman, of 3 low-pathogenic chicken-origin infl uenza viruses L.T. Daum, and D.L. Suarez. 2003. Develop- for chickens. Avian Pathology 23:345–352. ment of real-time RT-PCR for the detection of 84. Swayne, D.E., J.R. Beck, M. Garcia, M.L. Perdue, avian infl uenza virus. Avian Diseases 47:1079– and M. Brugh. 1998. Pathogenicity shifts in exper- 1082. imental avian infl uenza virus infections in chick- 72. Spackman, E., D.A. Senne, T.J. Myers, L.L. ens. In: D.E. Swayne and R.D. Slemons (eds.). Bulaga, L.P. Garber, M.L. Perdue, K. Lohman, Proceedings of the Fourth International Sympo- L.T. Daum, and D.L. Suarez. 2002. Development sium on Avian Infl uenza, May 29–31, 1997, of a real-time reverse transcriptase PCR assay for Athens, Georgia. Symposium on Avian Infl uenza, type A infl uenza virus and the avian H5 and US Animal Health Association: Richmond, VA, H7 hemagglutinin subtypes. Journal of Clinical pp. 171–181. Microbiology 40(9):3256–3260. 85. Swayne, D.E., J.R. Beck, and T.R. Mickle. 1997. 73. Stafseth, H.J. 1925. The new poultry disease. Effi cacy of recombinant fowl pox vaccine in Michigan Station Quarterly Bulletin 7(3):83. protecting chickens against highly pathogenic 74. Stegeman, J.A., A. Bouma, A.R.W. Elbers, M. van Mexican-origin H5N2 avian infl uenza virus. Avian Boven, M.C.M. de Jong, and G. Koch. 2005. Diseases 41:910–922. Effectiveness of control measures on the transmis- 86. Swayne, D.E., and D.A. Halvorson. 2008. Infl u- sion of avian infl uenza virus (H5N7) between enza. In: Y.M. Saif, J.R. Glisson, A.M. Fadly, L.R. fl ocks. In: R.S. Schrijver and G. Koch (eds.). Avian McDougald, and L. Nolan (eds.). Diseases of Infl uenza. Prevention and Control. Springer: Poultry, 12th ed. Blackwell: Ames, Iowa, in Dordrecht, the Netherlands, pp. 49–55. press. 75. Stubbs, E.L. 1925. Fowl plague. University of 87. Swayne, D.E., and R.D. Slemons. 1990. Renal Pennsylvania Veterinary Extension Quarterly Bul- pathology in specifi c-pathogen-free chickens letin 25(16). inoculated with a waterfowl-origin type A infl u- 76. Stubbs, E.L. 1925. Fowl plague in Pennsylvania. enza virus. Avian Diseases 34:285–294. Journal of the American Veterinary Medical 88. Swayne, D.E., and R.D. Slemons. 1994. Compara- Association 20:180–182. tive pathology of a chicken-origin and two duck- 77. Stubbs, E.L. 1926. Fowl pest. Journal of the origin infl uenza virus isolates in chickens: the American Veterinary Medical Association 21:561– effect of route of inoculation. Veterinary Pathol- 569. ogy 31:237–245. 216 Avian Influenza

89. Swayne, D.E., and R.D. Slemons. 1995. Compara- 94. U.S. Department of Agriculture. 1929. Outbreak of tive pathology of intravenously inoculated wild European fowl pest in New Jersey quickly eradi- duck- and turkey-origin type A infl uenza virus in cated. USDA Offi cial Record 8(33):8. chickens. Avian Diseases 39(1):74–84. 95. Villareal, C.L. 2006. Control and eradication strat- 90. Todd, C. 1928. Experiments on the virus of fowl egies of avian infl uenza in Mexico. Developments plague (II). British Journal of Experimental Pathol- in Biologicals 124:125–126. ogy 9:101–106. 96. Villareal, C.L., and A.O. Flores. 1998. The 91. Tweed, S.A., D.M. Skowroski, S.T. David, A. Mexican avian infl uenza (H5N2) outbreak. In: Larder, M. Petric, W. Lees, Y. Li, J. Katz, M. D.E. Swayne and R.D. Slemons (eds.). Proceed- Krajden, R. Tellier, C. Halpert, M. Hirst, C. Astell, ings of the Fourth International Symposium on D. Lawrence, and A. Mak. 2004. Human illness Avian Infl uenza, May 29–31, 1997, Athens, from avian infl uenza H7N3, British Columbia. Georgia. Symposium on Avian Infl uenza, US Emerging Infectious Diseases 10(12):2196–2199. Animal Health Association: Richmond, VA, pp. 92. United States Animal Health Association. 1994. 18–22. Report of the Committee on Transmissible Dis- 97. Webster, R.G., W.J. Bean, Y. Kawaoka, and D. eases of Poultry and Other Avian Species. Criteria Senne. 1986. Characterization of H5N2 infl uenza for determining that an AI virus isolation causing viruses from birds in live poultry markets in USA. an outbreak must be considered for eradication. Proceedings of the United States Animal Health Proceedings of the United States Animal Health Association 90:278–286. Association 98:522. 98. Wood, J.M., R.G. Webster, and V.F. Nettles. 93. U.S. Department of Agriculture. 1925. Source of 1985. Host range of A/chicken/Pennsylvania/83 fowl pest in introduced virus. USDA Offi cial (H5N2) infl uenza virus. Avian Diseases 29:198– Record 4(10):1–2. 207. 9 Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959, Excluding the Asian H5N1 Virus Outbreaks

Dennis J. Alexander, Ilaria Capua, and Guus Koch

INTRODUCTION unusual virus rather than the disease caused (15, 55). Highly pathogenic avian infl uenza (HPAI), then The outbreak goes unmentioned in the 1959 Report termed “fowl plague,” was fi rst recognized as a dis- on the Animal Health Service in Great Britain (37), tinct disease by Perroncito in 1878 (56), and reports possibly the occurrence of 2062 outbreaks of “fowl of outbreaks were common in many countries in pest” (Newcastle disease) in Great Britain in 1959 Europe and at least North Africa during a period understandably seeming more important and press- covering the last quarter of the 19th century and the ing. The outbreak occurred on a single chicken farm fi rst third of the 20th century (2). Very few reports in November/December 1959 (the fi rst virus isolate of “fowl plague” appear in the literature between being obtained on December 25); the exact location 1930 and 1959, although Wells, writing in 1963 of the farm has not been recorded, but it was close (65), considered the virulent disease to be still enzo- to the coast. Unpublished documents record the otic in Egypt and “not uncommon in other parts of clinical signs and pathology as typical of “fowl Africa, Asia and Eastern Europe.” plague.” In particular, it was noted that the birds that In this chapter, the reported outbreaks of HPAI died had severe cyanosis around the head (I. Lister, covering the period from 1959, when the fi rst known personal communication). On initial isolation, it was HPAI outbreak due to an infl uenza virus of H5 demonstrated that the virus isolated showed no anti- subtype was recorded, until the emergence of the genic relationships with “fowl plague virus” (i.e., AI Asian HPAI H5N1 virus and its spread through viruses of H7 subtype) or Newcastle disease virus, Asia and into Europe and Africa are described but it was some years before it was identifi ed as an (Table 9.1). infl uenza virus and its relationship with A tern/South Africa/1961 was recorded (15, 55). GREAT BRITAIN Little has been recorded relating to the epidemiology of the outbreak, but at the time there was some Scotland 1959 (H5N1) interest in the links between wild birds and New- The outbreak of highly pathogenic disease on a castle disease in poultry and it was noted that wild chicken farm near Aberdeen, Scotland, resulting ducks and sea birds had been seen feeding with the in the isolation of the fi rst known AI virus of H5 chickens on the affected farm (I. Lister, personal subtype and the fi rst known virulent AI virus that communication). Becker (14) reported a personal was not of H7 subtype, was surprisingly underre- communication from J. E. Wilson that large numbers ported. Reports of the occurrence appear to be of herring gulls (Larus argentatus) were present on limited to mention in papers concerned with the the farm at the time of the outbreak. In view of the

Avian Influenza Edited by David E. Swayne 217 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 Table 9.1. Summary of HPAI outbreaks in Africa, Asia, and Europe 1959–2006 excluding Asian H5N1 outbreaks. Number of farms Number of birds Year HPAI virus Subtype HA0 cleavage sitea HPAI conﬁ rmed affectedb

1959 A/chicken/Scotland/59 H5N1 PQRKKR*GLF- 1 Chicken Not known 1961 A/tern/S. Africa/61 H5N3 PQRETRRQKR*GLF None known 1000s of wild terns 1963 A/turkey/England/63 H7N3 PETPKRRRR*GLF 2 Turkey ∼29,000 Turkeys 1979 A/chicken/Germany/79 H7N7 PEIPKKKKR*GLF, PEIPKRKKR*GLF, 1 Chicken, 1 goose 600,000 Chickens, 80 PEIPKKRKKR*GLF, geese PEIPKKKKKKR*GLF 1979 A/turkey/England/199/79 H7N7 PEIPKKRKR*GLF, PEIPKRRRR*GLF, 3 Turkey 9,262 Turkeys PEIPKKREKR*GLF 1983 A/turkey/Ireland/1378/83 H5N8 PQRKRKKR*GLF 2 Turkey, 1 mixed, 1 8,000 Turkeys, 28,020 duck chickens, 270,000 ducks 218 1991 A/turkey/England/50–92/91 H5N1 PQRKRTR*GLF 1 Turkey 8,000 Turkeys (624 slaughtered) 1995 A/chicken/Pakistan/95 H7N3 PETPKRKRKR*GLF, PETPKRRKR*GLF, and PETPKRRNR*GLF 1997 A/chicken/Italy/330/97 H5N2 PQRRRKKR*GLF 8 (Chickens, turkeys, 6,601 geese, ducks, guinea fowl, pigeons, quail) 1999 A/turkey/Italy/99 H7N1 PEIPKGSRVRR*GLF 413 13,732,912 2003 A/chicken/Netherlands/2003c H7N7 PEIPKRRRR*GLF 255d >25,600,000 2003 A/chicken/Pakistan/2003 H7N3 Not known Not known Not known 2004 A/ostrich/S. Africa/2004 H5N2 PQREKRRKKR*GLF Not known >30,000 Ostriches 2005 A/chicken/N. Korea/2005 H7N7 PEIPKGRHRRPKR*GLF 3 Chicken 219,000 a Amino acid motif at the cleavage site (*) of the HA0 precursor protein of isolates of the causative virus. b Number of birds that died or were slaughtered. c Spread to Germany (one farm, 419,000 chickens) and Belgium (8 farms, 2,300,000 chickens). d 255 Flocks were confi rmed as infected, 233 commercial and 22 backyard or hobby fl ocks; in addition, 1126 commercial and 16,521 hobby fl ocks were culled preemptively. 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 219 die-off of terns in South Africa some 17 months later There was spread of the virus to one other farm. due to HPAI virus of H5N3 subtype (see later), This was associated with the movement of birds reports of mass mortality in kittiwakes (Rissa tridac- from one unaffected farm to another unaffected tyla) and fulmars (Fulmaris glacialis) in Scandina- farm. However, these birds were transported in a via and around the British coast from February to truck that had been used the previous day to take August 1959 could have had some signifi cance for turkeys from the primary affected farm to an abat- the Scottish outbreak, but the etiology of these toir. Although the truck had been disinfected, the die-offs was never investigated (14). cabin had been omitted. Also, the driver had failed The virus isolated was known initially as the to wear protective clothing on either day and had “Smith virus” after the farm owner and then “Wilson worn the same clothing on both days. Wells (65) virus” after J. E. Wilson, the head of the Lasswade reports that deaths of the birds introduced began 7 Laboratory, where it was isolated. It is now identi- days after their introduction and 17 of 19 died in the fi ed as A/chicken/Scotland/59 (H5N1). next 7 days. The total number of birds slaughtered While LPAI H5 viruses have the HA0 precursor on the second farm was not recorded, although it protein cleavage site motif -PQRETR*GLF-, most was reported that only partial slaughter of the birds HPAI H5 viruses have clear evidence of insertions on the farm was thought necessary (38). From the of basic amino acids at the cleavage site in keeping published data, it is not clear how much surveillance with the theory that the mutations to virulence arise was done in the locality of the outbreak, although as result of spontaneous duplication of purine trip- negative results with blood samples taken from terns lets due to a transcription fault by the polymerase and black-headed gulls at two wild bird sanctuaries complex, which results in the insertion of basic in the area were reported (38). amino acids at the HA0 cleavage site (54). However, The virus isolated from the affected turkeys now the cleavage site sequence for A/chicken/Scot- known as A/turkey/England/63 (H7N3) was initially land/59 is -PQRKKR*GLF-, as if the mutation identifi ed as the “Langham strain” (55). occurred by substitutions rather than insertions. In that regard it was similar to the later HPAI virus England 1979 (H7N7) A/chicken/Pennsylvania/83 (H5N2), which has the The outbreak of HPAI due to a virus of H7N7 motif PQKKKR*GLF (33). subtype in 1979 and the circumstances surrounding the occurrence were investigated in signifi cantly England 1963 (H7N3) greater depth than the preceding outbreaks in Great The second HPAI outbreak in the United Kingdom Britain. However, relatively little was reported in the during the period under review occurred in 1963. This public domain. The HPAI outbreaks again occurred HPAI outbreak was subjected to both a detailed inves- in turkeys on the north coast of Norfolk on three tigation and a report by Wells (65) that would serve farms a few kilometers from the coast in March as a model account of an outbreak on a single farm. 1979 (5). The HPAI H7N7 virus was isolated from The farm was situated in the north of the county diseased turkeys on three farms with 9-, 18-, and of Norfolk, England, some 2 miles from the coast 6-week-old turkeys, respectively. Two of the farms near the village of Langham. On the farm, turkeys were essentially on the same site (1), the third farm were kept in a series of outdoor folds and intensively was situated within 8 km of the other two, and all in two houses in a series of pens. The disease was three were under the same ownership. Alexander fi rst recognized in late May 1963 in one of the and Spackman (5) concluded that the disease houses in two pens, each containing 350 birds, occurred fi rst on one farm and was very quickly shortly after the birds had been transferred from the spread to the other two, presumably by the agency outdoor folds. Wells (65) stressed how slowly the of man. Surviving birds on the three farms were disease spread from pen to pen, stating that “evi- slaughtered under the stamping-out policy in place, dence points to spread within a house being depen- resulting in the recorded loss of 9262 turkeys and dent on man and bird-to-bird contact and not wind compensation payment of £52,309 (39). or other agencies.” All the birds on the turkey farm, Possibly the most remarkable feature of these out- totaling 29,000, were slaughtered some 23 days after breaks was the number of LPAI infections discov- increased mortality was fi rst noticed. ered in their vicinity. In total, during a period from 220 Avian Influenza

March to early May 1979, infections were discov- houses, which were chiefl y “pole barns” (essentially ered on 16 turkey farms—14 in Norfolk, 1 just houses with open sides), rendering them liable to across the county border in Suffolk (45 km south- wild bird invasion. Although there were no reports east, but under the same ownership as infected farms of waterfowl mingling with turkeys on farms, birds in Norfolk), and another much farther away in the such as sparrows and starling were present in large county of Hertfordshire. Alexander and Spackman numbers, one fl ock of starlings was thought to be (5) point out that in this brief period in 1979, more in excess of 1 million, and these readily invaded infections of poultry were detected than had been turkey houses searching for food. It is interesting reported in the preceding 16 years. On eight of the that Spackman et al. (unpublished manuscript) farms, infections were detected only serologically recorded that the previous time that weather condi- with no virus isolation. Seven of these were H7 tions in Norfolk were comparable to the severity of infections (six Norfolk and one Suffolk) and the the 1978–1979 winter was in the winter of 1962– other was H10. In addition to the isolations from the 1963, that is, the winter before the previous outbreak three farms infected with HPAI H7N7 virus, LPAI of HPAI. There is no record of surveillance for AI viruses of H7N2, H7N3 (2), and H1N1 subtypes infections of wild birds in Norfolk in 1979. Some were isolated from birds on Norfolk farms and surveillance was done on turkey farms in the area H10N4 from the farm in Hertfordshire. Noticeably, during 1979–1980; no virus was isolated from any the HPAI virus was H7N7 with all isolates giving farm, but birds on four farms (three H7 and one intravenous pathogenicity index (IVPI) values of H1) were serologically positive. These farms were 2.47 to 3.00; other H7 viruses had IVPI values of known to have been infected with LPAI virus in 0.00 to 0.12. Interestingly, the H10N4 virus gave March–May and, although sampled some months IVPI values in two separate tests of 1.34 and 1.68, later, turkeys surviving from that time were still which with today’s defi nitions (3) would have been present on the farms (4). classifi ed as HPAI. The H1N1 virus gave an IVPI The relationship between the 1979 HPAI out- value of 0.00. breaks in England and Germany are not clear due to The reasons for the apparently unprecedented the lack of information available on the German AI infections of turkeys in the county of Norfolk outbreaks (see later). Phylogenetic analysis of the appeared to be associated with the weather. In an representative viruses indicated that the HPAI unpublished manuscript, Spackman et al. (D. Spack- English isolates were closely related to the HPAI man, W.H. Allan, D.J. Alexander, and E. Borland. German isolates goose/Leipzig/79 and chicken/ 1980. Avian infl uenza in turkeys in England during Leipzig/79 (H7N7). The nucleotide sequences of the the Spring of 1979 and associated epidemiological HA gene of viruses available from these outbreaks factors. VLA Weybridge, unpublished manuscript) showed very close homology (greater than 98.6%), recorded the winter of 1978–1979 as particularly and there was close homology (greater than 97%) to harsh with severe snow and ice across continental the German wild bird LPAI isolates tern/Potsdam/79 Europe from late December 1978 to mid February (H7N7) and swan/Potsdam/81 (H7N3), an H7N7 1979 due to an anticyclone over mid Europe. This LPAI virus isolated from turkeys in England in 1977 resulted in most tracts of inland water being frozen and the LPAI H7 viruses isolated in Norfolk in 1979 over; even the Baltic Sea froze in some areas. This (10, 11, 59). It would appear that these HPAI viruses encouraged a westward movement of birds after the emerged from the same close common ancestor as normal winter migration had taken place and for the viruses of low virulence present in wild birds and birds that would normally over-winter in Germany poultry. Both the various German (PKKKKR* and the low countries to continue farther west, GLF, PKRKKR*GLF, PKKRKKR*GLF, and making landfall in east England. In the early part of PKKKKKKR*GLF) and English (PKKRKR*GLF, 1979, unusual numbers of birds were recorded in PKRRRR*GLF, and PKKREKR*GLF) H7N7 HPAI tidal estuaries and lakes close to the turkey farms. isolates obtained were shown to have variable HA0 In Norfolk, there had been a cold snap at the end of amino acid cleavage site motifs (10, 11, 59). While December lasting a few days, but in mid February none of the German isolates showed an identical 1979, there was a severe easterly blizzard lasting 8 motif to any of the English isolates, the marked vari- days. This blizzard damaged many of the turkey ability of the different isolates does not mean that 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 221 the viruses rose independently in the two countries, between A/goose/Guandong/96 (H5N1) isolates that and it is possible that there was one mutation to showed high and low virulence in chickens (35). virulence and that the HPAI virus transferred from The cleavage site motif of PQRKRTR*GLF was one location to the other, with wild bird movement considered unusual at the time as all other HPAI up being the prime candidate for this transfer. The to then had had the motif RXK/RR*GLF (6). direction of this move is unclear as the time of year However, some of the HPAI virus A/chicken/ of the outbreaks in Germany is not known. Mexico/94 (H5N2) isolates had an identical motif to the Norfolk 1991 isolates (54). England 1991 (H5N1) In December 1991, another HPAI outbreak occurred Discussion of Outbreaks in Great Britain in turkeys on a farm in the village of Weston All four HPAI outbreaks occurring in Great Britain Longville in Norfolk, England (7). This farm was since 1959 have been in farms relatively close to the farther inland than the previous outbreaks in 1963 coast and in areas where large numbers of shorebirds and 1979 but still less than 20 miles from the coast and/or waterfowl were active (Fig. 9.1). The three in an area where the presence of rivers and lakes outbreaks in England, although spanning 28 years, meant there was much waterfowl activity. There all occurred in a very small area of eastern England were several unusual circumstances associated with within 25 miles of each other and close to the coast. this outbreak and the viruses isolated. This part of England in the county of Norfolk is well The outbreak occurred in a single house of 8000 known for its lakes and other water features and is 18-week-old turkeys and apparently did not spread, a haven for migratory waterfowl and other birds. even to other houses on the same site that, although The outbreaks also seem to have had some relation- separated by a road, were part of the same enter- ship to the weather. The outbreaks in 1959 and 1991 prise. There were few clinical signs other than occurred mid-winter, the 1979 outbreak occurred in sudden death (in an 8-day period, 7129 of the birds early spring, but at a time of severe cold weather died), and this led early on to a suspicion of poison- with high winds and snow, and although the 1963 ing rather than infectious disease. The lack of spread outbreak occurred in June, it followed one of the was probably due to the enforced isolation of the longest and coldest winters on record in Great house and staff, that is, effectively applying strict Britain. biosecurity, while this was being investigated. There was considerable delay in diagnosis as, initially, despite killing embryos rapidly, the virus failed to hemagglutinate red blood cells, but eventually the virus was shown to be HPAI of H5N1 subtype. There were 624 surviving birds at the time of confi rmation that were slaughtered as part of the stamping-out policy, resulting in payment of £6202 compensation (40). These birds had H5 antibodies, confi rming that they had been infected. Two H5N1 viruses were isolated from a carcases that had been frozen. One from a respiratory tract swab was highly virulent for chickens with an IVPI of 3, while virus isolated from the brain had an IVPI of 0. Surprisingly, both viruses had the HA0 cleavage site of PQRKRTR*GLF, and, indeed, no difference that could account for the marked difference in virulence could be found in the HA gene sequence (67). Subsequent studies have also failed to fi nd an explanation (10). The low virulence virus does not Figure 9.1. The approximate positions of the have the same single amino acid changes in the NS1 HPAI outbreaks occurring in the British Isles 1959–1991. protein that were thought to be the sole difference 222 Avian Influenza

The four British outbreaks showed no or only with chicken/Scotland/59. The virus was initially extremely limited spread; for the three in turkeys in termed “tern virus” (13) and is now identifi ed as Norfolk, this seems to be due to the nature of the A/tern/South Africa/61 (H5N3). turkey industry, reasonably rapid diagnosis, rapid application of control policies, and good luck. It also South Africa 2004 (H5N2) seemed that if the viruses responsible originated as Confi rmation of HPAI of H5N2 in ostriches in two LPAI, the mutation to HPAI occurred very quickly, farms in the Eastern Cape Province in South Africa and there was no spread of LPAI virus prior to the following the investigation of clinical signs consist- mutation. ing of respiratory signs, excessive lacrimation, green diarrhea, depression, and death, fi rst seen in July, SOUTH AFRICA was made in August 2004. Of the 9000 ostriches involved, 1500 were showing signs of disease and South Africa 1961 (H5N3) 1000 had died (52). Olivier (53) reported that the Investigation of the cause of the death of thousands confi rmation of HPAI virus was based on the HA0 of common terns (Sterna hirundo) along the southern cleavage site motif (PQREKRRKKR*GLF) as the coast of South Africa in 1961 (60) resulted in the intravenous pathogenicity index in 6-week-old isolation of a highly virulent virus, which was identi- chickens was less than 1.2. Surveillance of healthy fi ed as an infl uenza A virus, A/tern/South Africa/61, ostrich fl ocks in the area revealed a number to have by Becker (14) and is now identifi ed as of H5N3 birds with H5 antibodies, but chicken fl ocks appeared subtype. This virus was important in that it was the to be free of infection. As a result of the stamping- fi rst reported isolation of an AI virus from wild birds out policy employed, about 30,000 ostriches were and for many years represented the only time sig- slaughtered on 37 infected and contact farms. Olivier nifi cant numbers of wild birds had been infected with (53) records that this represents 40% of the farmed HPAI virus (until the Asian H5N1 HPAI virus ostriches in the Eastern Province. South Africa entered the wild bird population). According to declared itself free of HPAI on September 13, Becker (14), the common tern is normally present in 2005. South Africa only during the months of October to Surveillance of ostrich farms by hemagglutination February, but in 1961 the migration north to Europe inhibition tests revealed antibodies to H5 virus on was delayed until May. Deaths were fi rst recorded in 42 farms in Western Cape Province, but no virus early April, but by the third and fourth weeks of the could be isolated or detected. month, large numbers of dead terns were reported The lack of apparent virulence for chickens of the over a 1000-mile stretch of the coast of the Cape 2004 HPAI H5N2 virus isolated from ostriches in Province from Port Elizabeth to Lamberts Bay. initial IVPI tests was somewhat unusual. The origi- Samples from the carcases of three common terns nal isolate gave an IVPI value of 0.63, but virus submitted to Cape Town University and two terns receiving a further passage in embryonated eggs submitted to Oonderstepoort Veterinary Laborato- gave an IVPI value of 1.19. When virus reisolated ries yielded the virus. Becker was able to reproduce from the chickens in the fi rst IVPI in embryonated the disease in captured healthy common terns infected eggs was tested, the IVPI value obtained had experimentally by the conjunctival or intramuscular increased to 2.73 (43). This is further evidence that routes, but swift terns (Sterna bergii) similarly viruses obtained from species remote taxonomically infected showed no signs of disease, although they from chickens require adaptation to chickens before produced antibodies to the virus. their potential virulence in pathogenicity index tests Pereira et al. (55) demonstrated the antigenic rela- is realized. tionship between the hemagglutinin of the virus The assumption that the precursor virus for the from terns and that of A/chicken/Scotland/59, and HPAI virus was an LPAI virus in wild birds was Allan et al. (9) demonstrated the antigenic relation- given considerable weight by the identifi cation of an ship between the neuraminidases of the tern virus infl uenza virus detected in an Egyptian goose (Alo- and A/turkey/England/63. Becker and Uys (15, 64) pochen aegypticus) culled on one of the antibody- demonstrated the high virulence of the virus for positive Western Cape farms in June 2004, 1 month chickens and the similar pathology to infections before the disease fi rst occurred, as an LPAI H5N2 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 223 virus. This virus was genetically close to the HPAI farm. The number of birds involved was reported as virus. 600,000 chickens and 80 geese (31). In June 2006, HPAI due to H5N2 virus was con- Sinnecker et al. (61) described the properties fi rmed in a fl ock of 58 4-month-old ostriches in the of an H7N7 virus isolated from a gull, A/gull/ Western Cape Province. The virulence of the virus Potsdam/79; the virus produced a highly pathogenic was determined by reverse transcriptase–polymerase disease in day-old and 3-week-old chickens infected chain reaction (RT-PCR) and sequence analysis. All experimentally. Whether the infected gull was in birds on the farm were culled as part of the stamp- close proximity, geographically and temporarily, ing-out policy. There was some evidence that this with the outbreaks in poultry was not revealed. virus was genetically distinguishable from the H5N2 There was a single outbreak of HPAI H7N7 virus responsible for the outbreaks in Eastern Cape affecting 419,000 chickens in Germany in 2003 as Province, but no data had been made available at the a result of spread from the Netherlands (see later). time of writing. Further farms were shown to be serologically or virus detection positive, and it IRELAND 1983 (H5N8) appeared that both HPAI and LPAI H5N2 viruses The outbreaks of HPAI in Ireland in November 1983 were present in the ostriches (52). caused by a virus of H5N8 subtype were important Infection of ostriches with avian infl uenza (AI) because the ability of commercial ducks to be virus of H5N2 subtype on two farms in Zimbabwe infected by and excrete HPAI virus without showing was reported to the World Organization of Animal clinical signs was demonstrated in the fi eld for the Health (Offi ce Internationale des Epizooties [OIE]) fi rst time. in December 2005. The virus had been detected as The outbreaks, which were described by Murphy a result of routine surveillance and no clinical signs (48), were seen initially as a disease in turkeys with had been seen in the ostriches. No data were sup- high mortality and clinical signs of depression, inap- plied on the virulence of the virus for chickens. All petance, diarrhea, and nervous signs on two closely birds on the farms were slaughtered as part of a situated farms in County Monaghan. The farms (I stamping-out policy. and II) were relatively small, consisting of 700 and Ostriches represent a unique problem in the epi- 4700 turkeys, respectively. A 5-km restriction zone demiology of HPAI in that they may be infected was set up, and surveillance of the 45 poultry fl ocks without showing clinical signs (25, 44) and possibly within that zone revealed another turkey farm (III), act as healthy carriers. Where clinical signs were 1.3 km from farm I, with suspicious signs. Farm III seen in the South African outbreaks, this may well had 2600 turkeys showing disease and 28,000 appar- have been due to secondary infections or exacerbat- ently healthy broiler chickens. All birds on the three ing conditions (53). farms and another small farm (120 turkeys, 20 chickens) with dangerous contact with the second GERMANY 1979 (H7N7) farm were slaughtered, and the carcasses were buried No contemporaneous reports of the outbreaks in on the farms. HPAI virus was isolated from samples poultry in 1979 in the then German Democratic from turkeys on farms I through III. Republic were placed in the public domain, and little Further surveillance resulted in the isolation of information has been published subsequently. Rohm an HPAI H5N8 virus from healthy 4- to 5-week-old et al. (59) recorded that an outbreak occurred on a Pekin ducks on a large commercial duck farm situ- chicken farm near Leipzig and that the chickens had ated between farms I and II. This farm had 270,000 shown peracute signs including edema and cyanosis ducks, representing about 97% of all the commercial of the head, necrotic lesions on the legs, and high ducks in the Republic of Ireland. These, too, were mortality. Isolates of HPAI virus of H7N7 subtype, slaughtered and buried on the farm. Investigations A/chicken/Leipzig/79 (H7N7), were obtained from failed to discover any evidence of contact between chickens on the farm. They record there was no the turkey farms and the duck farm (48). A total of spread of disease to other chicken farms but that 8000 turkeys, 28,020 chickens, and 270,000 ducks HPAI viruses of H7N7 subtype were isolated from were slaughtered as a result of the outbreaks. commercial geese showing “moderate FPV-like During the outbreaks in the Republic of Ireland, signs” situated 20 miles from the infected chicken an investigation of turkeys that had shown high 224 Avian Influenza mortality while being transported from the Republic Control measures consisted of (1) voluntary of Ireland to Northern Ireland resulted in the isola- depopulation, cleaning, disinfecting, and not restock- tion of the HPAI H5N8 virus (41). ing for 60 days (there was no compulsory stamping- Experimental infections with a turkey isolate, A/ out policy or compensation); (2) vaccination using turkey/Ireland/1378/83 (H5N8), and a duck isolate, an homologous vaccine; and (3) setting up a national A/duck/Ireland/113/84 (H5N8), from the outbreaks surveillance program. Two months after the peak confi rmed that while both viruses caused disease and mortality in the fi eld, the severity and incidence of deaths in chickens, turkeys, and quail, they failed to the disease started to decline. The vaccination cam- produce any clinical signs in ducks (8). paign covered 80% of the commercial layers and was extended to the breeding stock in the north of Pakistan H7N3 1995, 2003–2004 the country. Naeem and Siddique (50) considered The AI situation in poultry in Pakistan has been the limited spread of the HPAI H7N3 outside the extremely complex since 1995 as a result of circula- Karachi area to be attributable to the control tion of HPAI viruses of H7N3 and H5N1 subtypes measures. and LPAI viruses of H7N3 and H9N2. In addition, An H7N3 HPAI virus showing close genetic sim- there has been widespread use of H7 and H9 ilarity to the Pakistan viruses was isolated in 1998 inactivated vaccines (50). Naeem and Siddique (50) from a peregrine falcon (Falco peregrinus) dying in reported two major outbreaks of HPAI occurred in the United Arab Emirates (11, 45). There was no Pakistan—the fi rst in 1995 and the second in 2003– evidence of links to Pakistan. 2004, both caused by viruses of H7N3 subtype. The 1995 outbreaks of HPAI H7N3, which ITALY affected chickens primarily in northern Pakistan, resulted in the death of some 3.2 million birds, Italy 1997 (H5N2) mainly broiler breeders and broilers. During these Toward the end of October 1997, episodes of sudden, outbreaks, HPAI viruses with three different HA0 high mortality in backyard poultry fl ocks were cleavage site amino acid sequences were detected: recorded in northeastern Italy. Even though there PETPKRKRKR*GLF, PETPKRRKR*GLF, and had been no record of outbreaks of HPAI in Italy for PETPKRRNR*GLF (11). A vaccination campaign some 60 years (57), offi cial veterinarians at fi eld with a homologous vaccine coupled to an upgrading investigation centers suspected HPAI very early on, of biosecurity measures was implemented, and this and all possible control and preventive measures led to the control of HPAI (49). were implemented quickly. The primary concern for The AI situation in Pakistan was further compli- the offi cial and private veterinarians was to prevent cated by the emergence of H9N2 infections in 1998, the spread of disease into the densely populated which became endemic, and the reemergence of poultry area (DPPA) of the Veneto and Friuli H7N3 subtype virus, this time LPAI, in 2000 (50, Venezia-Giulia regions. 51). These infections caused considerable problems Capua et al. (23) reported that between October and vaccination was introduced using a bivalent H7/ 27, 1997, and January 11, 1998, eight outbreaks of H9 oil-based inactivated vaccine. HPAI were diagnosed, notifi ed, and stamped out HPAI virus of H7N3 subtype with an IVPI of 2.8 according to European Union (EU) Council Direc- was isolated in November 2003 from diseased tive 92/40/EC (27). A total of 6503 birds, 1846 chickens in the Karachi area of Pakistan, in which chickens, 1503 turkeys, 2270 ducks, 45 geese, 731 70% of the country’s commercial layers are reared guinea fowl, 98 quail, and 12 pigeons were involved (50). This had been preceded by numerous outbreaks in the outbreaks and died or were killed as part of of LPAI H7N3 virus in the area. Within 4 weeks, the stamping-out control policy. the HPAI virus had spread throughout commercial Common signs in galliform birds were weakness layer fl ocks covering an 80-km2 area with mortality and ruffl ed feathers, incoordination, and comb and usually in the region of 70% to 80% of the infected wattles that appeared cyanotic and swollen; most fl ock. Diagnosis was often complicated by the pres- birds presented with rales and coughing. Death took ence of LPAI viruses of H7N3 or H9N2 subtypes or place 24 to 48 hours after the onset of clinical signs. both on HPAI-infected farms. In one particular fl ock in which turkeys were 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 225 affected, a unique sign observed in these birds was accounted for approximately 65% of the Italian acute enteritis with whitish droppings. At necropsy, industrially reared poultry, particularly in the prov- birds exhibited swelling and cyanosis of the head, inces of Verona, Vicenza, Mantova, and Brescia comb, and wattles. The trachea often had hemor- (Fig. 9.2). This area had developed into a multispe- rhagic mucosa and was edematous. Air sacs appeared cies DPPA with the highest density of turkeys in thickened and lined with a fi brinous exudate. Italy and one of the greatest in Europe. Hemorrhages were present in the mucosa of the From a structural and functional point of view, proventriculus and the cecal tonsils, and catarrhal biosecurity levels were generally below standard hemorrhagic enteritis was also present. The spleen due to the absence of physical barriers between appeared enlarged, and in some cases fi brionus peri- establishments and the common practice of sharing tonitis was observed. staff and equipment among farms. Moreover, the Highly pathogenic H5N2 viruses with IVPI values local poultry industry had developed and grown into of 2.98 to 3.0 and with a deduced amino acid one that intensively reared a number of different sequence of PQRRRKKR*GLF at the HA0 cleav- avian species such as chickens, turkeys, guinea fowl, age site were isolated from all eight outbreaks. quail, and ostriches. Production circuits of these dif- Given the fact that the outbreaks affected either ferent birds often overlapped, because the feed mills backyard fl ocks or medium-small poultry traders, and slaughtering plants were owned by single com- the outcome of the epidemiological investigation panies that served a number of farms. was limited by the lack of accurate and precise infor- Despite the absence of legislative powers, because mation concerning the number and species present the LPAI viruses were causing real disease prob- in each group at any given time, including the dates lems, often with high mortality in meat turkeys, and numbers of birds introduced to the fl ocks and voluntary restriction policies aimed primarily at the precise dates concerning the onset of clinical avoiding the movement of infected birds were put signs and mortality. However, it was possible to in place. Although these initially appeared to have identify several risk factors associated with the out- some success with a marked decline in outbreaks breaks, such as rearing of mixed species—particu- during the hot summer months, the number of H7N1 larly galliforms and waterfowl, contact with wild LPAI outbreaks rose again during the autumn and birds and introduction of birds originating from live early winter of 1999. bird traders—who, by defi nition, obtain birds from In mid December 1999, a fi eld veterinarian sub- different sources and then sell them to small mitted samples from a suspected infl uenza outbreak farms. in a meat-turkey fl ock exhibiting high mortality. HPAI was diagnosed within 4 days of submission Italy 1999–2000 (H7N1) with the characterization of an H7N1 isolate with an IVPI of 3.0 and a deduced HA0 cleavage site amino The Emergence of H7N1 HPAI acid sequence of PEIPKGSRVRR*GLF. This motif, The H7N1 HPAI virus that caused the 1999–2000 although unusual, contained multiple basic amino epidemic in Italy emerged from an LPAI precursor, acids, typical of HPAI viruses. which had circulated in the poultry population of northeastern Italy for approximately 9 months (12, Spread of the H7N1 HPAI Virus 20). The LPAI virus had an IVPI of 0.0 in 6-week- Because the isolation of an H7 virus from turkey old SPF chickens and a deduced amino acid sequence fl ocks showing high mortality was not unusual at the for the HA0 cleavage site of the precursor of the time, the implementation of statutory control mea- hemagglutinin molecule of PEIPKGR*GLF, a sures, which would normally be used preemptively typical motif of LPAI viruses. At the time of occur- on suspicion had been delayed until the laboratory rence of the LPAI outbreaks, there was no legisla- confi rmation of HPAI, and this resulted in spread of tive basis to intervene with statutory measures to infection. From the epidemiological follow-up of control the spread of the LPAI virus, although the subsequent outbreaks, it appeared that at least 16 possibility of viral mutation to a highly pathogenic fl ocks were already infected on the day restriction form could not be ruled out. The disease occurred policies were implemented. The resulting loss of in the Veneto and Lombardia regions, which control of the infection culminated in 413 outbreaks 226 Avian Influenza

Figure 9.2. Distribution of poultry ﬂ ocks and ﬂ ocks infected during the 1999–2000 H7N1 HPAI outbreaks in northeast Italy.

notifi ed between December 17, 1999, and April 5, Clinical and Pathological Findings 2000 (Table 9.2). Although the clinical signs and pathological fi ndings Retrospective analysis of the outbreaks indicated were similar to those reported from HPAI outbreaks that infection was detected more frequently in turkey preceding 1999 or reported subsequently, the diver- (χ = 118.37, P < .0001) and layer farms (χ = 373.04, sity of species of poultry involved was probably P < .0001) than in any other type of farmed category. unprecedented and several unusual observations Layers and turkeys accounted for 73% of the out- were made. breaks. Other risk factors were the size of the fl ock, HPAI affected a greater number of establishments as larger fl ocks were shown to be more at risk (z = than LPAI and was often characterized by 100% 5.895, P < .0001), and location as poultry farms morbidity and high mortality in the affected fl ocks. located in the plains (altitude of 300 m or less) (χ = All intensively reared species were affected; fl ocks 37.27, P < .0001) were shown to be at higher risk. of turkeys, chickens, and guinea fowl often exhib- Tracing exercises carried out on affected premises ited 100% mortality rates within a few days (22, 26), allowed the identifi cation of the possible origin of the although as reported in other outbreaks, the speed of infection in 66.3% of the outbreaks. In particular, the spread within a fl ock was much quicker for birds origin of infection could be attributed to movement of reared on the ground compared with those in cages. animals (1.0%), indirect contacts at the time of In particular, in chicken fl ocks reared on litter, 100% loading for slaughter of female turkeys (8.5%), neigh- fl ock mortality was observed 48 to 96 hours from borhood spread (within 1-km radius) (26.2%), lorries the onset of the fi rst clinical signs. In contrast, in for the transport of feedstuff, litter and carcasses caged layers, the onset of mortality and clinical (21.3%), and other indirect contacts (i.e., exchange of signs was slower. In such fl ocks, severe depression manpower, machinery, equipment) (9.4%) (46). or mortality could be seen initially in only one bird Table 9.2. H7N1 highly pathogenic avian infl uenza in Italy (December 17, 1999, to April 5, 2000). Turkey Turkey Guinea Quail, duck, Broiler Backyard Region breeders meat type fowl pheasant Ostrich Broilers Layers breeders fl ocks Total

Veneto 2 102 2 1 2 14 21 8 6 158a Lombardia 3 72 7 4 1 25 97 21 4 234b Friuli Venezia Giulia — 3 — — — — 1 — 1 5 Piemonte — — — — — — 1 — 5 6 227 Trentino — — — — — — — — 1 1 Sicilia — — — — — — — — 2 2 Sardegna — — — — — — 1 — — 1 Emilia Romagna — — — — — — — — 5 5 Umbria — — — — — — — — 1 1 Total outbreaks (fl ocks) 5 177 9 5 3 39 121 29 25 413 Total birds 42,276 2,692,917 247,379 260,340 387 1,625,628 8,118,929 743,319 1,737 13,732,912 a The total includes 8 preemptively slaughtered fl ocks that proved to be virologically positive. b The total includes 21 preemptively slaughtered fl ocks that proved to be virologically positive. 228 Avian Influenza per cage in a restricted area of the house, and then found dead inside poultry houses or in their close progressed to neighboring cages, generally reaching proximity (24). the far end of the shed 10 to 14 days from the fi rst clinical signs. This different behavior in spread Infection of Mammals Including Humans between caged and litter-reared chickens was prob- Despite the large number and widespread nature of ably related to the amount of infected faeces in the outbreaks, no case of infection in mammals was direct contact with the birds. recorded throughout the duration of the outbreak. Guinea fowl (Numida meleagris) appeared to be Active and passive surveillance, including an inves- particularly susceptible to HPAI with 100% fl ock tigation in poultry workers, farmers, and veteri- mortality being observed 48 to 72 hours from the narians (58), failed to demonstrate interspecies onset of the fi rst clinical signs. transmission to humans. Varying degrees of resistance to the clinical disease were recorded in waterfowl (especially Eradication of H7N1 HPAI ducks), quail, and ostriches (22, 25). Efforts to eradicate the HPAI virus were focused The Italian H7N1 outbreak in ostriches appeared basically on the application of restriction to move- to be the fi rst recorded natural outbreak of HPAI in ments and stamping out of infected or suspected ratites. Clinical signs were observed only in juvenile infected farms. HPAI was eradicated in just over 4.5 (7 to 9 months of age) birds. The fi rst clinical signs months. As a result of mass mortality (due to the observed were anorexia and depression in a limited stamping out policy and implementation of pre- number of the young birds. Feed con sumption emptive slaughter), several establishments such as dropped, and the birds appeared sleepy and depressed. hatcheries, feed mills, abattoirs, processing plants, Within the next couple of days, the clinical condition and other connected activities were forced to inter- affected a signifi cant number of the young birds, rupt their activity. The resulting disruption of the although the adults appeared healthy throughout the marketing system caused unemployment and heavy episode. One notable sign was the brilliant green economic losses to the poultry industry and to the urine produced by affected ostriches. social community. Further economic losses also In Japanese quail (Coturnix coturnix japonica), occurred due to the export bans imposed on the mortality rates were probably infl uenced by their infected regions. confi nement in cages and were approximately 5% Delays in restocking cleaned and disinfected per day. Infection in these birds was characterized farms and, therefore, a return to normal commercial by a severe respiratory condition, which in a couple activities were caused by new outbreaks, which of days evolved into a clinical status characterized resulted in overlapping of the protection and surveil- by prostration, somnolence, listlessness, production lance zones imposed by Directive 92/40/EEC. of whitish diarrhea, and gasping prior to death. Because of the high population density in the area, Nervous signs such as opistothonus and torticollis the only option to avoid reoccurrence of the disease could also be seen prior to death. when restocking birds was to impose depopulation Although waterfowl are often described as resis- of uninfected birds in some zones, which inevitably tant to the clinical disease caused by HPAI, during caused further economic losses. the Italian H7N1 HPAI epidemic this appeared to Epidemiological analyses carried out on data col- apply only to domestic ducks (Anas platyrhynchos) lected during the epidemic indicated that control and clinical signs and mortality were recorded both measures were more effi cient in the Veneto region in geese (Anser anser var. domestica) and in despite a higher density of animals. The reason for Muscovy ducks (Cairina moschata) reared in a this lies in the application of restocking bans and of backyard fl ock. preemptive slaughter to a greater extent than what was carried out in Lombardy. The infection incidence Infections in Wild Birds rate (IR) was 2.6 cases per 1000 fl ocks in Lombardy Testing of wild and resident free-ranging birds and 1.1 in Veneto (42). This study also indicated that yielded positive results for H7N1 HPAI virus only viral transmission occurred over large distances and in two sparrows (Passer domesticus) and in a col- that turkey farms had a greater IR compared with lared dove (Streptopelia decaocto), which had been other avian species reared in the area. 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 229

Conclusions Mortality increased in one of the three fl ocks from The Italian H7N1 outbreak was the fi rst of a series 1% on February 22 to about 90% on February 28 of outbreaks of signifi cant magnitude that have (29). The diagnosis was confi rmed independently by occurred in recent times. The outbreaks of HPAI in staff of the Erasmus Medical Centre, who also deter- such an area highlighted several aspects of AI crisis mined the N type as N7 by sequencing (30). management that had not been fully pinpointed prior Virus was isolated and the subtype H7N7 con- to this outbreak. Possibly the most important fi rmed by conventional hemagglutination and neur- outcome of this epidemic was that it indicated to EU aminidase inhibition tests using standard reference legislators that viruses of the H5 and H7 subtype, sera on March 4, 2003. An IVPI of 2.94 was deter- regardless of their virulence for poultry should be mined in 6-week-old specifi c pathogen–free chick- included in EU legislation, and that therefore a leg- ens according to standard procedures (27). islative basis to manage LPAI infections caused by viruses of H5 and H7 subtypes was necessary. Route of Introduction Transmission of an H7N7 LPAI from wild birds to THE NETHERLANDS, BELGIUM, AND the free-ranged chickens and subsequent emergence GERMANY 2003 (H7N7) of an HPAI mutant was thought to have caused the outbreak. This hypothesis was supported by the fact The Beginning that 17 of 20 serum samples collected from the fl ock In 2003, the Netherlands experienced its fi rst out- that did not show major clinical signs were positive break of HPAI in 76 years (32). In February 2003, in HI tests with H7 antigens and no antibodies were serious suspicion of an infection of HPAI virus was detected in serum samples from the fl ock that showed raised on a free-range layer farm. One of the fl ocks high mortality (62). Later, serological testing showed on the farm showed a reduction in feed and water that only very few fl ocks with antibodies against H7 intake on February 22, followed by an increase in were detected in the infected area. Moreover, a mortality. On Monday, February 24, chickens were cross-sectional serological survey of Dutch poultry medicated with antibiotics and some chickens were farms excluded the possibility that an outbreak of submitted to the laboratory of the Dutch Animal H7N7 LPAI virus had preceded the H7N7 HPAI Health Service for testing for bacterial infections. outbreak, unnoticed. The serological survey on 1193 The Service was unaware of similar problems that randomly selected poultry farms was completed in developed later that week on fi ve other farms in the the second week of March 2003. Antibodies against neighborhood. The investigation developed into a H7 were only detected in a cluster of three farms, serious suspicion of HPAI on February 28, 2003, two free-range chicken farms and a turkey farm, all when a trachea smear stained positive for infl uenza located in the southwest region of the Netherlands. A antiserum. The veterinary authorities were noti- Epidemiological investigations on the three farms fi ed only after this positive test result. showed that one free-range layer farm had experi- Samples from all six suspect fl ocks were subse- enced a transient drop in egg production, some mor- quently submitted to the Dutch National Reference tality, and peritonitis with Escherichia coli in weeks Laboratory (Central Institute for Animal Disease 35 to 36 of 2002 and a 20% drop in egg production Control Lelystad, Wageningen University and and feed intake in weeks 49 to 51 of 2002, well Research Centre) by the veterinary authorities on the before the H7N7 HPAI outbreak. The other free- same day. On March 1, an H7 subtype infl uenza range layer farm showed a decrease in feed intake virus was confi rmed by RT-PCR in samples from all of 13%, a drop in egg production of 10%, and an six locations and differentiated as an HPAI strain increase in mortality up to 0.5%. The turkey farm by sequencing on the next day (29). The deduced showed increased mortality of 15% of females and amino acids at the HA0 cleavage site were 27% of males in week 50 and clinical signs between PEIPKRRRR*GLF. The layer farm, which ranged weeks 48 and 52. Based on the clinical signs, Orni- chickens freely, located in the central part of the thobacterium rhinotracheale and secondary E. coli Netherlands, in a region called the “Gelderse Vallei” infections were diagnosed. Based on laboratory (Gelder Valley), was considered to be the index testing, an airsacculitis caused by E. coli was diag- case. The farm was composed of three housed fl ocks. nosed. Some dead birds were frozen for demonstra- 230 Avian Influenza tion purposes by the students of the Faculty of Only if the Rh is smaller than 1 will the chain of Veterinary Medicine of the University Utrecht who infection be broken and the epidemic be extin- had assisted the veterinarian. In week 11 of 2003, guished. Stegeman et al. (62, 63) estimated that when the turkey fl ock was identifi ed as positive for control measures reduced the Rh from 6.5 (95% H7 in a serological cross-sectional survey, an H7N3 confi dence interval, 3.1–9.9) in the period before the infl uenza virus was isolated from the carcasses kept confi rmation to 1.2 (95% confi dence interval, 0.6– in the freezer. Based on the sequence near the hem- 1.9) in the period after the confi rmation of H7N7 agglutinin cleavage site, the virus was characterized HPAI in the laboratory. Thus, although control mea- as an LPAI virus. Because of the different N type, sures effectively reduced the between-fl ock transit was concluded that no direct relation existed mission, the effect was insuffi cient to halt the between the H7N3 epizootic outbreak in the South- epidemic. The epidemic in the Gelderse Vallei was west and the large H7N7 HPAI epidemic in the most likely contained only because all susceptible central-eastern region of the Netherlands in March fl ocks had been culled at the end of March rather 2003 (66). than because of the reduction in transmission rate. However, before the epidemic came to a halt in the Control Measures Gelderse Vallei, the virus had spread to another area On suspicion of disease on the fi rst farm, the Min- with high fl ock density in the southeast of the Neth- istry of Agriculture had issued a ban on movement erlands (Fig. 9.3) at the border of the provinces of of all chickens in the surrounding farms on the night Noord-Brabant and Limburg (henceforth indicated of Friday, February 28, to Saturday, March 1, 2003. as Limburg region). At the end of March, deroga- However, after confi rmation of an H7 virus on tions of some of the control measures were issued March 1, 2003, many more measures were imple- in the Limburg region, which may have contributed mented. Among others, a protection zone of 3 km to an Rh of 3.1. The Rh was reduced to 1.2 (95% and surveillance zone of 10 km were established. Within the established zone the movement of poultry, poultry manure and eggs were forbidden. Also, export of poultry and hatching eggs was suspended from the Netherlands. However, after confi rmation of an HPAI virus on Sunday, March 2, 2003, the decision was made to start culling birds on all farms within a 1-km zone around the infected farm. However, actual culling did not start until the suspicion was confi rmed by virus isolation. The culling in the fi rst week was relatively slow because of lack of culling capacity. The capacity was raised from 1000 birds per hour on March 4 to 160,000 per hour on March 8, but this was not suffi cient to keep pace with the quickly growing number of infected farms. Only at the end of the epidemic in May was the culling capacity, which had been raised to 800,000 per day, suffi cient to cull all infected farms within 24 hours and all farms considered at risk of infection from the infected farm within 48 hours of confi rmation. Mathematical models predicted that these time limits needed to be met to control the epidemic. The effectiveness of the control measures were evaluated by quantifying between-fl ock transmission characteristics of the virus using the reproduc- Figure 9.3. Distribution of fl ocks infected during the 2003 H7N7 HPAI outbreak in the tion ratio Rh, which is defi ned as the average number Netherlands. of secondary infections caused by 1 infected fl ock. 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 231 confi dence interval, 0.6–1.2) in the period after the In Belgium, eight farms were infected, resulting derogations were withdrawn and new zones were in the death or stamping out of 2.3 million birds. established. On May 9, an infection of H7N7 HPAI was con- The Gelderse Vallei and North-Limburg were fi rmed on a farm in Schwalmtal, Viersen region, in identifi ed retrospectively as a high-risk areas. A the German state of NordRhein Westfalen (NRW), high-risk area is defi ned as an area where the density close to the border with the Netherlands. The 32,000 of farms is so high that an introduction of HPAI broiler chickens on the farm were all destroyed. virus causes a major outbreak. A major outbreak is Slight increase of mortality had started on May 2. when more than 2.5% of the farms in the area are Although the farmer had a small shop selling agri- infected. In the Netherlands, two such areas were culture products located at a distance of 60 m from identifi ed the Gelderse Vallei (mean fl ock density, the chicken house, the route of infection was not greater than 4 fl ocks/km2) and a relatively small area established. In total, 419,000 birds died or were in the southeast of the Netherlands (18). Stegeman culled. et al. (62, 63) and van Boven et al. (19) concluded that outbreaks in high-risk areas can be effectively Infections of Humans controlled only by completely depopulating that A veterinarian who visited several farms with area. Alternative measures to reduce the probability infected poultry developed acute conjunctivitis on of an epidemic of this size that have been considered March 5. The symptoms started 30 hours after the are reduction of fl ock density or applying preventive last farm visit. An infl uenza A H7N7 was detected vaccination. in and virus isolated from eye swabs collected at During the outbreak in the Netherlands, a total of 60 hours after onset of the symptoms. Based on 255 fl ocks were confi rmed as infected, consisting of this fi nding, people involved in the outbreak were 241 (227 commercial and 14 backyard or hobby actively questioned about their health from March fl ocks) showing clinical signs in which virus was 10 onward. By June 9, 451 people reported illness detected by isolation or RT-PCR and 14, of which of some kind. In 89 of those cases, H7N7 virus was 6 were commercial and 8 were hobby fl ocks, that detected by RT-PCR and/or virus was cultured. Two were detected by serological screening. In addition, of the 89 cases developed infl uenza-like illness only; totals of 1126 commercial fl ocks and 16,521 hobby 83 developed conjunctivitis, of whom 5 also had fl ocks were culled preemptively. The total number infl uenza-like illness; and 4 did not meet the case of birds stamped out during the control of the out- defi nition (34). break was in excess of 25.6 million. Costs have been After the diagnosis of 19 cases, all people who estimated as 270 million Euros in direct costs, but had been exposed to poultry located in the surveil- in excess of 1 billion Euros in indirect costs (16). lance zone, including farmers and their families, received mandatory vaccination against human Spread to Belgium and Germany infl uenza and prophylactic treatment with the On April 7, the virus was detected in a turkey farm neuraminidase inhibitor oseltamivir (Tamifl u; in Koningsbosch near the German border. On April Hoffmann-La Roche Inc., Nutley, New Jersey, 16, the fi rst infected fl ock in Belgium was detected USA). Moreover, workers were equipped with in the village of Meeuwen-Gruitrode located 23 km personal protection equipment consisting of cover- from the Dutch border. Restriction zones and culling alls, P3 mask, and goggles. The majority of human of all poultry within a region of 3 km including H7N7 infections (56%) occurred before the intro- hobby poultry within a radius of 1 km around the duction of these measures. infected farm did not prevent the further spread Eighty-two of the 89 cases were considered as of the virus in Belgium westward to Antwerp and primary cases and 3 as secondary cases that were subsequently to the north to reenter the Netherlands believed to result from person-to-person transmis- in Wernhout at a distance of 3 to 4 km from the sion. The case register indicated that persons with Belgium border. The farm in Wernhout was the last health complaints lived at locations scattered over farm to be culled in the Netherlands on May 11, the Netherlands; eight of them had assisted in culling 2003. At that time, Belgium had already claimed operations in Gelderland but lived in the Limburg to have had the outbreak under control on May 9. region. Two had confi rmed H7N7 infections, and it 232 Avian Influenza is therefore conceivable that these cullers had con- Pig farms were divided into three categories: (1) tributed to the virus spread. Also, culling operations category 1: farms of mixed herds of poultry and called for more workers than were available locally infected poultry, (2) category 2: where poultry was and workers from other European countries were preventively culled, and (3) category 3: pig farms employed to assist in the cull. Some poultry workers with no poultry at all. who were confi rmed to be H7N7 infected returned A fi rst familiarizing, mainly serological survey to their country of origin during the period while still was started at 13 category 1 farms. Antibodies were infectious (34). More evidence for the occurrence detected using the hemagglutination inhibition test of person-to-person transmissions came from the with the H7N7 as antigen. The results of this fi rst results of a serological survey among cullers and surveillance showed more than one high titer (greater farmers and poultry workers and their family (17, than 40) in eight of these herds, indicating that the 28). Antibodies were detected using a modifi ed H7N7 virus had been introduced into the swine hemagglutination inhibition test with horse instead population. The prevalence of positive pigs in each of turkey erythrocytes (47). The study also indicated of these herds was 5.1%, 5.6%, 8.3%, 15.0%, and that the use of personal equipment had hardly any 26.0%. The fi ve mixed herds likely to be infected effect on the transmission to humans. Partly this were again sampled 11 days after the fi rst sampling might have been due to the lack of or late informa- to exclude virus transmission among pigs. The tion on the proper use of the equipment and partly results showed no signifi cant increase in seropreva- because wearing the equipment in a warm and humid lence in these herds (now 2.5%, 7.7%, 6.9%, 14.0%, poultry house is very uncomfortable. In contrast, the and 29.0%, respectively). Moreover, 60 oropharyn- prophylactic use of Tamifl u appeared to reduce the geal swabs collected in each herd for virus detection transmission to humans (17). by RT-PCR did not reveal evidence of virus On April 2, 2003, a veterinarian visited a farm in shedding. Teeffelen, the Netherlands, with a fl ock suspected In the fi rst three herds, the seropositive pigs were of an HPAI infection. The farm was well outside the found scattered across the farms, rarely more than surveillance zone, and therefore the veterinarian was one per pen. In the herd with the highest seropreva- not instructed to wear personal protective equipment lence, these seropositives pigs were correlated with and Tamifl u was not prescribed. On April 4, the feeding of broken eggs from infected poultry in two veterinarian had a high fever and severe headache. compartments. Paired blood samples from over 200 When he fi rst visited his family physician for per- individual animals showed fi ve conversions from sistent fever and headache 4 days later, no signs of positive to negative and three conversions from respiratory disease or conjunctivitis were observed, negative to positive. All other results were shown to and the man, therefore, still did not receive anti- be reproducible. In all cases, the titer of these con- infl uenza medication. The veterinarian was admitted versions was around the threshold value of 40, and to the hospital, where his condition deteriorated until test variation and nonspecifi c responses were most he died of respiratory insuffi ciency under intensive likely the cause of these “conversions.” care on April 17 (30). H7N7 virus was isolated from Category 2 included 21 herds and category 3 and detected by RT-PCR in lung tissue and bron- included 23 herds, all located in the protection zones, choalveolar lavage collected 3 days before he died. within a 3-km radius of an infected holding. The Virus isolated from lavage displayed 14 amino acids seroprevalence of 1.1% was detected with no sig- differences compared with the virus isolated from nifi cant difference between categories. Titers were the poultry index case. Some of the substitutions low at threshold level. Similar prevalence and titers are believed to affect the virus pathogenicity in were detected in herds located in the northern part mammals. of the Netherlands and sampled prior to the introduction of H7N7 into the Netherlands. Only sera Infections of Pigs with a positive HI titer from category 1 farms were In early April, a serological surveillance program confi rmed in the virus neutralization test. Based on was started to determine whether the virus was also the results, it was decided that all category 1 herds infecting pigs. Infections of pigs may have conse- in the surveillance zones should be tested serologi- quences for both animal and human health. cally according to the same representative sampling 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 233 schedule used during the familiarizing part of the isolated virus was produced (L. Sims, personal surveillance in pigs. In total, 46 category 1 herds communication). were identifi ed and tested, including the fi rst 13 There was spread to two farms about 4 km away “category 1” herds that had been tested during the from the index farm; all three farms were under the fi rst part of the surveillance. From these 46 herds, same ownership although managed separately. 13 herds had a signifi cant number of seropositive Sims reports that the farms had high biosecurity pigs, indicating that they were likely infected. In one and it was not clear how the initial introduction or of these herds having a high seroprevalence (35%), secondary spread occurred (L. Sims, personal oropharyngeal swabs for RT-PCR were taken from communication). all 63 pigs present. In none of these swabs could A representative virus, isolated at the Central Vet- viral RNA be detected (36). erinary Laboratory DPR Korea, was sent to the OIE The conclusions from these results indicated Reference Laboratory at CSIRO Australian Animal swine in mixed herds with infected poultry were at Health Laboratory, Geelong, Australia, where it was risk from the introduction of the HPAI, subtype shown to be of H7N7 subtype with an IVPI of 3.0. The H7N7, whereas swine in mixed herds with no HA0 cleavage site amino acid motif was shown to infected poultry or swine in herds with no poultry have the unusual sequence PEIPKGRHRRPKR*GLF were not at risk from becoming infected with the AI, (P. Selleck, personal communication). subtype H7N7. Most of the infections seemed to There were no further reports of outbreaks of have been transmitted from poultry-to-swine, either disease due to H7N7 HPAI virus, but it is not clear directly or indirectly and no evidence was obtained how much surveillance there was, especially in for transmission between pigs. Also, no evidence vaccinated fl ocks. was found that the virus was able to maintain itself for a long period in the swine herds after the removal CONCLUSIONS of the source of infection, namely the infected Conventionally, the emergence of HPAI outbreaks poultry. After experimental infection of pigs, evi- is considered to be the result of LPAI viruses of H5 dence of transient infection of a single pig and shed- or H7 subtype spreading from their reservoirs in ding of small amounts of virus in the pharynx in wild birds to poultry, where, via one of a number of a single pig for a single day were obtained with different mechanisms, mutation to virulence occurs no transmission of H7N7 to noninfected contact either immediately or after the LPAI virus has cir- animals. culated in poultry. In many respects, most of the 11 reported occurrences of HPAI viruses in Europe and DEMOCRATIC PEOPLE’S REPUBLIC OF Africa described in this chapter fail to conform to KOREA (NORTH KOREA) 2005 (H7N7) this dogma for outbreaks of HPAI. The H5N3 HPAI An outbreak of HPAI was reported in the Demo- outbreaks in terns in South Africa in 1961 (see cratic People’s Republic of Korea in early 2005. earlier discussion) seem to suggest that the assump- Disease was confi rmed on three farms near tion that mutation to virulence occurs only after Pyongyang, and 219,000 chickens died or were introduction of LPAI H5 or H7 viruses to poultry slaughtered. may not always be correct. Although an explanation The disease was fi rst recorded at a large layer could be that HPAI did occur in a small poultry fl ock farm consisting of 24 houses containing about and went unreported but led to spread to the terns 160,000 layers. Mortality was observed in one house directly or through some other intermediary. Inter- with a capacity of 19,000 to 24,000 birds on Febru- estingly, the H5N2 HPAI virus infecting ostriches ary 25, 2005, and mortality over February 25 to 28, in South Africa in 2004 has been reported as absent 2005, was 70, 200, and 300 birds (L. Sims, personal from other poultry, suggesting that ostriches may communication). The infected house was depopu- also serve as a vehicle in which LPAI viruses may lated, but not unaffected houses on the same farm. mutate to virulence for poultry. Remaining birds on the farm and those on related The European and African HPAI outbreaks give farms in the area were given an emergency autoge- no explanations of why and when H5 and H7 LPAI nous vaccine made from tissues of infected viruses may mutate to HPAI viruses, and if any- birds, and subsequently a vaccine made from the thing, the tern outbreak in South Africa and the 234 Avian Influenza possible wild bird spread between England and areas, the likelihood of spread and the overall impact Germany complicate the situation further. In all the of HPAI outbreaks were lessened compared with the outbreaks except Italy 1999–2000, mutation seems extreme density of poultry in the Dutch and Italian to have taken place rapidly (even at the primary site) outbreaks. Five of the HPAI outbreaks during the after introduction from wild birds. In the 1999–2000 period occurred in the British Isles, and while poultry Italian outbreak, the LPAI precursor virus circulated may be reared intensively in Ireland and the United in poultry for 9 months before mutating and the Kingdom, true DPPAs comparable to those in other detailed epidemiological and phylogenetic data countries have not been established so far. However, obtained from the careful monitoring and analyses the DPPAs in the Netherlands, Italy, and elsewhere of both the LPAI and HPAI outbreaks that occurred remain, and it seems important that surveillance and (12, 20) represent the best evidence to date that such control strategies should be put in place that will mutations do occur. However, it remains impossible allow early warning and rapid eradication of any to predict if and when this mutation will occur. future emergence of HPAI in these areas if similar Until the emergence of the Asian H5N1 virus and catastrophic outbreaks as those occurring in 1999– its spread to Europe and Africa in 2004–2006, the 2000 and 2003 are to be avoided. role of wild birds in the spread of HPAI had been considered unlikely and, except for the terns in REFERENCES South Africa in 1961, infections of wild birds had 1. Alexander, D.J. 1982. Avian infl uenza—recent been limited to birds found dead in the vicinity of developments. The Veterinary Bulletin 52:341– infected poultry. However, the similarity in the 359. viruses responsible for the outbreaks in England and 2. Alexander, D.J. 1987. Avian infl uenza—historical aspects. In: B.C. Easterday and C.W. Beard (eds.). Germany in 1979, the unusual wild bird movements Proceedings of the Second International Sympo- at the time of the Norfolk outbreak, and the reported sium on Avian Infl uenza, Georgia Center for isolations of the HPAI virus from wild birds suggest Continuing Education, the University of Georgia, they may well have been implicated in the move- Athens, Georgia, USA, September 3–5, 1986. ment of the HPAI virus between Germany and Symposium on Avian Infl uenza, US Animal Health England. It is a great pity that so little information Association: Richmond, VA, pp. 4–13. on the German outbreak has been placed in the inter- 3. Alexander, D.J. 2005. Avian infl uenza. In: Chapter national scientifi c literature or public domain. 2.07.12. Manual for Diagnostic Tests and Vaccines The 2003 HPAI epizootic in the Netherlands, with for Terrestrial Animals, 5th ed. World Organiza- limited spread to Belgium and Germany, resulted in tion for Animal Health: Paris. Available at http:// the death or slaughter of about 30 million birds, www.oie.int/eng/normes/MMANUAL/A_00037. htm. which was an unprecedented number in the history 4. Alexander, D.J., and W.H. Allan. 1982. Avian of the control of HPAI and has only been surpassed infl uenza in turkeys—a survey of farms in eastern in more recent years by the Asian lineage H5N1 England 1979–1980. British Veterinary Journal HPAI outbreaks. The reason for this massive loss of 138:473–479. birds is almost wholly attributable to the extremely 5. Alexander, D.J., and D. Spackman. 1981. Charac- high density and close proximity of poultry farms in terisation of infl uenza A viruses isolated from the affected area (up to 25 farms/km2 in the Gelder turkeys in England during March—May 1979. Valley). Not only did this result in diffi culties in Avian Pathology 10:281–283. containment, but it also meant there were large 6. Alexander, D.J., and G.W. Wood. 1994. Highly numbers of birds involved in the buffer zones when pathogenic avian infl uenza diagnosis: past experi- it was decided to depopulate those preemptively. ence, and future problems? In: Proceedings of the European Commission meeting on Virus Diseases The extensive Italian outbreak in 1999–2000 also of Poultry—New and Evolving Pathogens, Brus- occurred in the DPPAs in northeast Italy. The estab- sels, 1992. CEC: Brussels, Belgium, pp. 3–13. lishment of DPPAs in certain areas of Europe as part 7. Alexander, D.J., S.A. Lister, M.J. Johnston, C.J. of the industrialization and integration of poultry Randall, and P.J. Thomas. 1993. An outbreak of rearing has largely gathered momentum in the past highly pathogenic avian infl uenza in turkeys in 15 years; it may well be that while some of the Great Britain in 1991. The Veterinary Record 1959–1991 outbreaks occurred in poultry producing 132:535–536. 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 235

8. Alexander, D.J., G. Parsons, and R.J. Manvell. KAMERCORRESPONDENTIE/2003/ 1986. Experimental assessment of the pathogenic- BIJLAGEN/PAR03244A. ity of eight avian infl uenza A viruses of H5 subtype 19. Boven van, M., J. van der Goot, A.R.W. Elbers, G. for chickens, turkeys, ducks and quail. Avian Koch, G. Nodelijk, M.C.M. de Jong, T.S. de Vries, Pathology 15:647–662. A. Bouma, and J.A. Stegeman. 2005. Vaccination 9. Allan, W.H., C.R. Madeley, and A.P.K. Kendal. of poultry against avian infl uenza: epidemiological 1971. Studies with avian infl uenza A viruses: cross rules of thumb and experimental quantifi cation of protection experiments in chickens. Journal of the effectiveness of vaccination. In: R.S. Schrijver General Virology 12:79–84. and G. Koch (eds.). Avian Infl uenza: Prevention 10. Banks, J. 2001. Genetic variation in avian infl u- and Control. Wageningen UR Frontis Series enza viruses. PhD thesis, The Open University, Volume 8. Springer: Dordrecht, the Netherlands, England. pp. 85–92. 11. Banks, J., E.C. Speidel, J.W. McCauley, and D.J. 20. Capua, I., and S. Marangon. 2000. Review article: Alexander. 2000. Phylogenetic analysis of H7 hae- the avian infl uenza epidemic in Italy, 1999–2000. magglutinin subtype infl uenza A viruses. Archives Avian Pathology 29:289–294. of Virology 145:1047–1058. 21. Capua, I., and F. Mutinelli. 2001. A color atlas and 12. Banks, J., E.S. Speidel, E. Moore, A. Piccirillo, I. text on avian infl uenza. Papi Editore: Bologna, Capua, P. Cordioli, A. Fioretti, and D.J. Alexander. Italy. 2001. Changes in the hemagglutinin and the neur- 22. Capua, I., and F. Mutinelli. 2001. Mortality of aminidase genes, prior to the emergence of highly Muscovy ducks (Cairina moschata) and domestic pathogenic H7N1 avian infl uenza viruses in Italy. geese (Anser anser var. domestica) following Archives of Virology 146:963–973. natural infection with highly pathogenic avian 13. Becker, W.B. 1963. The morphology of tern virus. infl uenza of the H7N1 subtype. Avian Pathology Virology 20:318–327. 30:179–183. 14. Becker, W.B. 1966. The isolation and classifi ca- 23. Capua I., S. Marangon, L. Selli, D.J. Alexander, tion of tern virus: infl uenza virus A/tern/South D.E. Swayne, M. Dalla Pozza, E. Parenti, and F. Africa/1961. Journal of Hygiene 64:309–320. M. Cancellotti, 1999. Outbreaks of highly patho- 15. Becker, W.B., and C.J. Uys. 1967. Experimental genic avian infl uenza (H5N2) in Italy during infection of chickens with infl uenza A/tern/South October 1997—January 1998. Avian Pathology Africa/1961 and chicken/Scotalnd/1959 viruses. I. 28:455–460. Clinical picture and virology. Journal of Compara- 24. Capua, I., B. Grossele, E. Bertoli, and P. Cordioli. tive Pathology 77:159–165. 2000. Monitoring for highly pathogenic avian 16. Berenschot Groep B.V. 2004. Report De crisis infl uenza in wild birds in Italy. The Veterinary tussen mens en dier. Evaluatie bestrijding AI- Record 147:640. Crisis. Report for Dutch Ministry of Agriculture, 25. Capua, I., F. Mutinelli, M.A. Bozza, C. Terregino, Nature and Food Quality. Available at http:// and G. Cattoli. 2000. Highly pathogenic avian www9.minlnv.nl/pls/portal30/docs/FOLDER/ infl uenza (H7N1) in ostriches (Struthio camelus). MINLNV/LNV/STAF/STAF_DV/ Avian Pathology 29:643–646. KAMERCORRESPONDENTIE/ 26. Capua, I., F. Mutinelli, S. Marangon, and D.J. 2004/BIJLAGEN/PAR04109A. Alexander. 2000. H7N1 Avian Infl uenza in Italy 17. Bosman A., A. Meijer, and M. Koopmans 2005. (1999–2000) in intensively reared chickens and Final analysis of Netherlands avian infl uenza out- turkeys. Avian Pathology 29:537–543. breaks reveals much higher levels of transmission 27. CEC. 1992. Council Directive 92/40/EEC of 19 to humans than previously thought. Euro Surveil- May 1992 introducing community measures for lance 10:1. Available at http://www.eurosurveil- the control of avian infl uenza. Offi cial Journal of lance.org/ew/2005/050106.asp#2 the European Communities L167:1–15. 18. Boven van, M., G. Boender, A.R.W. Elbers, G. 28. Du Ry van Beest Holle, M., A. Meijer, M. Koop- Nodelijk, M.C.M. de Jong, A. Dekker, G. Koch, mans, and C.M. de Jager. 2005. Human-to- and J.A. Stegeman. 2003. Epidemiologische con- human transmission of avian infl uenza A/H7N7, sequenties van vaccinatie Bijlage 1 bij VVA03 the Netherlands, 2003. Euro Surveillance 10:264– 2553. Report for Dutch Ministry of Agriculture, 268. Nature and Food Quality. Available at http:// 29. Elbers, A.R.W., T.H.F. Fabri, T.S de Vries, J.J. de www9.minlnv.nl/pls/portal30/docs/FOLDER/ Wit, A. Pijpers, and G. Koch. 2004. The highly MINLNV/LNV/STAF/STAF_DV/ pathogenic avian infl uenza A (H7N7) virus 236 Avian Influenza

epidemic in the Netherlands in 2003—lessons 39. MAFF. 1981. Animal Health 1979 report of the learned from the fi rst fi ve outbreaks. Avian Dis- Chief Veterinary Offi cer. Her Majesty’s Stationery eases 48:691–705. Offi ce: London. 30. Fouchier, R.A., P.M. Schneeberger, F.W. 40. MAFF. 1993. Animal Health 1992 report of the Rozendaal, J.M. Broekman, S.A. Kemink, V. Chief Veterinary Offi cer. Her Majesty’s Stationery Munster, T. Kuiken, G.F. Rimmelzwaan, M. Offi ce: London. Schutten, G.J. Van Doornum, G. Koch, A. Bosman, 41. McNulty, M.S., G.M. Allan, R.M. McCracken, M. Koopmans, and A.D. Osterhaus. 2004. Avian and P.J. McParland. 1985. Isolation of a highly infl uenza A virus (H7N7) associated with pathogenic infl uenza virus from turkeys. Avian human conjunctivitis and a fatal case of acute Pathology 14:173–176. respiratory distress syndrome. Proceedings of the 42. Mannelli, A., N. Ferrè, and S. Marangon. 2006. National Academy of Sciences U S A 101(5):1356– Analysis of the 1999–2000 highly pathogenic 1361. avian infl uenza (H7N1) epidemic in the Mai 31. Harder, T.C., and O. Werner. 2006. Avian infl u- poultry production area in northern Italy. Preven- enza. In: B.S. Kamps, C. Hoffmann, and W. Preiser tive Veterinary Medicine 73:273–285. (eds.). Infl uenza Report. Flying Publisher. Avail- 43. Manvell, R.J., R.F. Horner, G. Akol, C. Abolnik, able at http://www.infl uenzareport.com/ir/ai.htm. M. Romito, and I.H. Brown. 2005. Isolation of an 32. Van Heelsbergen, T. 1927. Vogelpest. Tijdschrift infl uenza A virus subtype H5N2 from ostriches in voor Diergeneeskunde 54:516–519. South Africa in 2004. In: Proceedings of the 3rd 33. Kawaoka, Y., W.J. Bean, and R.G. Webster. 1987. International Ratite Science Symposium and XII Molecular characterisation of the A/chicken/Penn- World Ostrich Congress Madrid, 14–16 October sylvania/83 (H5N2) infl uenza viruses. In: B.C. 2005. E. Carbajo (ed.). Madrid, Spain, pp. 197– Easterday and C.W. Beard (eds.). Proceedings of 200. the Second International Symposium on Avian 44. Manvell, R.J., P.H. Jorgensen, O.L. Nielsen, and Infl uenza, Georgia Center for Continuing Educa- D.J. Alexander. 1998. Experimental assessment of tion, the University of Georgia, Athens, Georgia, the pathogenicity of two avian infl uenza A H5 USA, September 3–5, 1986. Symposium on viruses in ostrich chicks (Struthio camelus) and Avian Infl uenza, US Animal Health Association: chickens. Avian Pathology 27:400–404. Richmond, VA, pp. 197–214. 45. Manvell, R.J., P. McKinney, U. Wernery, and K. 34. Koopmans, M., B. Wilbrink, M. Conyn, G. Natrop, Frost. 2000. Isolation of a highly pathogenic infl u- H van der Nat, H. Vennema, A. Meijer, J. van enza A virus of the subtype H7N3 from a peregrine Steenbergen, R. Fouchier, A. Osterhaus, and A. falcon (Falco peregrinus). Avian Pathology Bosman. 2004. Transmission of H7N7 avian 29:635–637. infl uenza A virus to human beings during a large 46. Marangon, S., I. Capua, E. Rossi, N. Ferrè, M. outbreak in commercial poultry farms in the Dalla Pozza, L. Bonfanti, and A. Mannelli. 2005. Netherlands. Lancet 363:587–593. The control of avian infl uenza in areas at risk: the 35. Li, Z., Y. Jing, P. Jiao, A. Wang, F. Zhao, G. Tian, Italian experience. In: R.S. Schrijver and G. Koch X. Wang, K. Yu, Z. Bu, and H. Chen. 2006. The (eds.). Avian Infl uenza, Prevention and Control. NS1 gene contribute to the virulence of H5N1 Wageningen UR Frontis Series Volume 8. Springer: avian infl uenza viruses. Journal of Virology Dordrecht, the Netherlands, pp. 33–39. 80:1115–11123. 47. Meijer, A., A. Bosman, E.E. van de Kamp, B. 36. Loeffen, W.L.A., and G. Koch. 2003 Avian infl u- Wilbrink, M. du Ry van Beest Holle, and M. Koop- enza (H7N7) in Dutch pigs. CIDC: Lelystad, the mans. 2006. Measurement of antibodies to avian Netherlands. Available at http://www.promedmail. infl uenza virus A (H7N7) in humans by hemag- org/pls/promed/f?p = 2400:1202:44458149575892 glutination inhibition test. Journal of Virological 53518::NO::F2400_P1202_CHECK_ Methods 132:113–120. DISPLAY,F2400_P1202_PUB_MAIL_ID:X, 48. Murphy, T.M. 1986. The control and epidemiol- 21644. Accessed April 1, 2007. ogy of an infl uenza A outbreak in Ireland. In: J.B. 37. MAFF. 1961. Report on the Animal Health Service McFerran and M.S. McNulty (eds.). Acute Virus in Great Britain 1959. Her Majesty’s Stationery Infections of Poultry. Martinus Nijhoff: Dordrecht, Offi ce: London. the Netherlands, pp. 23–28. 38. MAFF. 1965. Report on the Animal Health Service 49. Naeem, K. The avian infl uenza outbreak in South in Great Britain 1963. Her Majesty’s Stationery Central Asia. 1998. In: D.E. Swayne and R.D. Offi ce: London. Slemons (eds.). Proceedings of the Fourth Interna- 9 / Highly Pathogenic Avian Influenza Outbreaks in Europe, Asia, and Africa Since 1959 237

tional Symposium on Avian Infl uenza, May 29–31, 59. Rohm, C., J. Suss, V. Pohle, and R.G. Webster. 1997, Athens, Georgia. Symposium on Avian 1996. Different haemagglutinin cleavage site vari- Infl uenza, US Animal Health Association: Rich- ants of H7N7 in an infl uenza outbreak in chickens mond, VA, pp. 31–35. in Leipzig, Germany. Virology 218:2153–2157. 50. Naeem, K., and N. Siddique. 2006. Use of strategic 60. Rowan, M.K. 1962. Mass mortality among Euro- vaccination for the control of avian infl uenza in pean common terns in South Africa in April–May Pakistan. Developments in Biology 124:145–150. 1961. British Birds 55:103–114. 51. Naeem, K., A., Ullah, R.J. Manvell, and D.J. Alex- 61. Sinnecker, H., J. Egert, H.J. Eggert, J. Gogolin, S. ander. 1999. Avian infl uenza A subtype H9N2 in Gumpel, C. Schrader, R. Sinnecker, and E. Zilske. poultry in Pakistan. The Veterinary Record 145:560. 1986. Studies of some biological and molecular 52. Offi ce Internationale des Epizooties. 2006. characteristics of avian infl uenza A/H7 (Hav1) Update on avian infl uenza in animals (type subtypes. Acta Virologica 30:25–34. H5). Available at http://www.oie.int/downld/ 62. Stegeman, A., A. Bouma, R.W. Elbers, M.C.M. de AVIAN%20INFLUENZA/A_AI-Asia.htm. Jong, G. Nodelijk, F. de Klerk, G. Koch, and M. Accessed October 14, 2006. van Boven, 2004. Avian infl uenza A virus (H7N7) 53. Olivier, A.J. 2006. Ecology and epidemiology of epidemic in the Netherlands in 2003: course of avian infl uenza in ostriches. Developments in the epidemic and effectiveness of control mea- Biology 124:51–57. sures. Journal of Infectious Diseases 190:2088– 54. Perdue, M., J. Crawford, M. Garcia, J. Latimer, 2095. and D.E. Swayne. 1998. Occurrence and possible 63. Stegeman, J.A., A. Bouma, R.W. Elbers, M. van mechanisms of cleavage site insertions in the avian Boven, M.C.M. de Jong, and G. Koch. 2005. infl uenza hemagglutinin gene. In: D.E. Swayne Effectiveness of control measures on the transmis- and R.D. Slemons (eds.). Proceedings of the Fourth sion of avian infl uenza virus (H7N7) between International Symposium on Avian Infl uenza, May fl ocks. In: R.S. Schrijver and G. Koch (eds.). Avian 29–31, 1997, Athens, Georgia. Symposium on Infl uenza, Prevention and Control. Wageningen Avian Infl uenza, US Animal Health Association: UR Frontis Series Volume 8. Springer: Dordrecht, Richmond, VA, pp. 182–193. the Netherlands, pp. 49–55. 55. Pereira, H.G., B. Tumova, and V.G. Law. 1965. 64. Uys, C.J., and W.B. Becker. 1967. Experimental Avian infl uenza A viruses. Bulletin of the World infection of chickens with infl uenza A/tern/South Health Organization 32:855–860. Africa/1961 and chicken/Scotland/1959 viruses. 56. Perroncito, E. 1878. Epizoozia tifoide nei gallina- II. Pathology. Journal of Comparative Pathology cei. Annali Accademia Agricoltura Torino 21:87– 77:167–179. 126. 65. Wells R.J.H. 1963. An outbreak of fowl plague in 57. Petek, M. 1982. Current situation in Italy. In: R.W. turkeys. The Veterinary Record 75:783–786. Bankowski (ed.). Proceedings of the First Interna- 66. De Wit, J.J., G. Koch, T.H.F. Fabri, and A.R.W. tional Symposium on Avian Infl uenza, April 1981, Elbers, 2004. A cross-sectional serological survey Beltsville, Maryland. Symposium on Avian Infl u- of the Dutch commercial poultry population for the enza, US Animal Health Association: Richmond, presence of low pathogenic avian infl uenza virus VA, pp. 31–34. infections. Avian Pathology 33:565–570. 58. Puzelli, S., L. Di Trani, C. Fabiani, L. Campitelli, 67. Wood, G.W., J. Banks, J.W. McCauley, and D.J. M.A. De Marco, I. Capua, J.F. Aguilera, M. Alexander. 1994. Deduced amino acids sequences Zambon, and I. Donatelli. 2005. Serological of the haemagglutinin of H5N1 avian infl uenza analysis of humans exposed to avian H7 infl uenza virus isolates from an outbreak in turkeys in viruses in Italy between 1999 and 2003 Journal of Norfolk, England. Archives of Virology 134:185– Infectious Diseases 192:1318–1322. 194. 10 Avian Influenza in Australia

Leslie D. Sims and Andrew J. Turner

INTRODUCTION The fi rst outbreak of HPAI in early 1976 repre- The global prominence of highly pathogenic avian sented a watershed for the poultry industry and infl uenza (HPAI) has grown dramatically over the animal disease authorities in Australia. At the time past 10 years from a relatively rare but serious of this outbreak, preparedness for such an event was disease of poultry to become the most publicized not well developed either in the fi eld or the labora- zoonotic infection. This has largely been the result tory. In addition, at that time there was still much of the spread of H5N1 HPAI viruses across three uncertainty regarding the molecular factors deter- continents with concurrent fatal disease in humans, mining the pathogenicity and emergence of HPAI and also from high-profi le, multifarm outbreaks in viruses, and these agents were regarded as exotic to Italy, the Netherlands, and Canada that resulted in Australia. This outbreak provided the fi rst indication the death and destruction of more than 50 million in Australia that poultry kept in nonbiosecure facil- poultry (11, 14, 34). The number of countries ities were vulnerable to HPAI without the need for affected by HPAI and the magnitude of outbreaks introduction of a virulent virus from another has increased dramatically during this period (13), country. yet Australia remains the only populated continent This, and subsequent outbreaks over the next 20 not to have experienced an outbreak of HPAI of any years, led to major changes in the understanding of type since 1997. HPAI has occurred in Australia avian infl uenza (AI); in emergency disease pre- in the past but outbreaks of the magnitude seen in paredness and funding arrangements for disease Asia, Europe, and North America have not been control operations; in training and awareness of recorded. roles and responsibilities of animal disease authori- Australia experienced fi ve outbreaks of HPAI ties; and in improvements to farm biosecurity. All between 1976 and 1997 (30, 41). Each of these out- of these have helped to protect Australia from this breaks involved only single farms or small clusters disease and led to rapid responses to each of the of farms, with limited spatial spread. In all cases, the outbreaks, limiting their spread and therefore the disease was eradicated through a combination of adverse effects of the disease. stamping out, strict movement controls, cleaning This paper briefl y reviews the history of AI in and disinfection of the infected premises, and inten- Australia, provides a description of each of the Aus- sive surveillance. All outbreaks were caused by tralian outbreaks of HPAI, explores some lessons highly pathogenic viruses of the H7 subtype (two learned from these, and examines issues relating to H7N7, two H7N3, and one H7N4). these outbreaks, including the reasons why Australia Despite the relatively small size of these out- has not had any cases of HPAI since 1997. breaks, they still caused signifi cant disruptions to trade, requiring mobilization of considerable human AVIAN INFLUENZA IN BIRDS OTHER and fi nancial resources. The outbreaks resulted in THAN DOMESTIC POULTRY curtailment of other veterinary activities in affected IN AUSTRALIA states until the disease was brought under control Studies on AI viruses in wild birds in Australia com- and freedom from infection was reestablished. menced in the 1970s when the role of wild aquatic

Avian Influenza Edited by David E. Swayne 239 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 240 Avian Influenza birds as natural hosts of AI viruses was fi rst estab- detected in a high proportion of emu chicks in this lished. Despite considerable testing, relatively few fl ock, but not in parent birds reared some distance AI viruses have been isolated from wild birds in away. Molecular characterization of the virus sug- Australia. This contrasts markedly with the fi ndings gested it was essentially identical to viruses from from Europe and North America where the preva- poultry farms in the vicinity affected by an outbreak lence of infection in wild birds is much higher (21, of HPAI in the vicinity (30). However, the intrave- 33), especially in places where juvenile ducks con- nous pathogenicity index (IVPI) of this virus (IVPI gregate prior to migration in late summer. = 1.3) was much lower than that of chicken isolates In one study in the 1970s, conducted in the north- from a neighboring infected farm (IVPI = 2.9) west, central coast, and southwest of Western Aus- despite having the same gene sequence at the cleav- tralia, only 42 of 6805 cloacal swabs from wild birds age site (see Origin of Viruses later). yielded infl uenza viruses (24). This low prevalence of infection in Australian wild birds has been con- OUTBREAKS OF HPAI IN fi rmed in other studies (29). AUSTRALIAN POULTRY Virtually all of the viruses from wild birds in HPAI has been recognized in only the three eastern Australia have been isolated from Anatidae [includ- states of Australia: Victoria (three separate out- ing Pacifi c black duck (Anas superciliosa), Austra- breaks), Queensland (one outbreak), and New South lian shelduck (Tadorna tadornoides), and Grey teal Wales (one outbreak) (Fig. 10.1). All of the affected (Anas graxilis)] and Charadriiformes [including farms had fl awed biosecurity systems that permitted Red-necked stints (Calidris rufi collis) and Lesser entry of infl uenza viruses, presumably from infected noddy (Anous tennirostris)], with some isolates wild birds, although the precise source of infection from Procelliformes (shearwaters) (24). There are in these outbreaks was not determined. Historical limited data available for some avian species (36). records do not suggest the occurrence of any out- The HA subtypes recorded in wild aquatic birds breaks of HPAI in Australia prior to 1976. Accord- include H1, H3, H4, H5, H6, H11, H12, and H15 ing to Turner (37), low pathogenicity (LP) viruses (3). were detected in poultry in northwestern Australia Surveys on infl uenza viruses in wild birds have continued intermittently over the past 35 years and the number of these studies has increased recently, due to concerns that wild birds could carry H5N1 HPAI viruses from Asia to Australia. Results from recent surveys provide no reason to alter the view that the prevalence of infection with AI viruses in wild aquatic birds in Australia is generally low (23). As of August 2006, HA sequences of only 28 AI viruses isolated from Australian birds have been uploaded into GenBank, of which six are from poultry. The only wild bird isolate of the H7 subtype among these was from a starling (Sturnus vulgaris) on a farm experiencing an outbreak of HPAI in Victoria in 1985. This virus was very similar to that found in the poultry on the infected farm, and it is likely that this bird was infected by poultry rather than being the original source of infection for the farm (26). No H5N1 HPAI viruses have been Figure 10.1. Location of outbreaks of highly isolated. pathogenic avian infl uenza in Australia. 1, One H7N4 virus was isolated from a farmed emu Keysborough (1976); 2, Bendigo (1985); 3, Bendigo (1992); 4, Lowood (1994); 5, (Dromaiius novaehollandiae) in New South Wales Tamworth (1997). (30). Serological evidence of H7 infection was 10 / Avian Influenza in Australia 241 in the 1970s, but the serotypes of the viruses involved proventriculus. An H7N7 virus was isolated from were not recorded. heart blood, trachea, and cloacal swabs. Inoculated embryonating eggs died approximately 30 to 36 1976—Keysborough Victoria H7N7 hours following inoculation. This virus was tenta- The fi rst outbreak of HPAI occurred in Australia in tively identifi ed as an AI virus using reference serum 1976 on a small mixed broiler and layer farm at and antigen (supplied by CSIRO, Melbourne/ Keysborough, about 26 km southeast of central Weybridge, UK), and its identity was confi rmed Melbourne, on the outskirts of the city (37, 38). at the international reference center for AI at The farm was a combined broiler and egg layer Weybridge. operation, with a poultry processing plant on site. The chickens on the farm were supplied with Broiler chickens and eggs were sold at a market in untreated water from a surface dam. Based on a lack the northern suburbs of Melbourne. The layers were of other likely pathways, water from the dam was reared in cages. Biosecurity on the farm was minimal. considered to be the most likely source of the virus. Wild birds could easily access the chickens and their This water was possibly contaminated by wild ducks feed and water. Water used on the farm was untreated that frequented the dam in September to October and derived from a surface dam adjacent to the farm. 1975. The property experienced mortalities that were All chickens on the farm were destroyed within 5 attributed to fowl cholera in October 1975. There days of the diagnosis. The cost sharing agreement were eight barns on the farm. One enclosed barn between Australian state and federal governments contained broilers and the other seven contained for eradicating specifi c diseases in Australia was layers. Mortality in one barn reached 25%. Mortality invoked. Compensation at full market value was in the other barns was lower but there was evidence, paid. serological and clinical, that birds in two other barns The chickens, eggs, and all litter were buried on had also been infected. site. The property was cleaned, disinfected, and left Specimens were examined on a number of occa- unoccupied, after which sentinel chickens were sions between October 1975 and January 1976 and placed in the farm. As these did not develop infec- the lack of response to treatment was ascribed to tion, repopulation was allowed. antibiotic resistance. Virus culture was not done Directly across the road from the chicken farm until early 1976 after signs typical of HPAI were was a duck farm that kept breeders in barns and detected during a farm visit. These signs, detected reared other ducks on open range. Hygiene condi- in layer hens, included grossly swollen combs, and tions on this farm were poor. Blood samples taken ecchymotic and petechial hemorrhages visible on from the ducks before the chickens on the adjacent featherless areas of the legs of affected chickens. property were killed were serologically negative in Layer cages contained gaps, presumably because HI tests using the virus isolated from the neighbor- poultry had died and been removed. Some poultry ing chicken farm. However, when sampled the fol- were developing clinical signs, others were severely lowing week, some ducks appeared to have low depressed, and some appeared to be recovering from levels of antibodies and shortly thereafter ducks with infection. antibody titers indicative of infection were detected. Disease was evident in a small number of broiler No clinical signs of infection were seen in the chickens that were kept in multiage groups on dry ducks. litter. A small number of broiler chickens died each At the time, it was assumed that the slaughter and day. Antibody to H7 virus was detected in the fl ock cleaning operations on the chicken farm had created suggesting apparent recovery from infection, sub- suffi cient plumes of dust to disseminate infection clinical infection, co-cycling of LPAI and HPAI across the road to the duck farm, a distance of less viruses, or cycling of a virus of LP for some time than 100 m. However, this conclusion was not prior to conversion to an HPAI strain. entirely supported by subsequent molecular studies, On postmortem examination, lesions highly sug- unless several strains of H7 virus were circulating gestive of HPAI were detected in dead chickens on the index farm. including hemorrhages throughout the musculature, The genes coding for the HA protein of the infl u- heart, and other internal organs, including the enza virus from the duck on this farm differed suf- 242 Avian Influenza

Table 10.1. Amino acid sequence around the proteolytic cleavage site of the hemagglutinin of Australian avian inﬂ uenza viruses of the H7 subtype. Virus Amino acids at cleavage site

A/duck/Victoria/76 (H7N?) PEIPK—KR GLF A/chicken/Victoria/75 (H7N7) PEIPKKREKR GLF A/chicken/Victoria/1/1985 (H7N7) PEIPKKREKR GLF A/starling/Victoria/1/1985 (H7N7) PEIPKKREKR GLF A/chicken/Victoria/1/92 (H7N3) PEIPKKK-KR GLF A/chicken/Queensland/667/95 (H7N3) PEIPRKR-KR GLF A/chicken/NSW/1/1997 (H7N4) PEIPRKR-KR GLF

fi ciently from that of the virus isolated on the chicken laboratory examination. This chicken had swelling farm, to indicate that there had been multiple virus around the head, although this was not readily introductions to the area (10). The duck isolate was noticeable on the small, poorly developed comb. On considered to be an LPAI virus based on the amino postmortem examination, internal hemorrhages acid sequence at the hemagglutinin cleavage site were detected and an H7N7 HPAI virus was iso- (Table 10.1) and did not produce disease in experi- lated. Despite intensive surveillance by both virus mentally inoculated chickens, turkeys, or ducks (41). isolation and serology, no further infection was This duck farm was also depopulated with all detected on this property. poultry slaughtered and buried on site, along with Young chickens dosed intranasally with the virus the material from demolished barns that could not from the fi rst farm all died within 48 hours, as did be cleaned and disinfected. The farm was not allowed 8 of 10 chickens placed in contact with infected to repopulate with ducks, to comply with local nui- birds 8 hours postinoculation. Ducks exposed to the sance requirements. virus intranasally did not develop infection (1). Within 5 days of suspecting infection, serological Subsequent studies using the original chicken testing of poultry on other farms commenced using virus and that from the third infected farm demon- the isolated virus from the index farm as the source strated that they were both HP, but not universally of the test antigen in HI tests. Poultry farms across fatal in chickens. In contrast to the earlier studies Victoria were tested, with 60 randomly collected (1), under the conditions of this experiment, these blood samples taken from each of these. A total of viruses could infect ducks (42). 76,000 blood samples were tested for antibody (up The declaration of HPAI infection resulted in all to 9000 per day) with positive results only being other Australian states and territories imposing detected on the known positive chicken and duck embargoes on poultry meat and eggs from Victoria farms. (39). These trade measures were particularly costly Active surveillance was also carried out by col- to the Australian poultry industry with losses to the lecting and testing dead and sick birds. For about 4 industry running into the hundreds of thousands of weeks after the outbreak, hundreds of dead poultry dollars. Farms producing fertile eggs were hit most from surrounding areas were tested for infection severely. All trade restrictions were lifted about 1 using culture of pooled cloacal swabs (fi ve samples month after the ducks had been slaughtered (39). from the same farm were pooled before egg inocula- The cost of the eradication program for compensation). In total, some 3400 swabs were tested for virus tion and cleaning was Au$220,000. isolation in eggs. Wild birds were shot around each infected farm, but no AI virus was isolated from any 1985—Bendigo Victoria H7N7 of these. The next outbreak of HPAI occurred in May 1985. In the third week of the eradication and surveil- It involved a multibarn farm in central Victoria some lance campaign, a dead pullet, from an egg layer 15 km southwest of the city of Bendigo (9, 15, 25). farm housing some 100,000 layers, was collected for This farm reared a mixture of 26,000 layers, 24,000 10 / Avian Influenza in Australia 243 broiler breeders, and 61,000 broilers. The farm had from loss of export sales estimated at about a poultry processing plant on site and a new slaugh- Au$600,000 per week (15). tering plant was under construction at the time of the outbreak. Biosecurity on the farm was relatively 1992—Bendigo Victoria H7N3 poor. The next outbreak occurred 7 years later on a broiler Broiler breeders on the farm had been under treat- breeder farm related to the farm associated with the ment for several weeks for a disease that was fi rst 1985 outbreak (18, 41). This farm was located on recognized as a drop in egg production. Farm visits the northern outskirts of the city of Bendigo about by veterinarians revealed respiratory distress as well 160 km northwest of Melbourne. The farm was as evidence of external parasites in some poultry; located directly opposite a duck layer farm. lesions and culture results indicating, among other The index farm comprised four barns containing things, infectious coryza. Rather than responding breeder poultry on litter. Biosecurity on this farm favorably to antibiotic therapy, the mortality rate was not well developed. The four barns were located increased dramatically within a few days of systemic within 50 m of each other and farm workers moved antimicrobial treatment, with 80% mortality occur- between the poultry houses. Severe disease with ring over 3 days in one poultry house. Surviving rapid onset of high level mortality and lack of birds in the affected barn were depressed and had response to antibacterial therapy was detected in one signs suggestive of chronic respiratory disease. of the four fl ocks. Chickens in this fl ock developed An HPAI virus of the H7N7 subtype was isolated clinical signs and lesions typical of HPAI including from dead chickens, and in two separate experi- subcutaneous edema of the wattles and combs. ments, the IVPI of this virus was 2.74 and 2.8 Serological testing conducted on poultry in the (17). other barns revealed that two of these had high It is possible that a virus of LP circulated in one prevalence of antibody to H7 virus; poultry in the or more fl ocks of chickens on this farm before con- remaining barn were seronegative. The birds in one verting to an HP virus, but insuffi cient serological seropositive fl ock had experienced reduced egg pro- testing was done to demonstrate the duration of cir- duction but no other clinical signs were observed in culation of viruses on the farm before severe disease any of these three houses. The source of the H7N3 occurred. Virological testing was not performed virus causing the outbreak was not established. Sub- until high mortality occurred. sequent serological testing of the duck fl ock on the The source of the virus was not identifi ed. Water neighboring farm suggested evidence of infection in from the farm was derived from a large pond that younger ducks with an H7 subtype virus, but no attracted considerable numbers of aquatic birds, and virus was isolated. it is speculated that contaminated water was the Diagnosis of disease in the affected house was source of infection. Other possibilities cannot be established through immunofl uorescence tests on ruled out, including carriage of the virus onto the pancreatic impression smears (32). Additional farm by construction workers building a new slaugh- immunofl uorescence tests were performed on pan- terhouse within the farm boundary. However, sub- creatic smears from other dead poultry around the sequent serological testing of other fl ocks in the infected property. A total of 15,000 blood samples region failed to reveal evidence of infection, sug- were also tested from poultry across the state. No gesting infection via this route was unlikely. further cases of infection were detected. The disease was handled by slaughtering all The outbreak was managed according to Ausvet- infected poultry on the farm and those of related plan, the national plan for the eradication of contract growers. Surveillance testing was con- emergency animal diseases. As with the previous ducted across the state of Victoria and intensive outbreak, chickens on the affected farm were testing of dead poultry from farms in the vicinity of destroyed using carbon dioxide, in this case by the index case was conducted. placing chickens in steel rubbish containers (skips) This case again resulted in bans on movement of prefi lled with carbon dioxide. Carcasses, litter, eggs, poultry from Victoria to other states. The direct and other contaminated material were buried in costs were estimated at approximately Au$2 million, deep pits. In controlling the outbreak, two poultry but this did not include other losses to the industry farms, one backyard fl ock, and one hatchery were 244 Avian Influenza depopulated involving 17,000 chickens, 6000 ducks, November/December 1997 (30). The disease was 105,000 day-old chicks, and 540,000 fertile eggs at fi rst detected on a large chicken broiler breeder farm a related hatchery. Although the hatchery was holding about 128,000 breeders. There were four located some distance from the infected farm, all separate units on this farm, each comprising four fertile eggs in the hatchery were destroyed because separate poultry houses. At the time of the outbreak, the eggs from the affected property could not be 15 of these were occupied with chickens reared on distinguished from those from other farms. litter. The cost of eradication was Au$1.34 million com- The initial clinical signs were diarrhea, gasping, prising Au$720,000 in compensation and the and cyanosis of combs. The disease was initially remainder for operating costs (exclusive of staff suspected of being fowl cholera, and treatment with salaries). Overall 335 people were involved in the antibiotics was initiated, after which daily mortality depopulation, cleaning, disinfection, tracing, and in this house declined after more than 40% of the surveillance program. All barns on the duck farm poultry in this house had died. By this time, the were destroyed. The owner was provided with com- mortality in other houses in this and other units had pensation and the duck farm was not allowed to increased. In some of these, there were no other reestablish due to local law nuisance provisions. obvious clinical signs apart from an increase in the number of dead birds. The mortality rate in these 1994—Lowood Queensland H7N3 barns varied, but all had some evidence of increased The next outbreak occurred in 1994 near the town mortality. The time between the detection of the fi rst of Lowood, 40 km to the west of Brisbane in clinical signs and stamping out was approximately Queensland, on a multiage layer farm housing about 2 weeks. Retrospective serological testing of samples 20,000 fowl aged between 6 and 40 weeks. collected in October 1997 showed no evidence of The outbreak occurred during a severe drought. A infection, suggesting the introduction of the virus river forming a border for the farm and from which was recent. water was drawn had attracted a large population of Two other farms were also found to be infected wild birds. It was speculated that the virus could through active surveillance conducted in early have entered the farm via contaminated water. Of December on surrounding farms. One was a broiler the small number of wild birds caught in the vicinity breeder farm some 3 km to the south of the index of the farm, none were positive for H7 infection farm. Infection on this farm was detected through either on culture or serologically (41). tests on “routine” dead poultry and was followed a The virus from this outbreak was an H7N3 virus few days later by an explosive outbreak of disease with an IVPI of 2.63. The cleavage site of this virus on this farm in which more than 50% of poultry in differed from that of the virus from earlier outbreaks some affected barns died. but was the same as that in the subsequent outbreak The other infected farm was a contract broiler in New South Wales (Table 10.1). farm that, at the time, was rearing some 260 3- Serological testing of poultry in the fl ock revealed month-old emu chicks. No clinical disease was limited seroconversion in the area around the initial detected in these chicks, but an H7N4 virus was cases of infection, suggesting that the virus had not isolated from cloacal swabs from these chicks and cycled in the barn for long before causing death in serological testing revealed low-level titers to H7 the poultry. The nearest commercial poultry farm virus in 26 of 30 chicks bled just prior to depopula- was located some 12 km away, and as the infected tion of this farm. farm only sold eggs locally, the risk of spread from The outbreak was handled in accordance with this farm was low. No additional foci of infection Ausvetplan at a total cost of Au$4.445 million, were detected (27). approximately half of which was for compensation. The disease was handled in accordance with Approximately 310,000 poultry were destroyed Ausvetplan. The total cost of eradication was along with more than 1.2 million fertile eggs. approximately Au$420,000. The only known contact between these farms was a vehicle that collected dead birds. Numerous star- 1997—Tamworth New South Wales H7N4 lings were found on the premises rearing the emus, This outbreak involved three farms near the city raising the possibility that these birds could have of Tamworth in northern New South Wales in spread the virus between farms. The other potential 10 / Avian Influenza in Australia 245 source of virus was the river from which water was All of the outbreaks of HPAI in Australia were drawn on the index farm. However, the owner of this much smaller than those that occurred recently in farm had installed a plant to treat water some 6 Canada in 2003, the Netherlands in 2002, and in months earlier because of concerns about the number Asia from 1997. This probably related to the low of wild birds frequenting the river and surrounding poultry density in the affected areas that reduced the watercourses. opportunities for spread of virus. This low transmis- As with the outbreaks in 1985 and 1992, there was sibility of HPAI viruses in places with low poultry a variable pattern of mortality, even within indi- density is well recognized (2). Spread was also pre- vidual farm units. The IVPI of isolates from the vented by rapid action on the affected farms once a three affected farms varied from 1.3 (emus) to 2.52 highly pathogenic virus was identifi ed. (index farm) and 2.90 (second farm). The lack of cases in the past 10 years is probably a refl ection of the low risk of virus incursion that LESSONS LEARNED has been further reduced by enhanced biosecurity One of the main lessons learned from outbreaks of measures implemented in the wake of previous out- HPAI in Australia was that the initial incursion of breaks of both AI and Newcastle disease. It may also virus (presumably an LPAI virus) into a farm was refl ect reduced wild bird numbers as a result of the not always recognized immediately and the disease prolonged drought in Australia (40). that occurred could be easily mistaken for or complicated by other endemic diseases such as infec- Variation in Clinical Disease tious coryza or fowl cholera. In retrospect, earlier Unlike most recent Asian-origin H5N1 HPAI serological testing in the fi rst two Australian out- viruses, the Australian HPAI viruses were not always breaks might have resulted in earlier detection of uniformly fatal in fl ocks of commercial chickens infection, perhaps before the virus had the opportu- reared on litter, and the mortality rate varied consid- nity to circulate in poultry and increase its virulence erably from house to house on individual farms. for chickens. The reasons for this variation in the disease pattern Rapid diagnosis of incursions of AI viruses of the between fl ocks are not known. The simplest expla- H5 and H7 subtypes is essential to prevent emer- nation is that the virus converted from LP to HP as gence of HPAI. Serological testing for H5 or H7 AI it moved from one house to another, as has occurred is now recommended for any unexplained or ill- elsewhere in the world, but only circumstantial evi- defi ned production diseases or apparent bacterial dence for this is available in the Australian out- diseases in poultry that do not respond to treatment. breaks. The evidence is most convincing for the Regular serological testing of poultry at slaughter 1992 outbreak where infection of two fl ocks of can also provide assurances regarding infection with poultry occurred, with no or minimal signs of LPAI viruses and should be considered for higher disease, before severe disease occurred in a third risk populations such as free range and periurban barn. poultry. Other possible reasons include differences in sus- Following the fi fth HPAI outbreak in Australia, a ceptibility of different groups of poultry and the role national review was instituted to make recommen- of other stressors. In the 1985 outbreak, there was dations on the steps necessary to limit the costs considerable (unresolved) debate about whether the associated with HPAI outbreaks. The review recom- very high mortality rate in one barn was due to mended that biosecurity measures to prevent entry additional stresses of antimicrobial injection per- and dissemination of AI viruses from commercial formed 3 days earlier. Some age-related variation in poultry farms should be instituted across the country. susceptibility was demonstrated experimentally with Before the review was completed in 1998, the fi rst H7N7 virus infection in chickens using a virus from outbreak of Newcastle disease, due to mutation of this outbreak (22). This could have contributed to an endemic low virulent virus, occurred (7). This the differences seen between houses. event provided further impetus for improvements to The variation in mortality between farms, between biosecurity practices in the poultry industry. The units on the index farm, and in individual poultry standard of biosecurity in the commercial poultry houses within the units of the 1997 outbreak was industry is now much higher than it was in the dramatic (30). The reasons for this variation are still 1990s. not clear, but the possibility of an HP virus with a 246 Avian Influenza low IVPI increasing its pathogenicity as it cycled detection of seroconversion but no signifi cant disease through chickens is the most attractive hypothesis. in broiler breeders in two barns on an infected farm This may not have involved changes at the cleavage in 1992 provides some evidence to support the site given an HP virus with a low IVPI was detected conversion of a virus from LP to HP, although the in an emu on one of the infected farms. possibility of this being due to variation in suscep- Variation also occurred in the 1976 outbreak with tibility in different fl ocks to an HPAI virus cannot mortalities in broilers on litter never reaching the be ruled out. levels seen in layers. Again, the reason for this was The isolation of an H7 LPAI virus from a duck not determined. One possible explanation is that farm that differed genetically from the HPAI virus viruses may have gained or lost glycosylation sites isolated on the adjacent chicken farm in 1976 (42) in the HA. The 1976 chicken isolates were found to suggested multiple incursions of viruses to that par- contain two variants. Both had the same basic amino ticular location but did not provide additional infor- acids at the cleavage site but they varied by one mation on the origin of the virus on the index farm. amino acid elsewhere (position 188) that resulted in Molecular studies indicate that there is limited formation of a glycosylation site near the globular variation in the HA gene of H7 viruses in Australia head of the HA protein (28) that appeared to be (Table 10.2) and that these viruses form a distinct associated with a change from nonvirulence to viru- sublineage within the Eurasian lineage (8). The lence. Similar fi ndings (involving a different glyco- detection of different NA subtypes in viruses pos- sylation site) have been reported in the United States sessing similar HA genes provide some evidence of during the 1983–1984 Pennsylvania outbreak in reassortment in these viruses. which presence of a glycosylation site in some Studies of wild birds have demonstrated the very strains of virus reduced the virulence of an H5N2 low prevalence of infection with infl uenza viruses in virus (16). Australian wild birds, and it may be that, for H7 viruses, this is so low that insuffi cient wild birds of Origin of the Viruses the right age in the appropriate locations have been There is still much speculation regarding the origin sampled in studies conducted so far. It has been of Australian HPAI viruses of the H7 subtype given argued (3) that, perhaps, this means wild Anatidae the absence (up to the end of 2006) of isolates of are not the origin and that other possibilities should this subtype from wild Australian water birds (3–5, be explored. 12, 40). The low prevalence of infl uenza viruses in wild The exact source of the viruses for these out- ducks could relate, in part, to the limited migration breaks is still not known. In line with experiences in undertaken by Australian Anatidae, which reduces other countries, it is generally presumed that the the opportunity for infection with a wider range of viruses originated from wild Anatidae as LP strains infl uenza viruses. These birds do not normally travel and then converted to HP after passage in chickens far beyond Australia, although some have been on the infected farms. Serological evidence of found in southeast Asia and New Guinea (36). infection with H7 viruses in a fl ock of free range Migration of northern hemisphere Anatidae does not commercial ducks adjacent to the outbreak site and normally extend south of the equator, limiting the

Table 10.2. Percentage similarity between HA genes of Australian H7 HPAI viruses.a Virus Ck/75 Ck/85 Ck/92 Ck/94 Ck/97

A/chicken/Victoria/75 (H7N7) xxxx 95.1 92.4 91.3 90.9 A/chicken/Victoria/1/1985 (H7N7) 95.1 xxxx 95.2 94 94 A/chicken/Victoria/1/92 (H7N3) 92.4 95.2 xxxx 96.7 95.5 A/chicken/Queensland/667/95 (H7N3) 91.3 94 96.7 xxxx 97.2 A/chicken/NSW/1/1997 (H7N4) 90.9 94 95.5 97.2 xxxx a Based on sequence data in GenBank comparison of matched sequences covering a minimum of 1638 and maximum of 1737 nucleotides. 10 / Avian Influenza in Australia 247 likelihood of these particular birds carrying infec- closest being those from New South Wales and tion to Australia. This is also refl ected in the NP Queensland with 97.2% similarity (Table 10.2). genes of selected Australian and New Zealand wild Up to the end of 2006, all but one of the H7 duck isolates, which form sublineages distinct from viruses isolated in Australia have the molecular sig- those detected in Asia and elsewhere, and suggests nature of HP viruses, even the virus isolated from a geographic isolation of these viruses (19). The healthy emu chick (30). The only H7 LPAI virus limited variation in the HA genes of Australian H7 detected in Australia was isolated from a commer- viruses suggests that a small local population of H7 cial duck in 1976 confi rming circulation of LP H7 viruses exists in this country that has not been viruses in Australia. The timing of detection of this replenished by viruses from Asia. virus and its molecular characteristics suggest it was Charadriiformes do travel from Asia to Australia probably not the precursor virus for the outbreak in but, so far, they appear to have played little, if any, chickens on the adjacent farm. role in the spread of Asia-origin H5N1 HPAI viruses based on the limited number of such birds, apart LIKELY APPROACH TO CONTROL OF from some gulls, that have tested positive for these HPAI TODAY viruses elsewhere in the world. In addition, very few Ausvetplan, the series of manuals setting out the isolates of H7 viruses have been reported from these response to emergency animal diseases, provides the birds globally. framework for management of outbreaks of HPAI. The potential role of ratites in the maintenance of This series of documents is revised regularly to take AI viruses in Australia is also an area warranting account of changes in the epidemiology of emer- further study. Experimental inoculation of emus gency diseases and advances in emergency disease with an Australian H7N7 HPAI virus (A/chicken/ management. The latest draft version of the AI Victoria 85) resulted in mild clinical signs and virus section of Ausvetplan was issued in 2005. The excretion for up to 10 days (20), demonstrating that approach adopted in any future outbreak would these birds could be short-term carriers of HPAI likely be similar to that used previously with the viruses if infected. destruction of poultry on the infected site—estab- The role of passerine birds in the local spread of lishment of a restricted area around the infected farm AI also needs to be explored, especially given recent surrounded by a larger control area. Destruction of experiences with H5N1 in Asia, where a range of poultry would normally be restricted to the infected passerines has been infected with highly pathogenic premises and, depending on risk assessment, certain viruses. In addition, a virus essentially identical to dangerous contact premises. Use of vaccination that found in a poultry barn in 1985 was isolated would not normally be considered unless the out- from a starling. Although experimentally this virus break involved multiple premises in an area with a was generally fatal in passerines, some inoculated high poultry density or spread could not be con- birds recovered and others excreted virus before trolled by stamping out. Current issues that are being dying. These birds were seen as less likely to trans- examined are appropriate methods for mass culling mit virus than infected ducks (26). and the potential for on site composting as used in Despite all of the outbreaks of HPAI being well Canada for handling of carcasses and litter. separated temporally and/or spatially, and therefore Diagnostic testing would rely on real-time reverse appearing to be independent, the amino acids at the transcriptase–polymerase chain reaction (RRT- cleavage site of the HA of the 1976 and 1985 viruses PCR) on cloacal and tracheal swabs as a fi rst-line were identical, as were those of the 1994 Queensland screening test, followed by culture of samples that and 1997 New South Wales viruses. This could be tested positive by RRT-PCR. This technique would due to parallel evolution of the viruses, but the pos- largely supplant the use of pancreatic smears, which sibility of persistence of HPAI in as-yet-unidentifi ed although an improvement over culture in 1992, were reservoirs, although unlikely, cannot be ruled out. In still extremely labor intensive, requiring opening of addition, the sequence data suggest that the farther poultry carcasses and collection of tissues. apart the viruses were isolated temporally, the Arrangements have now been developed and for- greater was the number of molecular changes. malized between the poultry industry and state and Nevertheless, all of the chicken isolates from 1976 federal governments for sharing of the cost of erad- to 1997 shared greater than 90% similarity, with the ication and control activities. The current formula 248 Avian Influenza would see the industry contribute 20% of defi ned because of the potential for incursion of virus either costs to control HPAI and 50% of the costs to control by wild birds or through illegally imported poultry LPAI. These cost sharing arrangements also place or products. This risk has been magnifi ed recently obligations on industry to implement appropriate by endemic infection with H5N1 HPAI viruses in disease prevention measures, including approved Indonesia, extending to the province of West Papua. biosecurity plans. Specifi c plans have been devel- This disease can also have indirect economic effects oped and adopted by the poultry industry in Austra- on the poultry industry via the adverse effects of lia (6). media reports about these viruses, which in other countries has led to reduced demand for poultry PUBLIC HEALTH ASPECTS products from consumers who erroneously believe The Australian outbreaks of HPAI all occurred or fear that these pose a food safety risk, even in before the serious public health risks of AI emer- places where infection has not occurred in domestic ged in Hong Kong in 1997 and therefore specifi c poultry. Australian quarantine authorities (AQIS) public health precautions were not used, beyond routinely maintain high levels of intervention on normal hygiene measures and changes of clothes incoming aircraft passengers and baggage. Addi- on entering and leaving infected premises. Those tional funds ($13 million) have been provided to involved in culling operations on infected farms AQIS to enhance this and to increase awareness for did not wear N95 masks and were not specifi cally travelers of the risks posed by the illegal importation vaccinated against human infl uenza (to prevent of poultry or poultry products (6). possible reassortment of viruses in the event of Avian infl uenza remains a continuous low-level dual infection of workers with avian and human threat to the Australian poultry industry that has infl uenza viruses). There were no reports of dis- been addressed through improved awareness of the ease consistent with AI in staff involved in farm disease and subsequent improvements in biosecurity operations. on most commercial farms. Australia is fortunate to The only reported human case of disease associ- be geographically isolated, but this does not provide ated with AI in Australia occurred in a laboratory room for complacency, especially given the proxim- worker exposed to an H7 avian-origin virus. This ity of H5N1 HPAI viruses, the magnitude and value resulted in an unusual, virus culture-positive kera- of illegal trade in wild birds and meat products glob- toconjunctivitis. No onward transmission of the ally that provide ongoing challenges for all national infection was detected (35). quarantine authorities, and the threat from local Any future outbreak of HPAI would be subjected H7 viruses of unknown origin, as demonstrated by to very close scrutiny by public health offi cials and the fi ve outbreaks of serious disease in poultry in plans have been made for monitoring and preventing Australia over the past 30 years. infection of persons undertaking eradication procedures (6). Conclusions The media would also play a signifi cant role in Five outbreaks of highly pathogenic avian infl uenza dealing with any HPAI outbreak, given concerns have been reported in Australia between 1975 and about the zoonotic potential of AI (which the media 1997, all involving viruses of the H7 subtype. These would refer to by the poorly defi ned and confusing all caused signifi cant disruption to trade in poultry appellation “bird fl u”). The importance of commu- in Australia but all were stamped out rapidly. Wild nications in outbreaks of emergency disease has bird survillance in Australia has demonstrated that been recognized and a number of measures imple- the incidence of infection with infl uenza viruses is mented to address the problems in communication low with few isolates of viruses of the H5 or H7 have been identifi ed in emergency disease exercises subtype recorded. This has raised some unanswered in Australia and in actual outbreaks in other coun- questions regarding the source of the viruses for the tries (6). outbreaks of highly pathogenic avian infl uenza. Genetic studies on Australian H7 avian infl uenza RISKS POSED BY H5N1 HPAI IN ASIA isolates demonstrate that these are all closely related H5N1 HPAI viruses currently circulating in Asia and belong to the Eurasian lineage. These form a remain a threat to Australia’s poultry industry distinct sublineage that does not appear to have been 10 / Avian Influenza in Australia 249 replenished by other isolates from the broader infl uenza viruses isolated during the Victorian region. This could refl ect the limited migration 1976 outbreak. Australian Veterinary Journal undertaken by Anatidae to and from Australia. 69(6):140–142. Australia is the only populated continent not to 11. Bowes, V.A., S.J. Ritchie, S. Byrne, K. Sojonky, have experienced an outbreak of HPAI since 1997 J.J. Bidulka, and J.H. Robinson. 2004. Virus characterization, clinical presentation, and pathology despite the apparent increase in incidence elsewhere. associated with H7N3 avian infl uenza in British The reason for this remains unknown but could Columbia broiler breeder chickens in 2004. Avian relate to enhanced biosecurity measures imple- Diseases 48(4):928–934. mented on commercial farms or, possibly, reduced 12. Bunn, C.M. (2004). The role of wild waterfowl in populations of wild birds due to extended drought. the epidemiology of avian infl uenza in Australia. Australian Veterinary Journal 82(10):644. REFERENCES 13. Capua, I., and D.J. Alexander. 2006. The challenge 1. Alexander, D.J., W.H. Allan, D.G. Parsons, and G. of avian infl uenza to the veterinary community. Parsons. 1978. The pathogenicity of four avian Avian Pathology 35(3):189–205. infl uenza viruses for fowls, turkeys and ducks. 14. Capua, I., S. Marangon, M. dalla Pozza, C. Terre- Research in Veterinary Science 24(2):242–247. gino, and G. Cattoli. 2003. Avian infl uenza in Italy 2. Allan, W.H. 1981. Uncomplicated infection with 1997–2001. Avian Diseases 47(3 Suppl.):839– virulent strains of avian infl uenza viruses. In: R.A. 843. Bankowski (ed.). Proceedings of the First Interna- 15. Cross, G. 1986. The status of avian infl uenza in tional Symposium on Avian Infl uenza, April 1981, poultry in Australia. In: B.C. Easterday and C.W. Bartsville, Maryland. Symposium on Avian Infl u- Beard (eds.). Proceedings of the Second Interna- enza, USAHA: Richmond Virginia, pp. 4–7. tional Symposium on Avian Infl uenza, Georgia 3. Arzey, G. 2004. The role of wild aquatic birds in Center for Continuing Education, the University of the epidemiology of avian infl uenza in Australia. Georgia, Athens, Georgia, USA, September 3–5, Australian Veterinary Journal 82(6):377–378. 1986. Symposium on Avian Infl uenza, US Animal 4. Arzey, G. 2004. The role of wild aquatic birds in Health Association: Richmond, VA, pp. 96–103. the epidemiology of avian infl uenza in Australia 16. Deshpande, K.L., V.A. Fried, M. Ando, and R.G. [reply]. Australian Veterinary Journal 82(10):644– Webster. 1987. Glycosylation affects cleavage 645. of an H5N2 infl uenza virus hemagglutinin and 5. Arzey, G. 2005. The role of wild waterfowl in the regulates virulence. Proceedings of the National epidemiology of AI in Australia. Australian Vet- Academy of Science U S A 84(1):36–40. erinary Journal 83(7):445. 17. Forman, A.J., I.M. Parsonson, and W.J. Doughty. 6. Australian Government Department of Agriculture 1986. The pathogenicity of an avian infl uenza Fisheries and Forestry. 2006. Avian Infl uenza. virus isolated in Victoria. Australian Veterinary Available at http://www.daff.gov.au/corporate_ Journal 63(9):294–296. docs/publications/word/animalplanthealth/chief_ 18. Forsyth, W.M., D.C. Grix, and C.A. Gibson. 1993. vet/avian_influenza_paper_april06.doc#HPAl. Diagnosis of highly pathogenic avian infl uenza in Accessed October 1, 2006. chickens: Bendigo 1992. Australian Veterinary 7. Australian Government Department of Agriculture Journal 70(3):118–119. Fisheries and Forestry. 2006. Newcastle dis- 19. Gorman, O.T., R.O. Donis, Y. Kawaoka, and R.G. ease. Available at http://www.daffa.gov.au/animal- Webster. 1990. Evolution of infl uenza A virus plant-health/pests-diseases-weeds/animal/ PB2 genes: implications for evolution of the newcastle. Accessed October 1, 2006. ribonucleoprotein complex and origin of human 8. Banks, J., E.C. Speidel, J.W. McCauley, and D.J. infl uenza A virus. Journal of Virology 64(10):4893– Alexander. 2000. Phylogenetic analysis of H7 hae- 4902. magglutinin subtype infl uenza A viruses. Archives 20. Heckert, R.A., M. McIsaac, M. Chan, and E.M. of Virology 145(5):1047–1058. Zhou. 1999. Experimental infection of emus 9. Barr, D.A., A.P. Kelly, R.T. Badman, A.R. Campey, (Dromaiius novaehollandiae) with avian infl uenza M.D. O’Rourke, D.C. Grix, and R.L. Reece. 1986. viruses of varying virulence: clinical signs, virus Avian infl uenza on a multi-age chicken farm. Aus- shedding and serology. Avian Pathology 28(1):13– tralian Veterinary Journal 63(6):195–196. 16. 10. Bashiruddin, J.B., A.R. Gould, and H.A. West- 21. Hinshaw, V., and R.G. Webster. 1982. The natural bury. 1991. Molecular pathotyping of two avian history of infl uenza viruses. In: A.S. Beare (ed.). 250 Avian Influenza

Basic and Applied Infl uenza Research. CRC Press: sion smears. Avian Diseases 47(3 Suppl.):1190– Boca Raton, FL, pp. 79–104. 95. 22. Hooper, P.T., G.W. Russell, P.W. Selleck, and 33. Stallknecht, D.E. 1997. Ecology and epidemiology W.L. Stanislawek. 1995. Observations on the rela- of avian infl uenza viruses in wild bird populations: tionship in chickens between the virulence of some waterfowl, shorebirds, pelicans, cormorants, etc. avian infl uenza viruses and their pathogenicity for In: D.E. Swayne and R.D. Slemons (eds.). Pro- various organs. Avian Diseases 39(3):458–64. ceedings of the Fourth International Symposium 23. Hurt, A.C., P.M. Hansbro, P. Selleck, B. Olsen, C. on Avian Infl uenza, May 29–31, 1997, Athens, Minton, A.W. Hampson, and I.G. Barr. 2006. Iso- Georgia. Symposium on Avian Infl uenza, US lation of avian infl uenza viruses from two different Animal Health Association: Richmond, VA, pp. transhemispheric migratory shorebird species in 61–69. Australia. Archives of Virology 151(11):2301– 34. Stegeman, A., A. Bouma, A.R. Elbers, M.C. de 2309. Jong, G. Nodelijk, F. de Klerk, G. Koch, and M. 24. MacKenzie, J., D. Britten, V. Hinshaw, and J. van Boven. 2004. Avian infl uenza A virus (H7N7) Wood. 1985. Isolation of avian infl uenza and epidemic in The Netherlands in 2003: course of paramyxoviruses from wild birds in Western the epidemic and effectiveness of control mea- Australia. In: A.J. Della Porta (ed.). Veterinary sures. Journal of Infectious Disease 190(12):2088– Viral Diseases—Their Signifi cance in SE Asia and 2095. Western Pacifi c. Academic Press: Sydney, pp. 35. Taylor, H.R., and A.J. Turner. 1977. A case report 330–335. of fowl plague keratoconjunctivitis. British Journal 25. Morgan, I.R., and A.P. Kelly. 1990. Epidemiology of Ophthalmology 61:86–88. of an avian infl uenza outbreak in Victoria in 1985. 36. Tracey, J.P., R. Woods, D. Roshier, P. West, and Australian Veterinary Journal 67(4):125–128. G.R. Saunders. 2004. The role of wild birds in the 26. Nestorowicz, A., Y. Kawaoka, W.J. Bean, and transmission of avian infl uenza in Australia: an R.G. Webster. 1987. Molecular analysis of the ecological study. Emu 104:109–124. hemagglutinin genes of Australian H7N7 infl uenza 37. Turner, A.J. 1976. The isolation of fowl plague viruses: role of passerine birds in maintenance or virus in Victoria. Australian Veterinary Journal transmission? Virology 160(2):411–418. 52:384. 27. Offi ce International des Epizooties. 1994. Offi cial 38. Turner, A.J. 1981. Avian infl uenza in Australia. In: disease report. Fowl Plague Australia 7(50):209– R.W. Bankowski (ed.). Proceedings of the First 210. International Symposium on Avian Infl uenza, 28. Perdue, M.L., J.W. Latimer, and J.M. Crawford. April 1981, Beltsville, Maryland. Symposium on 1995. A novel carbohydrate addition site on the Avian Infl uenza, US Animal Health Association: hemagglutinin protein of a highly pathogenic H7 Richmond, VA, p. 19. subtype avian infl uenza virus. Virology 213(1):276– 39. Turner, A.J. 1981. Economic impact of avian infl u- 281. enza in domestic fowl. In: R.W. Bankowski (ed.). 29. Peroulis, I., and K. O’Riley. 2004. Detection of Proceedings of the First International Symposium avian paramyxoviruses and infl uenza viruses on Avian Infl uenza, April 1981, Beltsville, Mary- amongst wild bird populations in Victoria. Austra- land. Symposium on Avian Infl uenza, US Animal lian Veterinary Journal 82(1–2):79–82. Health Association: Richmond, VA, pp. 114–115. 30. Selleck, P.W., G. Arzey, P.D. Kirkland, R.L. 40. Turner, A.J. 2004. The role of wild aquatic birds Reece, A.R. Gould, P.W. Daniels, and H.A. West- in the epidemiology of avian infl uenza in Austra- bury. 2003. An outbreak of highly pathogenic lia. Australian Veterinary Journal 82(11):713. avian infl uenza in 1997 caused by an H7N4 virus. 41. Westbury, H.A. 1997. History of highly patho- Avian Diseases 47(3 Suppl.):806–811. genic avian infl uenza in Australia. In: D.E. Swayne 31. Selleck, P.W., L.J. Gleeson, P.T. Hooper, H.A. and R.D. Slemons (eds.). Proceedings of the Fourth Westbury, and E. Hansson. 1997. Identifi cation International Symposium on Avian Infl uenza, May and characterisation of an H7N3 infl uenza A virus 29–31, 1997, Athens, Georgia. Symposium on from an outbreak of virulent avian infl uenza in Avian Infl uenza, US Animal Health Association: Victoria. Australian Veterinary Journal 75(4):289– Richmond, VA, pp. 22–30. 292. 42. Westbury, H.A., A.J. Turner, and L. Kovesdy. 32. Selleck, P.W., S.L. Lowther, G.M. Russell, and 1979. The pathogenicity of three Australian fowl P.T. Hooper. 2003. Rapid diagnosis of highly plague viruses for chickens, turkeys and ducks. pathogenic avian infl uenza using pancreatic impres- Veterinary Microbiology 4:183–195. 11 Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007)

Leslie D. Sims and Ian H. Brown

INTRODUCTION This particular genotype of H5N1 virus (H5N1/97- When a high pathogenicity avian infl uenza (HPAI) like) (42) was eradicated from Hong Kong SAR in virus of the H5N1 subtype was isolated from geese late 1997 but other H5N1 HPAI viruses, similar in Guangdong Province of China in 1996 (131), few to those fi rst found in geese in 1996, persisted in realized that this would portend a major panzootic* the region (99). Viruses derived from these caused of HPAI; the fi rst of this disease to affect poultry intermittent outbreaks of disease in poultry and across three continents. This panzootic has been wild birds in Hong Kong SAR from 2001 to 2003 noteworthy because of the extent of spread and also (33, 99). In late 2003 and early 2004, outbreaks for the breadth of species affected, not only chickens of HPAI caused by H5N1 viruses were reported but also domestic ducks, other poultry (94), a broad almost simultaneously in poultry in eight Asian range of nondomesticated avian species (33), and countries (China, Cambodia, Indonesia, Japan, some mammals (112, 117), including cats (52, 104), Korea, Lao PDR, Thailand, and Vietnam) (98). The dogs (105), civets (91), humans (7, 83), and mink impact of these viruses was particularly severe and stone marten (126, 117). in Thailand and Vietnam, where there was wide- In 1997, international interest in HPAI viruses spread disease in poultry and multiple fatal cases of increased dramatically following the occurrence of human infection. In these two countries alone, over fatal disease in poultry and humans in Hong Kong 100 million poultry were culled or died in 2004. associated with the H5N1 strain of HPAI virus (23). Disease was also reported in Malaysia in August These human cases provided the fi rst indications to 2004 in village poultry but was stamped out (75). the international community of the possibility of From May 2005, cases of disease associated with a H5N1 AI viruses being the precursor to a human novel, but closely related sublineage of H5N1 virus pandemic virus. These concerns were amplifi ed occurred in free-fl ying migratory birds in northwest because of the high fatality rate among those who China, centered around Qinghai Lake (17, 20). became ill, raising the specter of a human infl uenza Strains of this virus were then detected subsequently pandemic akin to that seen in 1918. These concerns across southern Russia, in Kazakhstan, and in north- persist today, even though there is, as yet, no direct ern Mongolia, affecting poultry and/or free-fl ying evidence to indicate that a strain of this virus will birds. In 2006, related viruses were reported in ever develop the capacity to transmit readily between Europe, Africa, the Middle East, Pakistan, and people (83). India (79). Concurrently, the number of human cases began to increase in previously infected countries, includ- * The term “panzootic” is used to distinguish between ing Indonesia, and China, and in newly infected disease in animals from a potential infl uenza pandemic countries such as Turkey, Egypt, Azerbaijan, and in humans caused by an avian-derived virus. Iran. Fortunately, the number of new human cases

Avian Influenza Edited by David E. Swayne 251 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 252 Avian Influenza fell in Thailand and Vietnam, which corresponded This paper provides a chronology of major events with control of infection in poultry (127). Further and some key features of this H5N1 HPAI panzo- outbreaks in poultry and/or humans in late 2006 and otic. It examines the viruses that have emerged and early 2007 resulted from introduction of virus to the effects of these on key countries and regions. It countries that had stamped out infection previously reviews information on the pathways of spread (Republic of Korea and Japan); occurrence of disease and discusses the control measures implemented in a number of countries that had not reported disease in selected countries including issues relating to in poultry (Bangladesh, Kuwait, Saudi Arabia, changes in rearing and marketing practices (often United Kingdom, Ghana, and Togo); cases of disease referred to as restructuring of the poultry industry) in parts of infected countries where disease had not necessary for long term control of this disease. It been reported previously (e.g., Russia) and addi- also assesses likely developments in this panzootic tional cases in countries where infection remains in the near future. endemic or has been recorded regularly in the past There are still many gaps in our knowledge relat- few years (e.g., Indonesia, Vietnam, Thailand, ing to H5N1 HPAI due in part to an absence of China, Nigeria, and Egypt). proper epidemiological studies of many outbreaks, From late 2003 to the end of March 2007, infec- especially those that occurred in the early phase of tion associated with H5N1 HPAI had been recorded the panzootic. Even in 2007, the source of individual in some 57 countries in poultry and/or nondomesti- outbreaks in many developing countries is not cated birds, and 285 laboratory confi rmed human traced. Much information on this disease has cases of infection and disease had been recorded appeared in the literature some years after the events (173 of which were fatal) from 12 countries (127). occurred, and links between many of these events This remains the most serious HPAI epizootic ever can only be explained retrospectively. Cases of experienced in terms of the number of infected infection in poultry are going unreported as demon- fl ocks and the geographical extent of the disease. strated by the presence of human cases in areas Furthermore, uncontrolled spread has led to the where no cases of infection are reported in poultry virus “spilling over” to other animals and humans (97). This failure to detect or to report infection also on a scale not detected in the past. hampers attempts to control this disease and under- The threat posed by these viruses to public health standing of the evolution of these viruses and the and to the livelihood of farmers and village com- diseases they cause. munities led to a concerted effort by authorities in infected countries, supported by international agen- GENETIC STUDIES AND NOMENCLATURE cies, to control and, in some places, to eliminate OF THE H5N1 infection. A number of Asian countries succeeded Although there are many AI viruses present in in eradicating the disease, notably Japan, Malaysia, Eurasia, the only ones considered in detail in this and the Republic of Korea, but these all have expe- chapter are HPAI viruses belonging to the H5N1 rienced reincursions of H5N1 virus. Disease was subtype (i.e., those with a hemagglutinin protein quickly eliminated from western Europe and also (HA) of the H5 subtype and a neuraminidase (NA) appears to have been eliminated from several African of the N1 subtype). Furthermore, the description countries with low poultry density and hot dry cli- here will only be concerned with H5N1 HPAI mates (e.g., Niger). Other countries have reduced the viruses that emerged from a common progenitor fi rst levels of infection, but most Asian countries and detected in Asia in the mid 1990s. several African nations have experienced repeated The one constant regarding infl uenza A viruses is outbreaks. It is now acknowledged that the virus is that they will change over time. This has certainly endemic in some countries and will be extremely been the case with Asian lineage H5N1 HPAI diffi cult, if not impossible, to eradicate globally. viruses isolated over the past 11 years. These viruses Local eradication is possible in countries or com- exhibit considerable genetic and antigenic heteroge- partments, but these will remain vulnerable to incur- neity as a result of drift in individual genes as well sions of virus from remaining infected areas, as has as genotypic variability through genetic reassort- been demonstrated in late 2006 and early 2007 in ment. Molecular studies have provided unique Asia and Europe. insights into the evolution of the H5N1 HPAI 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 253 viruses as they emerged, which has enabled tracking or antigenic variants. More than one genotype can of their movement around the globe. However, up exist within a clade. For example, the V genotype to June 2007, the nomenclature of clades/sublin- viruses from Japan and the Republic of Korea iso- eages, genotypes, and antigenic variants of these lated in 2003–2004 were grouped in clade 2 with Z viruses was not governed by an internationally con- genotype viruses from China and elsewhere. sistent set of rules beyond naming the subtype, the The latest system of nomenclature proposed by place of origin of the virus, the species of origin, the WHO/OIE/FAO H5N1 evolution working group and, usually, a laboratory reference number. Various (130) proposes 10 clades numbered from 0 to 9. systems to describe these viruses by different Viruses that previously fell within clade 3 are now research teams led to considerable scope for confu- referred to as clade 0; there is no longer a distinction sion (16, 42, 72, 92, 103, 119, 124). In 2005, an between clade 1 and clade 1′; and clade 2 is divided attempt was made to defi ne the various clades of into fi ve subclades (2.1 to 2.5) with the fi rst three virus, based on relationships of HA genes (124). essentially the same as described above except sub- This was further refi ned in 2006 and 2007 (125, clade 2.1 has been further divided into three sub- 129), and is currently subject to review under the groups (2.1.1 to 2.1.3) and subclade 2.3 into four auspices of a World Health Organization (WHO)/ subgroups (2.3.1 to 2.3.4). Subclade 2.4 comprises World Organization for Animal Health (Offi ce viruses found in southwestern China, and subclade Internationale des Epizooties [OIE])/Food and Agri- 2.5 comprises viruses found in Japan and the Repub- culture Organization of the United Nations (FAO) lic of Korea in 2003–2004. The other clades (clades working group (130). 3 to 9) consist mainly of viruses found in China, Under the initial WHO classifi cation in 2005 including Hong Kong SAR (Table 11.1). (124), three main clades (numerically assigned clades 1 to 3) were described, although these three Genetic Studies clades did not include all H5N1 HPAI viruses from Prior to 1996, only two HPAI viruses of the H5N1 Asia. This was later elaborated to include some subtype had been identifi ed—one from poultry in emerging variants within clade 2 (125). Clade 1 con- Scotland during 1959 (82), and one from turkeys in sisted of viruses isolated from Thailand, Vietnam, England (Norfolk) during 1991 (8). Although these Lao PDR, and Cambodia in 2004–2005, with a viruses were the same subtype as the Asian HPAI related clade 1′ comprising viruses isolated in (but viruses, they were only distantly related, forming not necessarily arising from) Hong Kong SAR. Clade part of the Eurasian H5 lineage. Similarly, a number 2 described a wide range of viruses covering those of low pathogenicity (LP) H5N1 viruses have been from China, Japan, Republic of Korea, Indonesia, isolated in Asia and elsewhere (e.g., Vietnam A/ and later those from Mongolia, Europe, the Middle Vietnam/342/2001) (101), but none of these are East, West Asia, and Africa. This clade was subdi- closely related to the current H5N1 strains. LPAI vided into a number of subclades including one viruses of the H5 subtype have not become estab- comprised of viruses from Indonesia (subclade 2.1), lished in terrestrial poultry in Asia (29). another including the viruses fi rst identifi ed in wild The 1996 H5N1 HPAI virus (A/goose/Guang- birds in northwestern China in 2005 and then subse- dong/1/96; hereinafter referred to as Go/GD/96) lies quently detected in Europe and Africa (subclade at, or near, the root of an Asian H5N1 HPAI lineage, 2.2), and the third consisting of a group of viruses comprising multiple distinct genotypes that have found in 2005 and subsequently in southeastern Asia emerged and, in many cases, disappeared during extending from Anhui Province in China to Malaysia subsequent years (16, 42, 43, 61). All H5N1 viruses (subclade 2.3). The original H5N1 viruses from isolated in the Asian H5N1 lineage since 1996, Guangdong in 1996 and Hong Kong in 1997 belonged regardless of the species of origin, meet the OIE to a different clade referred to as clade 3. However, defi nition of highly pathogenic viruses (intravenous this numbering system did not refl ect the date of pathogenicity index [IVPI] > 1.2) (6, 13, 16, 42, 43, origin of the clades, given clade 1 was derived from 61), although some variability in the intravenous clade 3. In addition, description by clade and sub- pathogenicity index has been described in some clade only related to the phylogenetic characteristics viruses, especially those from ducks and geese (16, of the HA and did not distinguish between genotypes 133). This indicates that HPAI viruses of the H5N1 254 Avian Influenza

Table 11.1. Information on clades of Asian-lineage H5N1 HPAI viruses. Clade/ subclade Distribution Comments

Clade 0 H5N1/97-like and Go/GD/96-like The fi rst H5N1 HPAI viruses to be recognized viruses from 1996 and 1997 in Asia, originally referred to as clade 3 and now clade 0 (130) Clade 1 First detected in Thailand, Still persisting in this region in 2007 Vietnam, Laos, Cambodia, and Malaysia in 2003–2004 Clade 1′ Hong Kong SAR in 2002–2003 Includes human and wild bird strains of Z+ genotype Isolated from humans in Hong Kong SAR but infection probably acquired from elsewhere in southern China (note clade 1′ is now considered to fall within clade 1 under the latest classifi cation scheme [130]) Clade 2 Viruses in this clade have now Subdivided into a number of subclades (2.1 to been isolated from virtually all 2.5) infected countries Subclade 2.1 Indonesia Some variation in Indonesian viruses within this clade across the country and further subdivision into 2.1.1 to 2.1.3 Subclade 2.2 First identifi ed in 2005 in Qinghai, Multiple “sublineages” have been described China, and subsequently within this subclade in African and European detected in Middle East, West viruses, including those described as EMA1, Asia, Europe, and Africa. Only EMA2, and EMA3 one reported isolate from southern China Subclade 2.3 Widespread in Asia from China to Referred to as Fujian-like viruses; now further Malaysia from 2005 onward subdivided into 2.3.1 to 2.3.4 Subclade 2.4 Viruses mainly from southern China Subclade 2.5 Viruses from Republic of Korea and Japan in 2003–2004 Clades 3 to Mainly comprises viruses from Includes recently recognized viruses from clade 9 China, including Hong Kong Shanxi (chicken/Shanxi/2/06) (18) (clade 7) SAR, but also some from and viruses from geese and ducks from Vietnam (and Myanmar) Guiyang province (clade 4) (102)

subtype have been circulating in Asia for at least 11 The origins and history of the Go/GD/96 virus are years. Apart from reports from the fi rst half of the not known (29). Presumably, like all AI viruses, it 20th century (4) and Pakistan in the 1990s with came from an unidentifi ed H5 LP precursor virus H7N3 viruses, this is the fi rst time that HPAI viruses circulating in wild aquatic birds that then crossed are known to have persisted in poultry and/or wild into domestic poultry where mutation to virulence birds for an extended period of time, and the only occurred, as is the case with many other H5 or H7 time this has occurred across such a wide geograph- HP strains (5). However, surveillance data prior to ical range. 1996 in poultry and wild birds in Asia especially in 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 255 the period from 1980 to 1996 are rare. An unrefer- assigned to specifi c clades or subclades using exist- enced statement (16) suggesting that “the pathoge- ing classifi cation schemes. Genetic studies indicate nicity of avian H5N1 infl uenza viruses to mammals that the strain of virus (clade 1) that caused disease has been evolving since the mid-1980s” provides the in Thailand, Laos, Cambodia, and Vietnam in 2004 only hint of a possible earlier isolation of related was clonal with limited variation between isolates. H5N1 viruses in Asia but is not supported by any This suggested either a single introduction or mul- published data. Other H5 LP viruses have been iso- tiple introductions of the same virus. The source of lated regularly in Eurasia, especially Europe, but none the introduced virus is not known. Some divergence of these the direct precursor. Likewise, the origin of has occurred in these viruses over time, and addi- the NA gene of Go/GD/96 virus is also unknown. It tional introductions have occurred to Vietnam, Thai- shares approximately 95% nucleotide similarity land, and Lao PDR (9, 22, 103). Currently, the with the NA of A/duck/Hokkaido55/96 (115), an prevailing strains in this region consist of both clade H1N1 LP virus, which again suggests only a distant 1 and subclade 2.3 viruses, indicating persistence of relationship between the NA genes of these viruses. viruses from 2003–2004 and introduction of new The H5N1 viruses that emerged in 1997 (H5N1/97- viruses from elsewhere (22, 103). like viruses) and caused severe disease in poultry A single introduction, estimated to be in 2003, is and humans in Hong Kong differed from those in also suspected for Indonesia with subsequent varia- 1996 in that they were reassortants with different tion occurring as the virus traveled across the archi- NA and internal protein genes to the H5N1 viruses pelago (103). All Indonesian viruses belong to fi rst identifi ed in geese in 1996 but with a closely subclade 2.1. Distinct subgroups (2.1.1, 2.1.2, and related HA gene (44). These H5N1/97-like viruses 2.1.3) within this subclade are now evident in dif- were considered to form a different genotype within ferent parts of Indonesia (103, 130). the H5N1 subtype. Subsequently, a wide range of Genetic studies of Japanese and South Korean genotypes emerged through reassortment, presum- viruses from 2003–2004 also suggest a common ably through multiplication in waterfowl. Different origin (but different from the viruses from Indonesia genotypes were assigned alphabetic characters (e.g., or Thailand). These were clade 2 viruses of the V V, X, Z) largely based on the confi guration of the genotype (67), and they belong to the newly genes they possessed encoding internal proteins, described subclade 2.5 (67, 130). Epidemiological although there was inconsistency in the nomencla- investigations conducted so far suggest, but do not ture and interpretation of the genetic fi ndings prove, wild bird introduction into both these coun- between different research groups (16, 42). These tries (66, 74, 122). The 2003–2004 virus isolates different genotypes retained the parent HA gene from farms in Japan and the Republic of Korea were derived from Go/GD/96-like viruses but even this very closely related (more than 99 per cent homol- has also varied somewhat over time through genetic ogy for all genes), suggesting a common origin (67). drift, resulting in the formation of the different Despite similarity of the isolates to a virus of the clades and subclades within this H5N1 HPAI lineage same V genotype from a chicken in Guangdong described earlier. This genetic drift is evident from Province (67), an identical precursor virus has not phylogenetic trees which demonstrate considerable been detected in domestic poultry or wild birds variation in the composition of nucleotides of the elsewhere. HA genes from the 1996 viruses to recent isolates. Most viruses isolated since May 2005 north and The phylogenetic trees produced from these studies west of Myanmar fall within subclade 2.2. This also demonstrate epidemiological linkages between includes all of the H5N1 viruses detected in wild these isolates (Fig. 11.1). birds or poultry in Europe, the Middle East, and Several useful observations have been made on Africa. These can be clearly distinguished from the genetic characteristics of these viruses. The wide other H5N1 HPAI viruses restricted geographically range of genotypes and sublineages found in China, so far to Asia (with the exception of those viruses including Hong Kong SAR, demonstrates that there detected in intercepted smuggled birds from Asia) have been signifi cant levels of virus activity over the (Fig. 11.2). past 11 years, in both poultry and wild birds (16, 43, Since the introduction of H5N1 HPAI viruses into 102). Until recently, many of these had not been Europe, West Asia, the Middle East, and Africa, a A/chicken/Nigeria/641/2006 A/chicken/Burkina Faso/5346-14/2006 80 A/turkey/Ivory Coast/4372/2/2006 A/chicken/Sudan/2115-12/2006 A/turkey/England/250/2007 A/whooper swan/Mongolia/3/2005 A/swan/Romania/1212/2005 75 A/turkey/France/06222/2006 A/chicken/Nigeria/SO493/2006 A/duck/Egypt/2253/3/2006

95 A/turkey/Turkey/1/2005 96 A/grey heron/Romania/1266/2005 Subclade 2.2 A/swan/Azerbaijan/107-K3-2/2006 A/swan/Austria/216/2006 A/swan/Bavaria/6/2006 A/whooper swan/Scotland/1430/2006 A/cygnus olor/Croatia/1/2005 A/chicken/Azerbaijan/107-K7-2/2006 A/chicken/Nigeria/BA211/2006 A/duck/Novosibirsk/56/2005 Clade 2 100 A/brown-headed gull/Qinghai/3/2005

84 A/swan/Azerbaijan/107/K2-2/2006 A/chicken/Moscow/2/2007 84 94 A/grebe/Tyva/Tyv06/1/2006 A/Cygnus cygnus/Iran/754/2006 A/chicken/Guangdong/174/2004

97 A/chicken/Korea/ES/2003 98 A/chicken/Yamaguchi/7/2004 Subclade 2.5

99 A/chicken/Yunnan/447/2005 A/duck/Guangxi/13/2004 Subclade 2.4 A/chicken/Yunnan/115/2004 98 A/chicken/Yunnan/374/2004 A/chicken/Hong Kong/YU324/2003

90 A/chicken/Indonesia/7/2003 90 Subclade 2.1 A/chicken/Indonesia/11/2003 A/Indonesia/CDC595/2006 A/Indonesia/CDC940/2006

97 A/duck/China/E319-2/2003

98 A/pheasant/Shantou/44/2004 A/duck/Hunan/5806/2003 98 A/duck/Hunan/114/2005 Subclade 2.3 89 90 A/chicken/Thailand/NP172/2006 A/chicken/Nongkhai/NIAH400802/2007 88 A/duck/Fujian/1734/2005 100 A/duck/Laos/3295/2006 78 A/common magpie/Hong Kong/645/2006 A/chicken/Malaysia/935/2006 A/chicken/Hong Kong/FY157/2003

84 A/egret/Hong Kong/757.2/2003 A/black headed gull/Hong Kong/12.1/2003 91 75 A/Hong Kong/213/2003 A/duck/Hong Kong/821/2002 87 A/chicken/Cambodia/022LC3b/2005 85 A/chicken/Vietnam/1/2004 A/chicken/Malaysia/5858/2004 Clade 1

98 A/chicken/Thailand/1/2004 A/duck/Vietnam/Ncvdcdc15/2005 A/chicken/Laos/7192/2004 A/Vietnam/1194/2004 A/mallard duck/Vietnam/3/2003 A/goose/Cambodia/28/2004 A/goose/Shantou/1621/2005 Clade 9 100 A/migratory duck/Jiangxi/1653/2005 A/chicken/Vietnam/Ncvd8/2003

99 A/duck/Vietnam/Ncvd1/2002 Clade 5/6 76 A/teal/China/2978.1/2002 A/chicken/Hong Kong/YU562/2001 Clade 3 A/duck/Hong Kong/380.5/2001 A/goose/Guangdong/1/1996 A/Hong Kong/156/1997 100 Clade 0

97 A/chicken/Hong Kong/220/1997 91 A/Hong Kong/483/1997 A/goose/Fujian/bb/2003

97 A/goose/Guiyang/1325/2006 Clade 4 100 A/duck/Guiyang/504/2006 A/goose/Vietnam/113/2001 A/chicken/Hunan/2292/2006 Clade 7 100 A/chicken/Shanxi/2/2006

0.005

Figure 11.1. Phylogenetic relationships of the HA1 domain of the HA gene of selected “Asian- lineage” H5N1 viruses based on Molecular Evolutionary Genetics Analysis (MEGA program) of nucleotide sequences. Presented as a Minimum Evolution tree (unrooted) with clades and subclades identiﬁ ed according to the classiﬁ cation proposed by the WHO, OIE, and FAO, H5N1 Evolution Working Group in 2007 (130). 256 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 257

Figure 11.2. Spatial distribution of clades and subclades of H5N1 HPAI viruses for selected countries, subregions, and virus isolates, as of May 2007. Clades and subclades were identiﬁ ed using the classiﬁ cations proposed by the WHO, OIE, and FAO, H5N1 Evolution Working Group in 2007 (130).

number of distinguishable sublineages have emerged, and/or wild birds. An additional closely related, but although these have not yet been defi ned by the distinct, grouping of viruses was associated with WHO/OIE/FAO working group, indicating a widespread infection in poultry in Turkey from late dynamic situation in which the viruses continue to 2005 (10), with subsequent spillover to humans. A evolve. Analysis of selected viruses, predominantly second sublineage (EMA2) includes viruses princi- from Africa, but also including recent strains from pally detected in wild birds in central and eastern Europe and the Middle East identifi ed at least three Asia and northern, central, and southeastern Europe sublineages (92) within subclade 2.2 (i.e., viruses during late 2005 and early 2006 (10). In addition, linked to those identifi ed in wild birds in Qinghai infections diagnosed in poultry in west Africa Province). One sublineage (referred to as EMA1) (Nigeria and Niger) and Romania later in 2006 were contains viruses that were detected in Mongolia, the due to related viruses. A third sublineage (EMA3) Russian Federation, several countries in the Middle contains viruses fi rst reported in wild birds and East, and Europe in late 2005 and early 2006, with poultry in late 2006 in the Russian Federation, the subsequent detection in west, north, and east Africa. Middle East, and a few European countries before The viruses in this sublineage from the Middle East more widespread detection in poultry in early 2007 and Africa were almost exclusively detected in (i.e., Kuwait and Turkey). To date, viruses from this poultry but other areas reported infection in poultry sublineage have not been found in Africa. 258 Avian Influenza

Some interesting changes have been observed Cleavage Site when mammals have been infected. Despite the fact For all infl uenza A viruses, the HA glycoprotein is that most cases in mammals are derived directly produced as a precursor, HA0, which requires post- from poultry, differences in the genes of these translational cleavage by host proteases before it is viruses have occurred, apparently following a single fully functional and virus particles are infectious. or limited passage in a mammalian host. The muta- The genetic motif at the HA0 cleavage site in HPAI tion in the PB2 gene at position 627 (E627K) has viruses typically contains multiple basic amino acids been reported as signifi cant but a number of other and can be found in all of the H5N1 Asian-lineage changes have also been associated with passage viruses. The original Go/GD/96 virus had the fol- through a mammalian host. These changes are prob- lowing confi guration, PQRERRRKKRGLF, but ably due to the fact that any population of infl uenza since then there has been considerable variation at viruses in a single host is a quasi-species and that, this site (Table 11.2). All these viruses remain HP in an aberrant host, a different “dominant” strain of for gallinaceous poultry with most isolates having virus is selected from this quasi-species due to dif- an IVPI in chickens of 3.0. ferences in the host environment. Resistance to amantadines has been identifi ed in Antigenic Change some H5N1 (and H9N2) viruses in Asia, suggesting Marked antigenic change in the HA protein of H5N1 selection pressure from use of this drug in the region, viruses has occurred since they fi rst emerged. Studies presumably in poultry, and this correlates with anec- using both polyclonal and monoclonal antibodies dotal reports of use of this drug in poultry in some demonstrate signifi cant differences between strains Asian countries. from different locations. This is probably being

Table 11.2. Variability in HA0 cleavage site motifs of selected H5N1 HPAI viruses. Virus Subtype Motif HP

Numerous LPAI Eurasian viruses H5N* PQRET—RGLF − A/chicken/Scotland/59 H5N1 PQRKK—RGLF + A/turkey/England/91 H5N1 PQRKR—KTRGLF + A/chicken/Hong Kong/990/97 H5N1 PQRERRRKKRGLF + A/Hong Kong/156/97 H5N1 PQRETRRKKRGLF + A/Hong Kong/486/97 H5N1 PQRRR-RKKRGLF + A/chicken/Thailand/04 H5N1 PQRERRRKKRGLF + A/chicken/Thailand/2059/04 H5N1 PQREKRRKKRGLF + A/duck/Thailand/14376/05 H5N1 PQRERRRKKRGLF + A/turkey/Turkey/05 H5N1 PQGERRRKKRGLF + A/chicken/Romania/6141/06 H5N1 PQGDRRRKKRGLF + A/chicken/Romania/8881/06 H5N1 PQGEKRRKKRGLF + A/swan/Azerbaijan/K2–2/06 H5N1 PQGERIRKKRGLF + A/chicken/Nigeria/06 H5N1 PQGERRRKKRGLF + A/pochard/France/1142–2/06 H5N1 PQGERKRKKRGLF + A/chicken/Turkey/7010–4/06 H5N1 PQGDRRRKKRGLF + A/chicken/Sudan/Lado4/06 H5N1 PQGEGRRRKRGLF + Examples of variability of amino acids at the cleavage site of the HA0 gene of selected H5N1 viruses, derived from published sequences and unpublished data from viruses submitted to the European Community Avian Infl uenza Reference Laboratory, Veterinary Laboratories Agency, Weybridge, UK. Abbreviations: HP, highly pathogenic; H5N*, various low pathogenicity viruses of the H5 subtype but variable N subtype; D, aspartic acid; E, glutamic acid; F, phenylanaline; G, glycine; K, lysine; P, proline; Q, glutamine; R, arginine; T, threonine. 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 259 driven by a combination of systemic circulation in in a much clearer understanding of the extent of ducks, which develop an immune response to these infection, although not all surveillance studies are viruses, interspecies transmission leading to strong reported, especially those with negative results. immune selective pressure and, possibly, some Political considerations still lead to withholding of impact from vaccination. Ongoing studies are needed timely information on some disease outbreaks, and to ensure that vaccine antigens remain appropriate farmers do not always recognize or report all cases for protection against the prevailing fi eld strains and of disease (97). to evaluate the impacts, if any, of antigenic drift in fi eld viruses. Emergence of H5N1 Viruses in Asia and Disease in Hong Kong in 1997 Deletions in Virus Proteins Highly pathogenic H5N1 viruses have been recog- Several protein deletions have developed as the nized in Asia since 1996 (131) when the fi rst highly H5N1 viruses have evolved. The original goose pathogenic Asian H5N1 virus (Go/GD/96) was viruses (Go/GD/96) did not have a deletion in the recovered from a sick goose in Guangdong Province NA stalk, but all of the 1997 strains did. This change in southern China. The fi rst reported cases of serious is generally regarded as an adaptation to gallina- disease associated with H5N1 HPAI virus in Asia ceous poultry. A similar, but not identical, 19- involved fatal disease in poultry and humans in 1997 amino-acid deletion occurred in the NA protein of in Hong Kong (23,99). The fi rst avian cases were most isolates from 2001 onwards (with the notable diagnosed on a farm in Hong Kong in March 1997 exception of some wild bird viruses from Hong and the fi rst human case was detected in May of that Kong SAR in 2002 and those from humans in year, although the identity of the virus in the human 2003 belonging to the Z+ genotype)(43). A fi ve- case was not confi rmed until 3 months later. No amino-acid deletion has also been identifi ed in the further cases were reported until November 1997 NS protein of many recent isolates. The signifi cance when additional human cases were detected, fol- of this is unclear but the maintenance of this change lowed by detection of avian cases in live poultry across a heterogeneous population of viruses implies markets and one farm. By the end of December a higher virus fi tness in key host species. It may also 1997, a total of 18 human cases had been detected, be a contributory factor in determining host range. 6 of which were fatal. Case-control studies suggested an association with visits to markets that sold HISTORY OF THE H5N1 PANZOOTIC poultry in the week prior to onset of illness (71). The following section describes the development of The causal H5N1 viruses were reassortants with the panzootic and is derived from a range of pub- an HA gene derived from a Go/GD/96-like virus, lished material, including gene databases. It does not and the other seven genes derived from different only refl ect offi cial notifi cations of confi rmed out- (non-H5) avian infl uenza (AI) viruses (121). This breaks to the OIE. Some fi ndings appear biologi- particular H5N1/97-like genotype has not been cally implausible, in particular, the lack of reports detected in the fi eld since it was eliminated late in of disease in poultry in places other than Hong Kong 1997 following the culling of all poultry in markets SAR from 2001 to early 2003, despite the presence and virtually all chickens on farms (99). A virus of of highly pathogenic H5N1 viruses in an arc extend- the same genotype was apparently detected on duck ing from Hanoi to Shanghai (98) and beyond to and goose eggs imported from Vietnam to China Hebei and Jilin Provinces of China, based on (62), but phylogenetic analysis of over 100 H5N1 sequences of viruses deposited in GenBank [e.g., viruses from poultry in different locations in Vietnam A/chicken/Hebei/718/01 (45) and A/chicken/Jilin/ from 2001 to 2006 has not confi rmed circulation of hd/2002 (58)]. this particular genotype in that country. Absence of data from countries with poorly devel- It is still not clear whether these H5N1/97 viruses oped veterinary infrastructure and surveillance arose in Hong Kong SAR or elsewhere. However, systems has sometimes been erroneously interpreted they were provided ample opportunity to multiply as lack of infection (97). Recently, most countries there largely unchecked, especially in live bird have improved their diagnostic and surveillance markets that sold a wide range of poultry, including capabilities and also their transparency, resulting terrestrial and aquatic species. Uncontrolled replica- 260 Avian Influenza tion and persistence of these viruses in these markets play a key role in the maintenance of these viruses would have provided considerable opportunity for and their spread to terrestrial poultry (39, 107, 108, genetic changes in these viruses. This could have 113). This expansion of host range may also have occurred through point mutations arising from the played a role in the spread of these viruses back to poor fi delity of infl uenza virus polymerases during wild birds given the close phylogenetic relationship virus replication or through reassortment following between domestic ducks and species of wild Anati- coinfection of poultry with other AI viruses known dae and their shared environment in many parts of to be present in these markets. These conditions Asia. were not unique to live poultry markets in Hong Kong. Similar conditions existed in markets in 2001–2002 mainland China and elsewhere in Asia at that time. Detection of infection in ducks and geese continued It has been suggested that some 20% of poultry into 2001 with an upsurge in the number of sub- in markets in Hong Kong SAR were affected with clinically infected domestic waterfowl detected in H5N1 viruses just prior to the culling of birds in consignments transported from mainland China to 1997 (95). High levels of infection were present at Hong Kong SAR (99). A small study of poultry in that time, but these samples were collected when the markets in Vietnam in 2001 resulted in the isolation demand for poultry had collapsed and therefore of two H5N1 viruses from 33 samples from geese poultry were kept in infected markets for an extended (73). Given the small size of the sample, it is period, providing ample opportunity for exposure extremely unlikely that these represented the only to virus. This widely quoted fi gure is a single infected geese in the country at the time. New cases point estimate of prevalence taken under unusual of infection in terrestrial poultry with H5N1 HPAI conditions and may not refl ect the true preva- viruses were reported in Hong Kong SAR and main- lence in markets in the months leading up to the land China in 2001, the fi rst since 1997 (99). Seven outbreak when no similar surveillance studies were distinct H5N1 genotypes (one Go/GD/96-like and conducted. the rest reassortant viruses) were identifi ed in ter- The H5N1 HPAI viruses isolated in Hong Kong restrial poultry in Hong Kong SAR and Guangdong during 1997 exhibited differences involving all Province of China in that year and at least another genes with variability in nucleotide homology fi ve genotypes were identifi ed in terrestrial poultry ranging from 97.9% to 100% within the eight gene in Hong Kong SAR in 2002 (42, 99). These viruses segments (134). This demonstrated the mutability caused outbreaks of disease in live poultry markets and rapid evolution of these viruses. Other H5N1 in May 2001 and in farms and markets in early 2002 viruses in this Asian lineage, but different from in Hong Kong SAR. In addition, H5N1 HPAI viruses those isolated in Hong Kong, were also apparently were isolated from chickens in northern China isolated from Hubei Province of China in 1997 (see, (Hebei Province) in 2001 and from a number of for example, A/chicken/Hubei/wh/1997) (59). provinces in 2002 extending from Jilin to Jiangsu Provinces (58, 60). Additional cases of infection 1998–2000 across southern China in apparently healthy ducks H5N1 HPAI viruses closely related to the original were also reported in 2001 and 2002 (16) extending genotype (Go/GD/96-like) continued circulating in from Guangxi to Shanghai. geese in southern China in the late 1990s (13). By Among the viruses isolated from terrestrial poultry 2000, multiple genotypes had also been detected in in Hong Kong SAR in 2002 was one referred to as domestic ducks (16, 42). Molecular studies suggest the Z genotype. This has subsequently become the that these reassortant viruses acquired new genes dominant (but not the only) genotype associated coding for internal proteins in various combinations with the panzootic. The fi rst known representative from unidentifi ed AI viruses, presumably circulating of the Z genotype virus was apparently isolated from in aquatic birds (42). a healthy duck in Guangxi Province, China, in 2001 This expansion of the host range of H5N1 HPAI (16).* These viruses have continued to evolve viruses from geese to ducks is considered a key event in the genesis of the subsequent panzootic. Recent studies strongly suggest that domestic ducks * Referred to here as the G genotype. 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 261

(through mutation in individual genes) such that Z it was controlled using a combination of limited genotype viruses isolated from the same location at depopulation and vaccination. The last H5N1 HPAI different times, and even those isolated from differ- virus isolate from live poultry markets in Hong ent countries at the same time have signifi cant Kong SAR was detected in November 2003, just genetic and antigenic differences based on gene prior to vaccination of all poultry from mainland sequencing and antibody profi ling (43, 61, 124, China destined for Hong Kong SAR (3). The early 125). part of 2003 saw the identifi cation of the next two The year 2001 also saw the fi rst reported cases of human cases of disease associated with H5N1 H5N1 HPAI viruses infecting mammals other than viruses. These were detected in Hong Kong SAR, humans. Pigs were found to be infected in Fujian but the patients developed clinical disease in Fujian Province in 2001. It is not clear whether clinical Province before returning to Hong Kong SAR (43, disease reported in some of these pigs was due to 85). These viruses were closely related to the Z+ infection with H5N1 virus (63). In addition, a tiger viruses detected in wild birds in late 2002 (i.e., no was found to be infected in Harbin in 2002. The amino acid deletion in the NA stalk). The HA gene virus isolated caused severe fatal disease in experi- of these viruses grouped within clade 1′. mentally infected mice (132). The concurrent emergence of severe acute respi- Infected duck meat was imported from China into ratory syndrome (SARS) in 2003 may have led to the Republic of Korea in 2001 (116), although no the misdiagnosis of some cases of severe H5N1- outbreaks of disease associated with this particular related disease in humans. This possibility was dem- virus or related strains of viruses were detected in onstrated by the detection of such a case in Beijing the importing country. This demonstrated the poten- in late 2003 (135). tial for spread of infection via contaminated or The fi rst offi cial report of disease associated with infected poultry products. H5N1 HPAI outside of Hong Kong SAR in 2003 An additional novel fi nding emerged in late 2002 was from the Republic of Korea. Results of investi- when wild birds in two Hong Kong SAR zoological gations suggest that subclinical infection in ducks collections developed fatal disease and were found preceded detection in domestic chickens (122). to be infected. The fi rst outbreak involved a zoo- Other countries where disease had already emerged logical collection in Penfold Park (Shatin, New Ter- by the end of 2003 include Indonesia, Vietnam, ritories) and a viral genotype referred to as Z+ was Japan, and Thailand, although, for some of these, the isolated (so called because of the absence of a 20- disease was not recognized or reported until 2004. amino-acid deletion in the stalk of the NA protein In 2003, a related H5N1 virus in a new lineage stalk) (43). This virus belonged to clade 1′. This was detected in Xinjiang autonomous region of park, in the center of a racecourse, contained a small China in geese (25), the fi rst report of infection in lake that was home to a collection of waterfowl. Free the north west of the country. Market studies confl ying Little egrets (Egretta garzetta) had access to ducted in a range of southern provinces identifi ed this site and at least one of these birds (found dead infection in China in both terrestrial and aquatic near the park) was infected with this virus (33, 43). poultry (61). Serological evidence of infection with The second outbreak in birds in a zoological collec- H5 virus in pigs was also reported in Fujian and tion occurred at Kowloon Park in late 2002 and early Guangdong Provinces. In addition, one H5N1 virus 2003, some 13 km from the fi rst outbreak. This was was isolated from a pig in an area in Fujian Province caused by a Z genotype virus. This outbreak was where infection in pigs had been detected previously halted using a combination of isolation, limited (63). Another case of infection apparently occurred depopulation, and vaccination and involved a wide in a tiger in Harbin (15). range of captive species. The source of the virus was not determined (33). 2004 In the fi rst 2 months of 2004, outbreaks of disease 2003 were reported in quick succession from a number of More cases of infection were detected in live poultry countries in Asia, including those listed earlier, as markets in Hong Kong SAR, and outbreaks of well as Lao PDR, China, and Cambodia. In August disease also occurred in several chicken farms where 2004, Malaysia also reported infection. The disease 262 Avian Influenza and the control measures implemented in the region of countries, suggesting either single introductions (based largely around stamping-out in a wide ring or multiple introductions of virtually identical around known infected premises with concurrent viruses. Viruses in Indonesia (subclade 2.1) differed movement controls) resulted in massive losses of from those in Thailand, Vietnam, Lao PDR, Cam- poultry. Some countries eradicated the virus (e.g., bodia, and Malaysia (clade 1), and from those iso- Japan, Republic of Korea, and Malaysia), but mea- lated in Japan and South Korea (subclade 2.5, sures used did not result in elimination of infection genotype V). The only exception was China, where from the region. Success was achieved in countries a range of virus genotypes and sublineages had where infection was detected relatively early, the already been identifi ed. Even within single prov- virus had not disseminated widely, and veterinary inces, there was variation in the viruses isolated in infrastructure was well developed. Lack of success 2004 (133). in eradication in other places was due to a range of factors including limited veterinary capacity, failure 2005—Qinghai Lake and the Westward by farmers to report or recognize disease, wide- Movement of Virus spread infection in dispersed nonbiosecure farms Events in 2005 were dominated by the detection and with complex market chains often involving sale of emergence of a new sublineage of H5N1 virus that birds through poorly regulated live poultry markets, was fi rst identifi ed in Qinghai Province in north- maintenance of virus in silently infected domestic western China in migratory birds (subclade 2.2). The waterfowl, and diffi culties in implementing appro- detection of this virus was preceded by the discovery priate movement controls (97). of H5N1 HPAI viruses with similar genes to those In 2004, no infection was reported in commercial found in Qinghai in wild ducks at Poyang Lake in poultry in Hong Kong SAR, but positive samples Jiangxi Province of China earlier in 2005 (20). The were obtained from free-fl ying birds including a per- origin of this subclade remains unclear with confl ict- egrine falcon (Falco peregrinus) and grey herons ing views expressed by different authors (17, 20). (Ardea cinerea) (3). H5N1 viruses were also iso- Viruses in this subclade differed from those isolated lated from free-fl ying tree sparrows (Passer monta- previously from wild birds or poultry in that virtu- nus) in Henan Province of China. These appeared to ally all of the viruses in subclade 2.2 isolated post be reassortants forming new genotypes (51). detection at Qinghai had an E627K mutation in the During 2004, there was a marked increase in the PB2 protein, a signature normally associated with number of human cases, especially in Thailand and viruses of mammalian origin. It is not clear how or Vietnam, as well as additional mammalian cases. where this mutation fi rst appeared as the initial One notable outbreak in tigers in a sanctuary in viruses isolated from Qinghai or those from Poyang Thailand led to the death of over 45 tigers and may Lake did not possess this mutation (17, 19), in con- have resulted in limited tiger-to-tiger transmission trast to virtually all other viruses in this subclade (49, 111). Most of the cases were probably the result isolated subsequently. These events appeared highly of feeding infected chicken carcasses, although it is signifi cant and raised concerns that H5N1 viruses now recognized that close contact between experi- with a signature for potential mammalian infection mentally infected domestic felids can lead to trans- and increased mammalian pathogenicity might be mission of infection (90). This conclusion is also spread by free-fl ying birds during their movement supported by subclinical cases in domestic cats in or migration. Strains of this subclade were subse- Austria in 2006 that occurred in an animal shelter as quently detected in wild birds and poultry in south- a result of exposure to infected wild birds kept in ern Russia and Kazakhstan and in wild birds in nearby pens. Spread of infection in this Austrian Mongolia (17). The outbreaks in southern Russia case was considered to be through close contact, and Kazakhstan predominantly involved small rather than ingestion of infected birds (57). poultry fl ocks in isolated locations where poultry Viruses isolated from the newly reported out- and wild birds shared a common environment. The breaks in 2003–2004 demonstrated considerable cases in wild birds in Mongolia occurred in areas genetic diversity of isolates from different countries, where there were no poultry farms. yet showed remarkable genetic homogeneity In early October 2005, a closely related virus between isolates within infected countries or groups forming part of subclade 2.2 was associated with 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 263 outbreaks of disease in poultry in Turkey and countries in west Africa including Niger, Burkino Romania. The outbreak in Turkey was brought Faso, Cameroon, Côte D’Ivoire, Ghana, and Togo. under control following “stamping out,” movement Detection in Nigeria was soon followed by detection restrictions, and quarantine, but infection spread in of infection in Egypt, Sudan, and Djibouti. The Romania where the virus became temporarily estab- disease in Egypt and Nigeria was present in both lished in village poultry in the Danube Delta. commercial and backyard poultry. Reported cases of disease in Romanian poultry were Disease, mainly in poultry, was also reported in confi ned mainly to small village fl ocks and several Myanmar, Pakistan, India, Afghanistan, Iraq, Iran, large farms between October 2005 and June 2006 Israel, Jordan, and Palestine in the fi rst quarter of (77). In addition, virus was reported in poultry and 2006. The high number of detections across Western wild birds in the Ukraine. Perhaps most signifi cantly Asia and the Middle East in a relatively short period in October 2005, virus was detected in Croatia in a of time indicated that the viruses were already wide- dead mute swan (Cygnus olor) in the absence of any spread across this region at this time. The source infection in poultry in the wider region (10). Fur- of infection and route of introduction for most of thermore, an H5N1 virus in subclade 2.2 was these countries was not determined but all viruses detected in a live wild teal in Egypt in late 2005 (32), examined to date, from these countries, except the fi rst report of an H5N1 HPAI virus from one from Myanmar (129), belong to subclade 2.2 Africa. (Fig. 11.2). Asian countries including China, Thailand, In European Union (EU) member states from Vietnam, Cambodia, and Indonesia continued to January to May 2006, H5N1 viruses were detected detect and report cases of infection and disease in or isolated in 748 individual dead wild birds from poultry and humans caused by strains of virus other over 60 species (89). The peak incidence occurred than those in subclade 2.2 (although some cases in in mid March, coinciding with adverse weather con- northern China were associated with viruses from ditions. Clusters of positive birds were detected in subclade 2.2). By 2005 at least three different sub- some areas such as the Baltic Sea and Danube Delta lineages of virus were present in Vietnam (72) sug- but incursions into poultry were limited, with only gesting additional introductions of virus from fi ve member states (Denmark, France, Germany, elsewhere (103). The fi rst viruses of subclade 2.3 Hungary, and Sweden) reporting outbreaks. These were detected in China in 2005 (125). were mainly in outdoor production systems, involv- One dead H5N1 HPAI virus–infected Chinese ing various types of poultry (89). By early July, pond heron (Ardeola bacchus) was found in Hong infection had been reported in 26 European coun- Kong SAR through a dead bird surveillance program tries: 25 with affected wild bird populations and (3), but no cases of infection were detected in 11 reporting outbreaks in poultry. Among these poultry. Additional mammalian cases were detected 11 countries, 4 successfully contained single out- in 2005 in captive Owston’s banded palm civets breaks (Denmark, France, Germany, and Sweden). (Chrotogale owstoni) in northern Vietnam (91). However, extensive spread in poultry was reported in Hungary, Romania, the Russian Federation, 2006–2007 Turkey, and Ukraine, predominantly in outdoor pro- In 2006, infection with H5N1 viruses of subclade duction systems. The presence of virus in wild birds 2.2 extended their range through Africa, western in many countries in the absence of reports of disease Europe, west Asia, and the Middle East, affecting in poultry provided further evidence for probable wild birds, poultry, humans, and other mammals. introduction of virus to countries via wild birds. Additional cases of infection and disease were also Further to these episodes in poultry and wild found in Asia, including cases in countries where birds, fi ve countries in the Middle East and Africa disease had not been reported for 3 years, and those reported human infections/fatalities as follows: that had never reported disease. Many of these were Azerbaijan (12/4); Djibouti (1/0); Egypt (14/6); Iraq due to viruses belonging to subclade 2.3. (3/2), and Turkey (12/4) (80, 127). All cases appar- In Africa, infection and disease associated with ently occurred in places where there was close H5N1 viruses of subclade 2.2 were reported in association between poultry and humans. Some poultry, initially in Nigeria (30) and later in other cases in Azerbaijan were possibly associated with 264 Avian Influenza defeathering of dead wild swans (128), suspected of sequences of one virus from this outbreak deposited being infected with H5N1 HPAI virus. in GenBank (18), the HA protein of this virus was Infection in poultry in Indonesia remained only 92.7% similar to that of Go/GD/96. The precise endemic. Genetic analyses of virus isolates revealed origin of this strain remains unknown, although it they all grouped together within subclade 2.1 but still appears to be within the Go/GD/96-like lineage. were relatively heterogeneous, refl ecting evolution Based on recent genetic analysis, this virus falls as they spread across the country (103). By the end within a new clade referred to as clade 7 (130). of 2006, infection had spread as far east as West Market samples from southern China were also Papua and had involved 29 of 33 provinces. Consis- found to be positive for H5N1 viruses in the fi rst tent with widespread infection in Indonesian poultry, half of 2006, predominantly, but not only, subclade an increasing number of human cases were identi- 2.3 viruses (102). The rate of recovery of virus from fi ed. By August 2006, Indonesia had recorded more swabs collected in markets conformed largely to human fatalities from H5N1 HPAI viruses than any patterns seen in previous years in which the rate of other country. This included at least one large cluster isolation increased in the winter, although, in this of cases in Sumatra in which limited human-to- particular survey, positive samples from chickens human transmission, probably occurred, although it were identifi ed in all months in the fi rst half of the was diffi cult to prove that there were no other year unlike the situation in previous years. Surveil- sources of exposure in this and other clusters (83). lance conducted by the Ministry of Agriculture in Further human cases in Asia in 2006 were reported China detected H5N1 virus in live bird markets in from China, Thailand, and Cambodia (127). Guangdong Province (69). New cases of disease in poultry were reported in Surveillance in Hong Kong SAR led to the isola- Thailand (after a period of more than 6 months tion of viruses from dead free-fl ying birds from 15 without a reported case) and in Lao PDR and Cam- locations in the fi rst quarter of 2006 and 14 locations bodia. Some of the cases in Thailand were due to in the fi rst quarter of 2007, using similar surveil- viruses from clade 1, indicating that these viruses lance strategies (2, 3). Two smuggled chickens were were still circulating in the region. However, other also positive for H5N1 virus in the fi rst quarter of human and poultry infections in Thailand and Lao 2006. Viruses examined in 2006 belonged to sub- PDR were due to viruses from subclade 2.3 indicat- clade 2.3. Virus was isolated from a range of pas- ing introduction of a different lineage from the ones serine species and also birds of prey. In 2007, no isolated in 2004 (22). virus-positive dead water birds were found, in con- Vietnam (which adopted a series of control mea- trast to previous years in which dead water birds sures, including blanket vaccination) detected virus such as herons, gulls, and egrets were found to be through targeted surveillance of unvaccinated ducks, infected. A seasonal pattern was apparent with all indicating that the risk of infection of terrestrial cases in 2006 and the majority of cases in 2007 poultry remained high. No outbreaks of disease were detected between January and March, despite sur- reported in poultry in Vietnam until December 2006 veillance on dead birds being conducted throughout when unvaccinated ducks in the south of Vietnam the year. in the Mekong Delta developed clinical disease. Further cases of infection and disease in mammals Sporadic cases of disease have been detected in were reported including cats (Germany, Austria, and early 2007, mainly in unvaccinated Pekin and Indonesia), stone marten (Germany), and mink Muscovy ducks. (Sweden). All incidents were believed to result from In the fi rst half of 2006, infection in wild birds close exposure to infected poultry or wild birds or was reported again in northwest China, largely in from feeding on dead birds. Xinjiang, Tibet, and Qinghai (79). New outbreaks of In February 2007, disease occurred in a turkey disease occurred in poultry in several northern prov- farm in Suffolk, England. Analysis of genes of inces, mainly in layer farms. One of these outbreaks these viruses demonstrated almost 100% similarity in Shanxi Province in June 2006 resulted in the with viruses from concurrent outbreaks in geese in culling of more than 1.7 million poultry and was Hungary. Further investigations revealed that unpro- caused by a novel virus, antigenically and phyloge- cessed turkey meat from Hungary was being trans- netically distinct from earlier strains (76). Based on ported to a related processing plant adjacent to the 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 265 turkey farm. However, links between this meat and rogate birds, such as Muscovy ducks, in parts of infected farms in Hungary have not been established. Asia) could conceivably become infected via expo- Biosecurity breaches were detected on the turkey sure to infected feces contaminating the surface of farm (28). the egg or the environment in which the chicks are Outbreaks of disease have also been reported hatched. If transport containers for day-old chicks widely in Bangladesh in early 2007, a country that are reused or are contaminated once they leave the had not reported infection previously. Disease also hatchery, exposure and infection of chicks after affected a number of layer farms in Kuwait where it hatching could occur. In many parts of Asia, trade was managed by stamping-out of most of the coun- in day-old chicks is poorly controlled with consider- try’s layer fl ock. The disease was fi rst detected in able mixing of poultry from different sources. Most February 2007 in backyard poultry but then affected of the global trade in live poultry involves move- commercial poultry. ment of day-old chicks. Trade in these birds, when conducted and regulated in accordance with interna- SOURCES OF INFECTION AND REASONS tional animal health regulations (78), is unlikely to FOR SPREAD pose a signifi cant threat. It has proved diffi cult to determine the precise origin The extent of trade in live poultry was used in one for most outbreaks. This section reviews some of the study as a possible indicator of risk of introduction factors that are likely to have been involved in of virus from infected to uninfected countries spread and persistence of these viruses. The single (50). However, as this is mostly trade in day-old biggest threat for spread of H5N1 HPAI viruses is chicks, it does not appear to be a particularly reliable mechanical transfer of infective feces that may indicator. contain high concentrations of virus. Therefore, any There is also considerable illegal trade in poultry, infected bird or any item or commodity that is con- especially across borders between countries where taminated with infective feces can be a source of there are signifi cant differences in the market price virus for a susceptible population. As a result, poorly of poultry. For example, illegally imported live regulated trade in live poultry, as occurs in many poultry move across the border between China and countries, represents a very high risk, especially if Vietnam, and this trade is driven by the higher price this occurs between infected and uninfected areas. available for poultry in Vietnam. This trade is noto- riously diffi cult to eliminate, and the volume of Trade in Poultry and Other Birds— illegal trade is hard to estimate, but it remains a Anthropogenic Factors signifi cant risk factor for introduction of AI viruses. Historically and conventionally, the main route of It has been hypothesized that the spread of virus spread of HPAI has been through trade in poultry or from Asia to Europe was largely anthropogenic with items related to the poultry industry. This has almost routes such as the Trans-Siberian railway suggested certainly been a major factor in many outbreaks of as possible modes of dissemination (38). However, H5N1 HPAI, especially in places where virus is until these hypotheses are subjected to rigorous epi- already endemic. For example, in Hong Kong SAR, demiological analysis, no conclusions can be drawn the high-level trade in live poultry was regarded as on the contribution of this railway and other possible a major factor in the recurring outbreaks. On several factors to the spread of this disease. It is noteworthy occasions, virus introductions via traded live ducks that many of the cases in Siberia occurred in isolated and geese were detected (99). settlements located between lakes distant from rail International trade in day-old chicks has been sug- links and to which no poultry had been introduced gested as a potential means of spread of infection. recently. Day-old chicks hatched in properly managed H5N1 HPAI viruses were detected in legally mechanical incubators are unlikely to be infected imported duck meat in South Korea (116) and Japan. when they hatch. Any virus contaminating the shell Trade in infected meat could contribute to the spread of fertile eggs is unlikely to survive the incubation of infection if uncooked meat scraps are fed to process and fertile eggs laid by infected hens, incu- poultry, although none of the strains of virus found bating disease, are also unlikely to hatch. However, so far in imported duck meat have been associated naturally hatched chicks (often incubated using sur- with cases of disease in these two countries. One 266 Avian Influenza

2001 isolate of H5N1 HPAI virus from duck meat Although the possibility of some cross-protective imported into the Republic of Korea is very similar cell-mediated immunity afforded by previous infec- genetically to viruses found in ducks in and around tion with AI viruses of a different subtype allowing Shanghai, the place of origin of the meat (16). multiplication of virus without disease cannot be Trade in songbirds and birds for religious release ruled out (47, 93). Multiple species of birds, both in Asia has also been proposed as a potential route wild and domestic, are held in live poultry markets of spread of infection (68). Experimental studies on in many parts of Asia, and this practice can facilitate the infection dynamics of H5N1 HPAI viruses in the exchange and spread of viruses. Contaminated passerine species have been limited to HK/97-like equipment and infected birds that are moved between strains (88). Dead birds representing species likely markets and farms represent a signifi cant threat. to be used for religious release in Hong Kong SAR It is possible to operate live bird markets in a have been shown to be infected, but it is unclear how manner that prevents infection with these viruses as they acquired this infection. Extensive testing of has been demonstrated in Hong Kong SAR since healthy, legally imported song birds in markets did 2003. However, even with stringent restrictions on not detect virus until a single case in June 2007. The sources of poultry to these markets and improved possibility of illegal imports has been suggested. market hygiene, infection with H5N1 HPAI viruses The threat of introduction of H5N1 virus via trade in Hong Kong SAR was only prevented by ensuring in captive birds was demonstrated by two incidents all poultry entering markets came from vaccinated in Europe prior to the detection of virus in free living fl ocks and all consignments were tested for evidence birds or poultry on this continent. The fi rst occurred of protective antibodies against H5 AI virus. when H5N1 HPAI virus was detected in two Crested However, the risks associated with live poultry Hawk Eagles (Spizaetus nipalensis) that were con- markets are exacerbated in those places with large fi scated at Brussels airport following smuggling numbers of poultry reared in poorly biosecure facil- from Thailand. The recovered virus was genetically ities, as is the case in many Asian countries where closely related to those isolated in Thailand (clade there is a mismatch between the threat of virus 1) (109, 118). A second incursion occurred when incursion to poultry fl ocks and the levels of biosecu- routine investigation of deaths in quarantine of rity practiced on farms. captive cage birds imported from Taiwan to the UK showed them to be due to infection with H5N1 Domestic Ducks HPAI virus. The viruses recovered were genetically In 2000, changes in the genetics of H5N1 HPAI most closely related to viruses from southern China viruses corresponded with an expansion of the host (subclade 2.3) (26). In both cases incursion beyond range into ducks. Prior to this, H5N1 virus had been the point of detection was prevented but, as a result found in ducks in Hong Kong SAR in 1997, but only of the demonstrated risk posed by these cases, legal at a time when the prevalence of infection was very importation of captive birds from outside the EU high (95). Experimental studies with a Hong Kong/ was banned. The possible role of illegally imported H5N1 virus demonstrated that this virus was not falcons in spread of infection in the Middle East well adapted to ducks but grew and was shed in low warrants further study. titers from respiratory and alimentary tracts (87). Since 2002, H5N1 HPAI viruses have continued Live Bird Markets to be found in domestic waterfowl in the region (61) Live bird markets are well recognized as important with some, but not all, being associated with disease places for the maintenance and exchange of AI (113). The reasons for this variation in pathogenicity viruses (53), including the H5N1 HPAI viruses and apparent preferential tropism for the respiratory found regularly in large markets in the region. tract of avian species are poorly understood (33, Detection of H5N1 HPAI viruses in markets was 107, 108). These differences are strain related and only occasionally associated with detectable signs of the mortality is age related, that is, higher lethality disease or reported increases in mortality (61, 99), in younger ducks (81). Surveillance in live bird making them apparent silent reservoirs of virus. markets in southern China in early 2004 revealed Much of this is considered to be due to underreport- high infection rates in clinically normal domestic ing of disease and rapid turnover of poultry (53, 97). ducks, with some 25% of samples yielding H5N1 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 267

HPAI virus (61). Mallard ducks experimentally (37). To date, the only isolates of virus from appar- infected by intratracheal challenge with H5N1 ently healthy “wild” birds are those from ducks at viruses excreted virus asymptomatically for 17 days Poyang Lake (19), hunted Anseriformes in Russia (46), demonstrating that ducks can contaminate the (48, 65), mute swans (Cygnus olor) in Poland (PCR environment with these viruses. They are a potential positive only and positive H5 serology) (70), one source of infection for chickens and other birds, teal in Egypt (32), and two gulls in Europe (89). including wild birds. Failure to detect infection in surveillance studies of The existence of high levels of infection in clini- healthy free-fl ying birds could be due to absence of cally normal domestic ducks is regarded as an virus in the tested population. However, it could also important factor that contributed to the epidemic, refl ect the low prevalence of infection and its transi- especially in countries such as Thailand and Vietnam tory nature. This means that very large numbers of and in southern China, where ducks are commonly samples must be collected at the appropriate time to present in households, ducks are unprotected from be confi dent of detecting virus. Given it may only wild birds, and ducks range freely on ponds and rice take a single infected bird to introduce virus to a paddies. China and Vietnam alone rear some 75% susceptible population, even low prevalence of of the world’s ducks. infection in wild birds is potentially signifi cant. The timing of bird migrations does not always Wild Birds match the timing of initial disease reports in poultry Wild birds are now recognized as having played a (68). However, this does not rule out these birds as role in the long distance spread of H5N1 HPAI, a potential source of infection as the timing of although the relative contributions of migratory disease reports does not always refl ect the date of birds and anthropogenic factors associated with the the initial viral incursion (98). Virus could spread poultry industry remain unclear. This uncertainty is from wild birds to domestic waterfowl (or resident compounded by limited knowledge of wild bird host wild duck populations), with subsequent silent factors including the range of susceptible species, amplifi cation, before infection of terrestrial poultry, infection dynamics in these birds, and precise details resulting in a lag between movements of infected of their migratory and other movement patterns. wild birds and detection of disease. “Bridge” species The role of wild birds in medium- to long-dis- between migratory birds and poultry populations tance spread is best demonstrated by the introduc- could also play a role in virus dissemination (36). tion of virus to places such as Western Europe and Although not yet proved to occur, relay transmis- Mongolia where there were few poultry or no cases sion of virus may be signifi cant. Under this scenario, of infection in poultry. It is still not clear which infected free-fl ying birds move over relatively short species were involved in spreading the virus, but distances to stopover sites where they mix with and Anatidae remain high on the list of suspicion given infect other birds, some of which then transport the H5N1 HPAI viruses have been found in some appar- virus to another location (35). This could help to ently normal wild ducks (19) and the capacity of explain the pattern of spread of viruses from Asia to mallards to be silently infected, at least for short Europe, given no wild birds are known to migrate periods of time (11). directly over this particular route and the relatively The spread of H5N1 HPAI from Russia and slow speed of spread, which is not consistent with Kazakhstan to the Black Sea basin is consistent in spread via migration (38). Much of the discussion space and time with migratory movement of ducks by ornithologists centered on the question of whether (40). Furthermore, as surveillance programs in migratory birds can carry virus over long distances poultry in Europe are generally well developed, the rather than the more important question of whether presence of H5N1 HPAI virus in dead wild birds in wild birds can transport virus from one location to the absence of infection in poultry appears signifi - another, which has been demonstrated in western cant. However, the missing link in many parts of the Europe and Mongolia. Recent cases in the Republic world is the detection of H5N1 HPAI virus in healthy of Korea and Japan are also considered likely to be wild birds in areas without poultry or without infec- due to introduction by wild birds given the location tion in poultry. For example, no H5N1 viruses were of the affected farms, the timing of the outbreaks, detected in recent wild bird surveillance in Africa and the molecular characteristics of the isolates. 268 Avian Influenza

In a number of cases, such as crows in Japan (67), (i.e., the fi rst reported case was not necessarily the and magpies in South Korea (54), infected wild fi rst case of infection) and the multitude of potential birds detected during targeted surveillance were pathways for introduction of virus, many of which believed to have been infected by exposure to dis- are illegal and diffi cult to trace (97). eased poultry (or material contaminated by infected poultry) rather than having been the source of virus Intensively Reared Poultry Versus for poultry. Spread to vulture populations provided Free-Range Poultry the fi rst evidence of spillover from poultry to local There has been much debate regarding the role of wild bird populations in Africa (31). In other cases, the intensive poultry industry in the emergence of free-fl ying birds were thought to be infected by H5N1 HPAI. Some of the earlier cases of HPAI captive birds in zoological collections (33). Never- occurred in large intensive farms but this may refl ect theless, the interface between wild birds and domes- reporting (ascertainment) bias. Disease in these tic waterfowl in shared environments is suffi ciently farms is far more obvious than it is in village fl ocks extensive in many parts of Asia and also in Europe and therefore it is more likely to trigger a report. In to provide signifi cant opportunity for exchange of addition, the high-level mortality produced is diffi - viruses (in both directions) between wild birds and cult to hide unless poultry are sold in the early stages domestic poultry, especially where ducks are free- of an outbreak. ranging or raised on open ponds. In some areas, such Disease has affected both large and small farms. as eastern Europe, transmission in both directions is Although numerically, many more cases have implicated by genetic data (10). occurred in poorly biosecure smallholder and village There is still insuffi cient evidence to determine fl ocks, this does not prove small farms are more the exact means of introduction of virus to Nigeria, susceptible (due to the lack of denominator data on the fi rst African country to report infection and the number of unaffected farms). Cases that occur disease. Initially, wild birds were presumed to be the in large farms have a devastating effect due to the source of infection (30). However, this hypothesis high mortality, but disease in these farms can also was countered by another suggesting that the virus be prevented through proper management including was introduced via illegal or poorly regulated trade enhanced biosecurity and, in high-risk locations, the in day-old chicks. Insuffi cient evidence is available use of concurrent vaccination, as demonstrated in to support or rule out either option. Nevertheless, the Hong Kong SAR. Ultimately, the risk of infection viruses from Europe, the Middle East, and Africa of a fl ock of poultry is determined by a combination show a close genetic relationship (92) and all belong of the likelihood of exposure to virus and the quality to subclade 2.2, despite the fact they were collected of the composite range of risk mitigation strategies from a broad geographic region covering three con- put in place. Not all intensively reared poultry farms tinents. In addition, viruses from this subclade are implement appropriate biosecurity measures com- not widely found in commercial poultry in Asia. If mensurate with the risk of exposure. trade in poultry from Asia was the source of infec- It has been suggested that native chickens may be tion it is diffi cult to explain why introduction did not more resistant to infection than commercial poultry occur earlier than 2005 and why only viruses from (41), but so far no experimental studies have been subclade 2.2 emerged in Europe, the Middle East, conducted to support this claim. Field evidence of and Africa rather than other clades or subclades lower rates of clinical disease in village fl ocks com- more closely associated with poultry and found pared with intensively reared chickens could refl ect widely across Asia for a number of years. Genetic levels of exposure in dispersed poultry rather than mapping of virus strains in Nigeria suggest at least innate resistance to the virus. So far, all gallinaceous two independent introductions of virus (30, 92), birds infected experimentally with these viruses although it is still not possible to rule out mutation have proved remarkably sensitive to them (33, 86). of viruses through extensive multiplication after However, there is a threshold dose of virus below introduction. which infection does not appear to occur. Turkeys In many infected countries, including some in are more susceptible than domestic chickens and can Africa, epidemiological investigations have been be infected with a much lower dose of virus complicated by late detection of infection in poultry (I. Brown, unpublished data). 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 269

Seasonal Effects and Black Sea regions (a wintering area) in a west- erly and southerly direction (27, 35). This large Temperature and Festivals displacement of birds brought many species into Disease patterns have varied between countries. The close contact on an atypical scale, which may have peak of reports of the Asian H5N1 epidemic occurred increased the extent of transmission within these in winter 2003–2004. Earlier surveillance studies of populations. In addition, some populations of birds markets in China, including Hong Kong SAR, were displaced to areas outside of their regular showed that H5N1 HPAI viruses were more fre- wintering range. quently isolated during the winter months (61). This could be attributed to better survival of virus in cold PATHOLOGY OF H5N1 HPAI temperatures, combined with the increased move- The pathology of HPAI caused by H5N1 viruses in ment and trade in poultry associated with winter avian species and selected mammals has been well festivals. However, outbreaks of disease have described (33, 56, 86, 87, 90, 110). Experimental occurred in Asia in all seasons. The fi rst outbreaks studies of Asian H5N1 HPAI viruses in chickens of H5N1 HPAI in chicken farms in Hong Kong were have demonstrated that infection is systemic and reported in March, April, and May 1997 (99). The causes severe damage to endothelial cells and fi rst post-1997 outbreak of HPAI in terrestrial parenchymal organs. poultry in live bird markets in Hong Kong SAR However, not all naturally infected chickens that occurred in May 2001, although H5N1 HPAI virus died of H5N1 HPAI developed classic signs such as had fi rst been detected in a market in February of swelling of the head and edema of the subcutis and that year (99). Low temperatures do not appear to severe hemorrhage. These lesions were found in a have been a major factor in the emergence of disease small number of dead chickens, but in many cases in Thailand where cases have been detected through- the only changes found in the carcass of dead poultry out the year, although late summer and early autumn were congestion and cyanosis of the wattles and appears to be a period of greater susceptibility combs, dehydration, some subcutaneous and subse- (perhaps associated with storm activity). rosal hemorrhages, serous fl uid exuding from the nares, congestion and edema of the lungs, and con- Seasonal Bird Migrations gestion and enlargement of the spleen. Pancreatic Cases in wild birds in northwestern China have cor- necrosis was not a prominent feature in chickens but responded with the migratory movements of these was reported in other species such as magpies in birds with most cases occurring in birds as or just South Korea (54). Cecal tonsils were usually slightly after they return to and congregate on breeding enlarged and hyperemic (T. M. Ellis, personal com- grounds after winter (e.g., those in Qinghai in 2005). munication, March 10, 2007). Histologically, multi- The fi rst reported incursions of H5N1 HPAI viruses focal necrosis and infl ammation were evident in the into the Russian Federation in summer 2006, fol- spleen, brain, pancreas, and heart. Viral antigen was lowed by spread to Europe in autumn of the same detectable in most tissues with high concentrations year, coincided with seasonal patterns of migration in vascular endothelium and cardiac myocytes and dispersal of some wild bird species. This alone (56, 86). does not provide defi nitive evidence of spread by Waterfowl and other wild birds that died from wild birds, although movements of ducks to the H5N1 HPAI had nonspecifi c gross lesions including Black Sea basin in autumn were consistent with dirty ruffl ed feathers (typical of changes seen in H5N1 HPAI spread in the region at that time (40). birds with neurological disease that cannot groom These movements of birds will also be infl uenced properly), dehydration, and congestion of visceral by weather patterns. For example, movement of organs (K. Dyrting, personal communication, Feb- Anatidae usually coincides with or precedes the fi rst ruary 25, 2007). Hemorrhages were found in cranial autumn frosts in the Western Palaearctic (40). The bones. Some mild edema of the lungs and increased spread of virus to western, northern, and southern fl uid in the pharynx and trachea have been seen in Europe in 2005 was associated with unusually severe naturally infected waterfowl and other wild birds in weather conditions at the time that resulted in excep- Hong Kong SAR. Corneal opacity has been reported tional movements of aquatic birds from the Caspian in a number of cases in ducks in Asia. Experimental 270 Avian Influenza infection of Pekin ducks resulted in a range of solution for histological and immunohistochemical lesions including dehydration, splenomegaly, and examination. thymic atrophy. The intestines of inoculated birds Culture in 9- to 11-day-old embryonated chicken were empty (81). A yellowish nasal discharge was eggs is the gold standard technique for isolation detected in some ducks. Histological lesions identi- of H5N1 HPAI viruses, followed by HA and NA fi ed included multifocal nonsuppurative encephalitis subtyping. Provided suitable eggs are available and malacia, multifocal myocardial degeneration from either specifi c pathogen–free or infl uenza and necrosis accompanied by minimal infl amma- antibody–negative fl ocks, virus isolation can be a tion, ulcerative rhinitis and degeneration, and necro- reasonably rapid diagnostic tool because most sis of pancreatic and adrenocortical epithelial cells. embryos inoculated with positive clinical samples In the lungs, congestion and interstitial infl amma- die within 24 to 48 hours, which reduces the lag time tion, and in the spleen, lymphoid depletion and normally associated with virus isolation, at least on necrosis were detected in experimentally infected positive cases. It also provides a source of virus for ducks (81). subsequent molecular studies and pathogenicity None of these gross or histological lesions are testing. pathognomonic of H5N1 HPAI virus infection To speed up diagnosis, a number of different tech- and detection of virus is required to confi rm the niques for detection of specifi c AI viral antigen or diagnosis. nucleic acids have been used. Rapid antigen detection tests have proved useful for clinical cases in DIAGNOSTIC ASPECTS gallinaceous birds, particularly when applied to mul- The OIE terrestrial manual of diagnostic tests and tiple birds from a suspected fl ock. Few false-positive vaccines (6) provides recommendations for appro- results have been achieved, and while not as sensi- priate tests for diagnosis. Methods should be well tive as culture, it was still capable of detecting infec- standardized and appropriately validated and have tion if three or more dead or sick chickens from an been shown to be fi t for purpose. Diagnosis can be infected farm or market were tested (using cloacal made by the detection of infectious virus, viral and tracheal swabs) (21). These tests are not suitable antigen, or viral nucleic acid. The use of serology for screening of clinically normal poultry such as for H5N1 HPAI in susceptible birds where there are fecal samples from markets and are of limited use clinical signs is not recommended because the for testing cloacal or tracheal swabs of infected course of infection is usually very short, leading to waterfowl due to the low sensitivity. This relates to death before a detectable immune response is the low concentration of virus in these samples. induced. Detection of antibody is an appropriate Various molecular techniques have also been method for detection of previous exposure in domes- used to detect infection. Standard reverse transcrip- tic waterfowl because many cases of infection are tase–polymerase chain reaction (RT-PCR) was used subclinical. originally in many countries, but is now being replaced by real-time RT-PCR (RRT-PCR) target- Appropriate Samples ing matrix gene conserved among all infl uenza A Cloacal and tracheal swabs have been used as viruses. This allows detection of any AI virus and samples of choice for fi eld samples as the carcass of such assays for specifi c detection of H5 and N1 affected birds did not have to be opened, reducing viruses are also in routine use (100, 106). Given the the risk of infection for those handling sick or dead changes to HA gene of H5N1 HPAI viruses over the animals. Recent strains of H5N1 HPAI virus appear past few years, it has been necessary to update to be excreted at higher levels via the respiratory primers and probes to ensure all isolates are detected. route than the cloaca, and therefore it is advisable to These tests are highly sensitive with a sensitivity collect samples from both sites. In places where equivalent to that of virus isolation but with the tissue samples can be collected safely, a range of advantage they can deliver results in about 4 to 6 organs (including the brain) should be collected into hours. All cases where type A infl uenza is detected specifi ed viral transport medium and kept cool (4º C) should be cultured to determine whether infectious or frozen (−50º C). Tissue samples should be virus is present, which can then be used to perform collected into 10% neutral buffered formalin standard characterization tests. 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 271

DISEASE CONTROL outbreak. The 1997 outbreak resulted in the depop- Control strategies used for H5N1 HPAI have varied ulation of virtually all commercial chickens on Hong considerably, demonstrating that there is no single Kong farms and in live poultry markets but spared approach appropriate to all situations for control. several fl ocks containing valuable genetic stocks. Individual control plans must match the local disease This was backed by the closure of live poultry situation, the likely risk of reinfection, and the avail- markets for 7 weeks and a ban on the importation of able resources. live poultry during this period. All depopulated There are six basic “tools” used to control and farms and markets were thoroughly cleaned and dis- prevent AI: enhancement of farm biosecurity, infected and were not reopened until they passed a stamping-out, cleaning and disinfection, movement stringent veterinary inspection. Once trade in poultry management, restructuring/modifi cation of industry resumed in 1998, only certain farms in the mainland practices, and vaccination. None of these measures were allowed to supply the market. Poultry from used alone is likely to lead to elimination of infec- these were subjected to inspection and serological tion. Control and eradication of AI also depend on testing at border entry points. Rapid infl uenza A a fully functional surveillance system that allows detection tests were performed on cloacal swabs early detection of infection and disease. This requires from dead or sick birds if present in the shipment. a well-resourced and trained veterinary service. A segregation policy was introduced that separated Control programs should be backed by public educa- domestic waterfowl rearing, transporting, and tion and behavioral change campaigns to provide slaughter in separate sites from terrestrial poultry. A accurate and timely information on the nature of the dedicated waterfowl slaughterhouse was established disease to groups at risk. It is important that pro- with the bagged carcasses being sold at retail grams fully engage all stakeholders to ensure markets. Market stalls that were previously licensed success. to sell wild birds had their permits revoked. Wooden This section briefl y reviews the control measures cages used for transport were replaced by plastic used in selected countries and places special focus cages that could be more readily cleaned and disin- on Asia where the mix of measures used has varied fected, and special cage washing equipment was and the disease (or threat of infection) has been installed in the main wholesale market. Affected present for up to 11 years. farmers and traders were paid generous compensation and ex gratia allowances to cover the loss of Hong Kong SAR poultry and lost business. Hong Kong in 1997 had approximately 1000 sepa- These measures apparently prevented virus from rate retail markets selling live poultry scattered reestablishing in markets until 2001, when several across the territory between wet markets and indi- H5N1 virus genotypes were detected. Markets were vidual shops. There were also 200 poultry farms, again depopulated and closed for 1 month. Imports largely located in the northern and northwestern part of live poultry were banned. As there were no outlets of the New Territories which supplied about 20% of for live poultry reared on local farms (there was no the live poultry sent for sale in markets. The remain- central poultry slaughterhouse in Hong Kong SAR ing 80% came from mainland China. There were for terrestrial poultry), market weight poultry on two large wholesale markets through which most farms had to be destroyed. Again, compensation and poultry were sold. There were few controls on the allowances were paid to affected traders and farmers. movement of poultry between farms and markets New measures were introduced to improve market and even from markets to farms. Hygiene standards hygiene including monthly rest days in retail markets in markets were generally poor, especially in older that required a total depopulation of market stalls for markets. A range of poultry were sold in individual 24 hours on one day every month (synchronized to stalls, including live chickens, ducks, geese, quail, prevent movement of poultry from stall to stall). A chukar, pheasants, and, in some cases, wild birds segregation policy was also introduced for quail. such as owls and wild ducks. Further outbreaks of disease in 2002 and 2003 in The outbreaks of disease in Hong Kong resulted farms and markets resulted in the introduction of in the introduction of a range of control and preven- vaccination along with enhancements in farm bio- tive measures and these differed from outbreak to security. These outbreaks were controlled by limited 272 Avian Influenza culling of poultry in the affected area and use of ring there is still a substantial smallholder sector, includ- vaccination. Since these outbreaks, additional mea- ing a signifi cant number of grazing ducks that feed sures have been implemented to enhance market in recently harvested paddy fi elds. hygiene including an additional rest day and strict Disease in poultry due to HPAI in Thailand was limits on the number of poultry allowed in market fi rst reported in 2004, but it is unlikely that this was stalls. No new cases of infection have been detected the fi rst case of infection given the fi rst human case in commercial poultry since late 2003 once a full occurred earlier (113). Several waves of disease vaccination policy was implemented covering both have been reported with the second wave in October local and imported poultry, despite outbreaks occur- 2004 having the most cases (but also more intense ring in poultry in Guangdong Province (the source surveillance than during the fi rst wave). Thai author- of imported poultry) in subsequent years and the ities have used intensive door-to-door surveillance presence of wild bird cases in Hong Kong SAR. This (“x-ray” surveillance) to detect cases, and this has conclusion is supported by an intensive surveillance played a key role in reducing the levels of infection program covering poultry in markets and on farms, through enhancing early detection (12). The “x-ray” including unvaccinated sentinel chickens in all survey detected more than 750 separate infected batches of vaccinated chickens on local farms. fl ocks in 51 provinces between October 1 and The experiences in Hong Kong SAR provide December 9, 2004. This large number probably some valuable lessons for other countries, although refl ects the introduction of intensive surveillance as they cannot all be directly transferred given the much as it refl ects larger numbers of disease out- unique features of the Hong Kong poultry industry breaks (12). Several movement restrictions have and the fi nancial resources available. In particular, aided in control: (1) Movement restrictions pre- these experiences demonstrate the need to use vented long distance transport of grazing ducks, and multiple measures, to use an iterative approach (2) Controls on fi ghting cocks were introduced, to control, to modify production and marketing including the development of a fi ghting cock pass- systems, and to tightly regulate sources and move- port. However, the use of vaccination against HPAI ment of poultry. These experiences also demon- is banned in Thailand, although some consignments strated that in areas at high risk of exposure to virus, of smuggled vaccine have been intercepted, suggest- such as live poultry trade, enhanced farm biosecurity ing that a demand for the product exists. alone was not suffi cient to prevent recurrence of So far more than 62 million poultry have been infection. This resulted in the decision to use pre- destroyed or died in Thailand (12). Extensive culling ventive vaccination (99). has reduced the levels of infection but has not yet The unexpected economic consequences of certain eliminated the virus from the country for an extended control measures were also demonstrated following period. Stamping out initially involved culling in a the implementation of the duck and goose segrega- wide zone around infected premises, but this zone tion policy. Chilled carcasses produced at the dedi- was reduced to the affected farm or village. No cases cated duck and goose market and slaughterhouse of disease have been reported in large industrialized established in 1998 in Hong Kong SAR could not farms during the period from 2005 to 2007. Com- compete against cheaper chilled carcasses from partmentalization is currently being considered for mainland China once trade in the latter was permit- large integrated operations (if its implementation ted. This market/slaughterhouse has since closed will facilitate trade in unprocessed poultry products, along with all local duck farms. especially if infection repeatedly returns to the smallholder sector). The Thai export industry is now Thailand based solely on cooked produce, which is less Among the countries in Southeast Asia, Thailand affected by the presence of HPAI in the country than had the most to lose from outbreaks of H5N1 HPAI fresh or chilled meat sales. because of the dependence of its poultry industry and economy on poultry exports. Prior to the out- Vietnam breaks in 2004, the poultry industry in Thailand had By late 2003, H5N1 HPAI virus infection was grown at a remarkable rate (24). Much of the indus- already established in Vietnam with clade 1 and try was vertically integrated with the majority of other viruses. By 2004, the disease had affected 24% poultry grown in the industrial sector. Nevertheless, of communes and 60% of towns. By March 2004, 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 273

17% of the poultry population had been culled or 3-km zone are vaccinated, which includes revacci- died from disease, amounting to about 45 million nation of some previously vaccinated poultry. Such birds. This initial “wave” of infection and disease action is only taken when clinical disease occurs. In was followed by other less severe waves of out- addition, progress has been made in enhancing sur- breaks, in 2004 and 2005. Stamping out reduced the veillance capacity and in improving market hygiene. levels of infection but did not eliminate the virus. Nevertheless, the massive size of the Chinese poultry After the fi rst wave, the stamping-out policy was sector with some 50% of poultry reared by small- changed from a wide ring around infected premises holders presents enormous challenges to veterinary to affected farms only. The major cities banned the authorities. rearing and sale of live poultry and closed urban Considerable postvaccination surveillance is con- wholesale markets. This led to a shift to the sale of ducted by national veterinary authorities, but as with chilled carcasses in these urban centers. many countries in Asia, passive surveillance systems A national poultry vaccination program was initi- require strengthening, as demonstrated by the detec- ated for poultry in high-risk areas in 2005. The fi rst tion of some cases of zoonotic disease in humans round was conducted from October 2005 to January before detection of the source of infection in 2006 and resulted in the delivery of 166.3 million poultry. doses of vaccine to chickens and 78.1 million doses of vaccine to ducks. This was followed by three Cambodia further rounds of vaccination in high-risk areas in Cambodia has experienced intermittent cases of 2006–2007. No outbreaks of HPAI in poultry were disease in both poultry and humans since 2004. Gen- reported from December 2005 until late 2006, when erally, the country has low poultry density, less than 2 disease was detected in unvaccinated ducks in the 100/km , with most poultry being reared in village or south of the country. Additional cases were detected smallholder fl ocks. The main control measure used in May 2007, predominantly in unvaccinated Pekin was stamping out, but in early campaigns no com- and Muscovy ducks. Stamping-out was used on the pensation was offered to support this process. Vac- affected farms. The precise contribution of vaccina- cination was not permitted and considerable resources tion in controlling the disease is not known given it have been expended recently on enhancing public was implemented with other measures. However, awareness and improved disease surveillance. since its introduction, there has been only one reported human case (as of May 2007). Vaccination Republic of Korea will be repeated in 2007, and it is expected that the In 2003–2004, 19 infected farms were detected in vaccination program in the future will be modifi ed the Republic of Korea, including a number of duck to ensure sustainability. The vaccination program is farms that were detected by enhanced, targeted sur- backed by a surveillance program that has demon- veillance, usually in the absence of severe clinical strated seroconversion in many but not all vacci- disease. Veterinary authorities in the Republic of nated poultry and has also detected viruses Korea consider it likely that domestic ducks were circulating in unvaccinated duck fl ocks. infected with H5N1 HPAI some time prior to the infection of chickens, possibly via wild birds (122). Mainland China The virus was eliminated through a combination of China has used a multifaceted approach to the stamping out, movement controls, and rigorous control of HPAI. Vaccination has been widely used tracing studies. through regular compulsory blanket vaccination Disease was detected again in late 2006 and early campaigns. Vaccines are produced locally from reg- 2007 and has been handled in a similar manner to istered vaccine plants with over 10 billion doses of outbreaks in 2003–2004. vaccine delivered in 2006 to a standing population of some 4 billion chickens and just under 1 billion Japan domestic waterfowl including geese. Four farms and a processing plant were found to be In the event of an outbreak of disease in poultry, affected in Japan, in the southern part of Honshu a 3-km infected zone is established around the island and in one prefecture in the north of Kyushu infected premises and all poultry within this zone in late 2003 and early 2004. The outbreaks in the are culled. Poultry within a 5-km ring around this different parts of the country appeared not to be 274 Avian Influenza directly related and only one secondary case was and other captive birds in areas deemed to be at high identifi ed, on a farm in Kobe prefecture (74). The risk of infection based on location of previous out- disease was stamped out, and this was assisted by breaks, proximity to fl yways of migratory waterfowl, the remote location of the affected farms and strin- and the nature of the enterprise (backyard versus gent movement controls. Similar methods were used commercial). A total of 150 million doses of local to control outbreaks in 2007. vaccine were administered between March and June 2006 (V. Irza, personal communication, October 1, Malaysia 2006). The number of cases was reduced and appar- Malaysia escaped HPAI infection until August 2004, ently only occurred in nonvaccinated or improperly when the fi rst case was identifi ed in a village some vaccinated populations. Where infection was 20 km from the Thai border, an area containing only reported, stamping out was used. Further incursions village and smallholder poultry at low average into previously unaffected areas, including suburban poultry population density. Field evidence suggested Moscow, have been reported in the fi rst quarter of that the illegal entry of a fi ghting cock was the most 2007 and were also handled by stamping out. likely source of infection. The outbreak in Malaysia was detected quickly and rapid, effective response Turkey by the offi cial veterinary services enabled the elim- The fi rst reported outbreak in Europe was in turkeys ination of infection. New incursions of virus in 2006 in western Turkey during October 2005. The out- and 2007 were also stamped out. break was swiftly controlled by stamping out, movement restriction, quarantine measures, and dis- Indonesia infection. Further cases occurred in poultry and wild Infection in Indonesia was present in 2003, persisted birds in late 2005 and early 2006, principally in through 2004, and is still endemic in many parts of backyard production of eastern Turkey. Similar the country in 2007. Disease occurred in both poultry control measures to the fi rst outbreak in October and humans. A range of control measures have been 2005 were deployed. No further cases occurred until used including vaccination, stamping out, a partici- February 2007, when outbreaks were reported in patory approach to disease detection and control, backyard poultry in southeastern Turkey and were and a ban on poultry production in the capital city, again dealt with through stamping out. Genetic data Jakarta. Vaccines are used widely in the commercial support the new introduction of virus rather than sector and have reduced the impact of the disease in reemergence of the 2006 viruses. these farms. Mass vaccination campaigns are being developed for implementation in selected provinces. Egypt Long-term control will require signifi cant restructur- Egypt has used a combination of measures including ing of the commercial poultry production and stamping out, restrictions on poultry movement, and marketing systems. closure of live bird markets. This has been augmented by the use of vaccination. As of May 2007, Russia H5N1 HPAI disease remains endemic and will not The fi rst reported incursions of H5N1 HPAI virus be controlled unless there is considerable restructur- into Russia occurred in July 2005, initially in western ing of the poultry industry. Siberia, and almost exclusively affected poultry at the village level. Many of these communities were Nigeria remote in places where wild birds and poultry shared The main method used for control in Nigeria was environments. Stamping out was deployed together culling of affected populations. Vaccination has with movement restrictions, quarantine measures, been offi cially banned, but there are some press and disinfection. During late 2005 and early 2006, reports of illegal use. As of May 2007, the disease there was spread to southern and central European has not been eliminated. regions of the Russian Federation. By the beginning of March 2006, in excess of 1 million poultry had Côte D’Ivoire died or been slaughtered (64). At this time, targeted Outbreaks of disease in Côte D’Ivoire have been vaccination was introduced for free-range poultry controlled by a combination of measures including 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 275 vaccination. There have been no new cases reported least four factors: (1) the capacity of veterinary between December 2006 and May 2007, but the authorities to detect infection and control trade in country remains at risk due to the large volume of potentially infected poultry from within and outside poorly controlled cross border trade in poultry. the country; (2) the extent of establishment of infection in “reservoirs” such as domestic ducks or live Pakistan poultry markets within the country; (3) whether The fi rst reported outbreaks occurred between Feb- viruses are established in wild bird populations ruary and July 2006 in various poultry sectors and that do not respect international borders; and (4) wild birds. Control was by stamping out, local how well veterinary authorities and farmers can movement restrictions, and quarantine measures. protect poultry from infection in high-risk places Vaccination is permitted. Further outbreaks have through measures such as enhanced biosecurity and occurred since February 2007. vaccination. Strengthening of veterinary services in affected Kuwait and at-risk countries so that they have the capacity Measures taken include stamping out, movement to recognize the disease at an early stage and to restrictions, zoning, quarantine, and disinfection. rapidly implement appropriate control measures is Vaccination has been permitted. Much of the coun- vital and a key element of most aid programs for try’s layer fl ock was destroyed in early 2007 follow- developing countries. However this is a diffi cult ing incursions of virus. task, especially in places with large, dispersed poultry populations such as China, Vietnam, Egypt, Countries of European Union and Indonesia (96). For example, in Vietnam, there A total of 750 wild birds from 14 member states are over 7 million households rearing poultry. tested positive for H5N1 HPAI between February Trade and wild bird movements can contribute to and July 2006. Zones were established around each the spread of H5N1 HPAI viruses. Both pathways positive location, poultry were confi ned indoors, must be considered when developing and imple- biosecurity measures were enhanced, and surveil- menting national and regional plans for control of lance in both poultry and wild birds was increased. this disease, even if the relative contributions or the Community control measures included stamping exact modes of spread of virus may not be known. out, zoning, movement restrictions, enhanced sur- Veterinary authorities will have to make decisions veillance, cleansing and disinfection, and controlled on the basis of incomplete information and then repopulation (14). Infection in Hungary, which refi ne their control programs as more information on started in June 2006, was predominantly in outdoor the disease and the viruses that cause it becomes geese and resulted in 29 outbreaks that were rapidly available. brought under control. Pilot preventive targeted vac- It is possible that cycles of infection largely inde- cination programs under the control of the veterinary pendent of poultry are established in wild birds and authority were approved for use in three member therefore repeated incursions of virus along migra- states. These programs were conducted under strict tory or wild bird movement routes in Asia and else- requirements for ongoing surveillance in vaccinated where may occur. This means that poultry reared in populations. Despite continued high levels of sur- areas where wild birds congregate will remain at risk veillance in all poultry sectors and wild birds, only of infection unless appropriate measures are taken two further incursions of H5N1 HPAI (Hungary and to protect them. Local spread of infection is then United Kingdom) occurred in the fi rst half of 2007. facilitated by trade in poultry and poultry products New cases involving wild birds and some poultry and associated traders. Similarly, infection is still farms have been reported in the Czech Republic, present in countries with large populations of free Germany, and France (wild birds only) in July 2007. ranging domestic ducks and continues to occur in The source of these viruses remains unknown. places with extensive trade in live poultry through poorly regulated live poultry markets. THE FUTURE Most village-level and many small commercial The potential for further outbreaks of HPAI in fl ocks have not implemented preventive measures infected and at-risk countries or areas depends on at appropriate to the risk of virus incursion, due to the 276 Avian Influenza nature of the low input production systems prac- complex market chains that interfere with disease ticed. For this reason, vaccination has been used as control. a supplementary measure in these fl ocks in high-risk In Africa, the emergence of further infections in parts of Russia, Vietnam, and China. Changes to Egypt and Nigeria in early 2007 indicated uncer- production and marketing systems will be needed to tainty over immediate prospects for control, particu- reduce reliance on vaccination and to reduce these larly in countries with large human and poultry risks, but in implementing these changes, national populations. Other places in Africa with limited authorities need to consider the social, economic, numbers of ducks, low poultry densities, and high and environmental implications and technical feasi- temperatures that are not conducive to viral survival bility of any changes they propose given the adverse outside of hosts may fare better in controlling this impacts these can have, especially on poor farmers disease, but prevention still depends on having and other vulnerable households in urban and peri- appropriate veterinary services and early detection urban areas. Until changes can be implemented, and reporting of disease. other risk reduction measures will be necessary. For Progress has been achieved in minimizing the this reason, vaccination will continue to be used in impact of H5N1 HPAI. However, infection remains the medium to long term for control of H5N1 HPAI widespread in some countries as the ongoing detec- and may even be required indefi nitely in some tion of infection and disease in poultry and, tragi- places. cally, humans attests. There is a real and continuing Experiences from the Republic of Korea and risk of disease resurgence and viral incursion into Japan in the winter of 2006–2007 suggest that bios- previously free countries or parts of countries as has ecurity measures in industrialized poultry farms in been demonstrated by the upsurge in cases in late these two countries were not able to prevent virus 2006 and early 2007. incursions. This raises concerns regarding overreli- One lesson learned from this panzootic is that ance on any one measure to prevent and control this prediction of behavior of H5N1 HPAI viruses is disease and the need to look at all available control diffi cult. Prior to 2005, when H5N1 HPAI viruses measures when implementing control and preven- were fi rst detected in wild birds in northwestern tive programs. China, few predicted the movement of the virus Control measures already implemented globally across Russia into Europe and Africa. By contrast, will gradually reduce the levels of infection, although many in 2004 were predicting that it was only a seasonal peaks of varying intensity are expected to matter of time before Taiwan and the Philippines occur, at least in the short to medium term. Global were infected, through either trade or wild bird eradication remains a distant and unlikely possibil- movements. Yet as of March 2007, these two places ity. Nevertheless, stepwise eradication can be apparently remain free from infection, although achieved, at least locally, in certain production intercepted smuggled infected poultry in Taiwan sectors, compartments or even whole countries have been detected (55). (especially in nations with few or no land borders Spread of H5N1 viruses across Asia and to other with other countries). Nevertheless, these will countries was probably inevitable given these viruses remain vulnerable to reinfection. were well established in poultry in parts of Asia for In Asia, the key to control of this disease depends some years and international trade in poultry and on the actions taken in places with large numbers of poultry products was expanding rapidly. Avian smallholder (small commercial) poultry populations, infl uenza viruses of the H9N2 subtype infl uenza pro- notably China, Indonesia, Vietnam, Bangladesh, vided a precedent for such spread, having moved Pakistan, and India, which are either infected or across Asia to the Middle East, and beyond, in the at high risk of infection. These countries face 1990s. It is important to build on the lessons pro- many different obstacles in controlling this disease, vided by this and other transboundary diseases. including limited resources for disease control It has been postulated that the range of H5N1 and provincial autonomy that can limit the capacity viruses will eventually extend to the Americas (50). to implement national control programs. Not all Others are cautiously optimistic that this will not disease occurs in small farms, but the presence occur through wild bird movement given the appar- of many smallholders is usually an indicator of ent separation of American and Eurasian lineages of 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 277

AI viruses (123). Nevertheless, the increased global in other host populations may have an impact on trade in poultry and captive wild birds, much of virus perpetuation and subsequent behavior of the which occurs illegally, provides multiple potential disease. routes of long distance spread for these viruses, The implementation of effective control mea- apart from migratory birds (50). sures, and programs for surveillance and diagnosis The potential for the emergence of a human pan- of infection will help to reduce the risks to animal demic strain cannot be accurately assessed owing and human health. Uninfected countries will remain to the current lack of knowledge regarding the cor- susceptible to the entry of disease from infected relates for transmissibility within the human popula- areas. Despite the considerable gains made in the tion, rendering high levels of uncertainty. Already, past few years towards enhanced surveillance, H9N2 viruses have developed many of the charac- reporting and disease management, some countries teristics that would be expected to prestage a pan- still lack the animal health and veterinary public demic as they are now isolated regularly from health infrastructure to support effective implemen- pigs (84) in some parts of Asia and have the capac- tation of control measures and subsequent surveil- ity to bind to human receptors in the upper res- lance. There are also many impediments to changes piratory tract (83). However, to date, an H9N2 to industry practices, not the least of which is the pandemic strain has not emerged despite a substan- need to balance disease control with livelihood tial interface between infected poultry, pigs, and issues for the rural poor, especially given the role of humans. Clearly, there is still much that we do not poultry in assisting people, especially women and know about the evolution and emergence of human children, to improve their income and, potentially, infl uenza A pandemic strains. It is possible that the their nutrition. 1918 H1N1 virus was introduced, perhaps from an avian source, to humans some years before the pan- CONCLUSIONS demic began. A period of adaptation to the new host Asian-lineage HPAI viruses of the H5N1 subtype may have been necessary before full transmissibility have been circulating continuously for over 11 years was acquired. since they were fi rst detected in China in 1996. They Despite the uncertainty of a pandemic strain have spread to three continents and are now well emerging, the high impact of such an event provides entrenched in a number of countries in Asia and suffi cient justifi cation to prepare for such an even- Africa. These viruses have caused considerable tuality. Reduction of risk for human infection losses to the poultry industry, and loss of human life requires control of infection at source in the animal and continue to raise concerns about the potential reservoirs. This action is appropriate regardless of for emergence of a human infl uenza pandemic strain the human health implications because this disease of virus. remains a serious veterinary problem that has marked Although local elimination of H5N1 HPAI viruses consequences on poultry producers including many has been achieved from some countries, prospects poor farmers and on national and international trade of global eradication are poor unless major changes in poultry and poultry products. occur in these viruses or signifi cant modifi cations Natural extinction of H5N1 HPAI viruses is still are made to methods used for rearing and marketing a possibility given this has occurred with other poultry in endemically infected countries and those infl uenza viruses (120), but the likelihood of this at high risk of infection. As the poultry industry in seems remote based on their remarkable persistence many of these places utilizes many high-risk prac- and evolution for over 11 years, the dramatic increase tices, such as sales of broiler chickens through in their geographic range and their capacity to infect poorly regulated live poultry markets, and supports ducks without necessarily causing disease. A number large numbers of poor smallholders and associated of variant strains of H5N1 HPAI virus have already traders, rearing poultry under conditions of minimal emerged and disappeared in the past 11 years, pre- biosecurity, this will take many years to achieve. sumably replaced by fi tter versions, but the most This is compounded by the relatively weak veteri- frequently encountered genotype (the so-called Z nary services, administration, and governance in genotype) has circulated continuously since 2001. most of these countries, which limit provision In addition, the potential capability to establish of appropriate veterinary preventive care and 278 Avian Influenza premarket checks for the millions of poultry fl ocks Tripodi, is greatly appreciated. We are grateful to B. in infected places. Z. Londt for assistance with the preparation of This panzootic has gradually resulted in a shift in Figure 11.1. Dr. David Swayne has provided excel- attitudes towards measures for control of this disease. lent support and information on AI whenever The classic emergency response used to control required. Many of the virus sequences were made infection involving stamping out and movement available through the submission of samples to the restrictions is less appropriate in endemically Weybridge Reference laboratory. The countries pro- infected countries than in countries with recent viding these are gratefully acknowledged. infection. Instead, control in these places requires REFERENCES longer term measures to change high-risk industry 1. Agriculture, Fisheries and Conservation Depart- practices, including enhancements in farm biosecu- ment, Hong Kong SAR. 2006. H5N1 infected rity and market hygiene. Where this is not possible wild birds found in Hong Kong in 2006. Avail- other measures to mitigate these risks are required, able at http://www.afcd.gov.hk/english/ such as vaccination, improvements in market man- quarantine/qua_vetlab/qua_vetlab_ndr/fi les/ agement, and species segregation during rearing, posth5n128_03_06.pdf. Accessed March 20, 2007. transportation, and sale. 2. Agriculture, Fisheries and Conservation Depart- There is still much that remains unknown about ment, Hong Kong SAR. 2007. H5N1 infected wild these Asian-lineage H5N1 viruses, particularly the birds found in Hong Kong in 2007. Available at role of wild birds in the maintenance of virus infec- http://www.afcd.gov.hk/english/whatsnew/what_ tion. For example, it is not clear yet whether an qua/fi les/common/h5n1_2007.pdf. Accessed March independent cycle of infection is present in wild 25, 2007. 3. Agriculture, Fisheries and Conservation Depart- birds and if there is whether this will persist. ment, Hong Kong SAR. 2007. Quarterly Animal Based on current knowledge, it appears that the Disease Reports to OIE. Available at http://www. world will have to learn to live with these viruses, afcd.gov.hk/english/quarantine/qua_vetlab/qua_ at least in the medium term. This will require imple- vetlab_ndr/qua_vetlab_ndr_adr/qua_vetlab_ndr_ mentation of measures that reduce the risk these adr.html. Accessed March 20, 2007. viruses pose to poultry and public health while at the 4. Alexander, D.J. 1987. Avian infl uenza—histori- same time balancing the social, economic, and envi- cal aspects. In: B.C. Easterday and C.W. Beard ronmental effects of the control programs used. (eds.). Proceedings of the Second International It is expected that H5N1 HPAI viruses will con- Symposium on Avian Infl uenza, Georgia Center tinue to circulate and, in doing so, will continue to for Continuing Education, the University of mutate, potentially leading to the emergence of new Georgia, Athens, Georgia, USA, September 3–5, 1986. Symposium on Avian Infl uenza, US Animal H5N1 sublineages or genotypes with unknown bio- Health Association: Richmond, VA, pp. 4–13. logical properties. Infected countries may experi- 5. Alexander, D.J. 2000. A review of avian infl u- ence intermittent resurgence of infections that could enza in different bird species. Veterinary Micro- be infl uenced by a variety of factors, including sea- biology 74:3–13. sonality and fl ock immunity after vaccination. Incur- 6. Alexander, D.J. 2004. Highly pathogenic avian sions to countries currently free from infection will infl uenza. In: Manual of Standards for Diagnostic continue. Based upon past experiences, it is also Tests and Vaccines. OIE: Paris. 5th ed. Available possible that some new, unexpected manifestations at http://www.oie.int/eng/normes/mmanual/A_ in this unprecedented panzootic will arise. 00037.htm. Accessed February 10, 2007. 7. Alexander, D.J. 2006. Avian infl uenza viruses ACKNOWLEDGMENTS and human health. Developments in Biologicals (Basel) 124:77–84. The information in this paper was collected from 8. Alexander, D.J., S.A. Lister, M.J. Johnston, C.J. many sources. The assistance provided by colleagues Randall, P.J. Thomas. 1993. An outbreak of in Vietnam, China, Hong Kong SAR, and FAO, highly pathogenic avian infl uenza in turkeys in especially Drs. Lai Thi Kim Lan, Do Huu Dung, Great Britain in 1991. Veterinary Record 132: Hoang Van Nam, To Long Thanh, Guo Fusheng, 535–536. Kitman Dyrting, Trevor Ellis, William Wong, Liu 9. Boltz, D.A., B. Douangngeun, S. Sinthasak, P. Kwei-Kin, Malik Peiris, Jeff Gilbert, and Astrid Phommachanh, S. Rolston, H. Chen, Y. Guan, J. 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 279

S. Peiris, J.G. Smith, and R.G. Webster. 2006. Kawaoka. 2006. Properties and dissemination of H5N1 infl uenza viruses in Lao People’s Demo- H5N1 viruses isolated during an infl uenza out- cratic Republic. Emerging Infectious Diseases. break in migratory waterfowl in western China. 12:1593–1595. Journal of Virology 80:5976–5983. 10. Brown, I.H, B.Z. Londt, W. Shell, R.J. Manvell, 18. Chen, H., Y. Li, J. Shi, and H. Zhao. 2006. A/ J. Banks, R. Gardner, L. Outtrim, S.C. Essen, M. chicken/Shanxi/2/2006(H5N1) Residues 1 to Sabirovic, M. Slomka, and D.J. Alexander. 2006. 568. GenBank accession number ABK34764. Incursion of H5N1 “Asian lineage virus” into Available at http://www.ncbi.nlm.nih.gov/entrez/ Europe: source of introduction? In: FAO/OIE viewer.fcgi?db=Protein&id=117414798. International scientifi c conference on avian infl u- National Center for Biotechnology Information: enza and wild birds Available at http://www.fao. Bethesda, MD. org/ag/againfo/subjects/en/health/diseases-cards/ 19. Chen, H., G.J. Smith, K.S. Li, J. Wang, X.H. Fan, conference/documents/I.Brown.pdf. Accessed J.M. Rayner, D. Vijaykrishna, J.X. Zhang, L.J. December 15, 2006. Zhang, C.T. Guo, C.L. Cheung, K.M. Xu, L. 11. Brown, J.D., D.E. Stallknecht, J.R. Beck, D.L. Duan, K. Huang, K. Qin, Y.H. Leung, W.L. Wu, Suarez, and D.E. Swayne. 2006. The susceptibil- H.R. Lu, Y. Chen, N.S. Xia, T.S. Naipospos, K. ity of North American ducks and gulls to H5N1 Y. Yuen, S.S. Hassan, S. Bahri, T.D. Nguyen, highly pathogenic avian infl uenza viruses. Emerg- R.G. Webster, J.S. Peiris, and Y. Guan. 2006. ing Infectious Diseases 12(11):1667–1670. Establishment of multiple sublineages of H5N1 12. Buranathai, C., A. Amonsin, A. Chaisigh, A. infl uenza virus in Asia: implications for pandemic Theamboonlers, N. Pariyothorn, and Y. Poov- control. Proceedings of the National Academy of orawan. 2007. Surveillance activities and molec- Sciences U S A 103:2845–2850. ular analysis of H5N1 highly pathogenic avian 20. Chen, H., G.J. Smith, S.Y. Zhang, K. Qin, J. infl uenza viruses from Thailand, 2004–2005. Wang, K.S. Li, R.G. Webster, J.S. Peiris, and Y. Avian Diseases 51:194–200. Guan. 2005. Avian fl u: H5N1 virus outbreak in 13. Cauthen, A.N., D.E. Swayne, S. Schultz-Cherry, migratory waterfowl. Nature 436:191–192. M.L. Perdue, and D.L. Suarez. 2000. Continued 21. Chua, T.H., T.M. Ellis, C.W. Wong, Y. Guan, circulation in China of highly pathogenic avian S.X. Ge, G. Peng, C. Lamichhane, C. Maliadis, infl uenza viruses encoding the hemagglutinin S.W. Tan, P. Selleck, and J. Parkinson. 2007. gene associated with the 1997 H5N1 outbreak in Performance evaluation of fi ve detection tests for poultry and humans. Journal of Virology avian infl uenza antigen with various avian 74:6592–6599. samples. Avian Diseases 51:96–105. 14. CEC. 2006. Council Directive 2005/94/EC of 20 22. Chutinimitkul, S., T. Songserm, A. Amonsin, S. December 2005 on Community measures for the Payungporn, K. Suwannakarn, S. Damrongwata- control of avian infl uenza and repealing Directive napokin, A. Chaisingh, B. Nuansrichay, T. Chieo- 92/40/EEC. Available at http://eur-lex.europa. chansin, A. Theamboonlers, and Y. Poovorawan. eu/LexUriServ/site/en/oj/2006/l_010/l_ 2007. New strain of infl uenza A virus (H5N1), 01020060114en00160065.pdf. Accessed Thailand. Emerging Infectious Diseases 13:506– December 15, 2006. 507. 15. Chang, S., X.T. Zheng, S.T. Yang, X.H. Zou, 23. Claas, E.C., A.D. Osterhaus, R. van Beek, J.C. De Z. Ding, and X.Z. Xia. 2006. Determining Jong, G.F. Rimmelzwaan, D.A. Senne, S. Krauss, the distribution and contents of high pathogenic K.F. Shortridge, and R.G. Webster. 1998. Human avian infl uenza virus from tiger in mice [in infl uenza A H5N1 virus related to a highly patho- Chinese]. In: Progress in Veterinary Medicine genic avian infl uenza virus. Lancet 351:472– 5. Abstract available at http://scholar.ilib.cn/ 477. A-dwyxjz200605015.html. Accessed May 15, 24. Costales, A. 2004 A review of the Thailand 2007. poultry sector. Available at http://www.fao.org/ 16. Chen, H., G. Deng, Z. Li, G. Tian, Y. Li, P. Jiao, AG/AGAINFO/resources/en/publications/ L. Zhang, Z. Liu, R.G. Webster, and K. Yu. 2004. sector_reports/lsr_THA.pdf. Accessed December The evolution of H5N1 infl uenza viruses in ducks 5, 2006. in southern China. Proceedings of the National 25. Cui, S.J., C.H. Lei, J.X. Cheng, F. Fu, J.F. Wang, Academy of Sciences U S A 101:10452–10457. C.Y. Wu, G.Z. Tong, and X.G. Kong. 2006. 17. Chen, H., Y. Li, Z. Li, J. Shi, K. Shinya, G. Deng, Result and analysis of the cloning and sequencing Q. Qi, G. Tian, S. Fan, H. Zhao, Y. Sun, and Y. of the HA gene of the Xinjiang goose origin 280 Avian Influenza

H5N1 subtype avian infl uenza virus (A/Goose/ in Hong Kong in late 2002. Avian Pathology XJYL/10/2003) [in Chinese]. Livestock and 33:492–505. Poultry Industry. Abstract available at http:// 34. Ellis, T.M., L.D. Sims, H.K.H. Wong, L.A. www.ilib.cn/A-xqy200606001.html. Accessed Bissett, K.C. Dyrting, K.W. Chow, and C.W. February 10, 2007. Wong. 2005. Evaluation of vaccination to support 26. DEFRA. 2005. Epidemiology report on avian control of H5N1 avian infl uenza. In: R.S. Schri- infl uenza in a quarantine premises in Essex. jvers and G. Koch (eds.). Proceedings of the Available at http://www.defra.gov.uk/animalh/ Frontis Workshop on Avian Infl uenza: Preven- diseases/notifi able/disease/ai/pdf/ai- tion and Control, Wageningen, The Netherlands, epidemrep111105.pdf. Accessed October 1, 13–15 October 2003, pp. 75–84. Available at 2006. http://library.wur.nl/frontis/avian_infl uenza/09_ 27. DEFRA. 2006. HPAI H5N1 situation in Europe ellis.pdf. Accessed October 5, 2006. and potential risk factors for the introduction of 35. Feare, C.J. 2007. The role of wild birds in the the virus to the United Kingdom. International spread of HPAI H5N1. Avian Diseases 51:440– Animal Health Division, 1A Page Street, London, 447. SW1P 4PQ, United Kingdom. Version 1, Released 36. FAO. 2006. Empress Watch, October. Evolution 06 July 2006, p. 26. Available at http://www. of highly pathogenic avian infl uenza type H5N1 defra.gov.uk/animalh/diseases/monitoring/pdf/ in Europe: review of disease ecology, trends and hpai-h5n1-developments060706.pdf. Accessed prospects of spread in autumn-winter 2006. January 30, 2007. Available at http://www.fao.org/docs/eims/ 28. DEFRA. 2007. Outbreak of highly pathogenic upload/214878/EW_europe_oct06_hpa.pdf. H5N1 avian infl uenza in Suffolk in January 2007. Accessed December 1, 2006. Available at http://www.defra.gov.uk/animalh/ 37. Gaidet, N., T. Dodman, A. Caron, G. Balança, S. diseases/notifi able/disease/ai/pdf/epid_ Desvaux, F. Goutard, G. Cattoli, F. Lamarque, W. fi ndings050407.pdf. Accessed May 2, 2007. Hagemeijer, and F. Monicat. 2007. Avian infl u- 29. Duan, L., L. Campitelli, X.C. Fan, Y.H. Leung, enza viruses in water birds, Africa. Emerging D. Vijaykrishna, J.X. Zhang, I. Donatelli, M. Infectious Diseases 13(4):626–629. Delogu, K.S. Li, E. Foni, C. Chiapponi, W.L. Wu, 38. Gauthier-Clerc, M., C. Lebarbenchon, and F. H. Kai, R.G. Webster, K.F. Shortridge, J.S. Peiris, Thomas. 2007. Recent expansion of highly patho- G.J. Smith, H. Chen, and Y. Guan. 2007. Char- genic infl uenza H5N1: a critical review. Ibis acterization of low-pathogenic H5 subtype infl u- 149(2):202–214. enza viruses from Eurasia: implications for the 39. Gilbert, M., P. Chaitaweesub, T. Parakama- origin of highly pathogenic H5N1 viruses. Journal wongsa, S. Premashthira, T. Tiensin, W. Kal- of Virology 81(14):7529–7539. pravidh, H. Wagner, and J. Slingenbergh. 2006. 30. Ducatez, M.F., C.M. Olinger, A.A. Owoade, S. Free-grazing ducks and highly pathogenic avian De Landtsheer, W. Ammerlaan, H.G. Niesters, infl uenza, Thailand. Emerging Infectious Dis- A.D. Osterhaus, R.A. Fouchier, and C.P. Muller. eases 12:227–234. 2006. Avian fl u: multiple introductions of H5N1 40. Gilbert, M., X. Xiao, J. Domenech, J. Lubroth, V. in Nigeria. Nature 442:37. Martin, and J. Slingenbergh. 2006. Anatidae 31. Ducatez, M.F., Z. Tarnagda, M.C. Tahita, A. migration in the western Palearctic and spread of Sow, S. de Landtsheer, B.Z. Londt, I.H. Brown, highly pathogenic avian infl uenza H5N1 virus. D.M. Osterhaus, R.A. Fouchier, J.B. Ouedraogo, Emerging Infectious Diseases 12(11):1650– and C.P. Muller. 2007. Genetic characterization 1656. of HPAI (H5N1) viruses from poultry and wild 41. Grain. 2006. Fowl play. The poultry industry’s vultures, Burkina Faso. Emerging Infectious central role in the bird fl u crisis. Available at Diseases 13(4):611–613. http://www.grain.org/briefi ngs_fi les/birdfl u2006- 32. Earhart, K.C., B.R. Boynton, M.D. Saad, and M.R. en.pdf. Accessed October 1, 2006. Monteville, 2007. The importance of monitoring 42. Guan, Y., J.S. Peiris, A.S. Lipatov, T.M. Ellis, of H5N1 infl uenza in Africa. Microbe 2(3):113. K.C. Dyrting, S. Krauss, L.J. Zhang, R.G. 33. Ellis, T.M., R.B. Bousfi eld, L.A. Bissett, K.C. Webster, and K.F. Shortridge. 2002. Emergence Dyrting, G.S. Luk, S.T. Tsim, K. Sturm-Ramirez, of multiple genotypes of H5N1 avian infl uenza R.G. Webster, Y. Guan, and J.S. Peiris, 2004. viruses in Hong Kong SAR. Proceedings of the Investigation of outbreaks of highly pathogenic National Academy of Sciences U S A 99:8950– H5N1 avian infl uenza in waterfowl and wild birds 8955. 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 281

43. Guan, Y., L.L. Poon, C.Y. Cheung, T.M. Ellis, R.C. Fleischer, P.P. Marra, and P. Daszak. 2006. W. Lim, A.S. Lipatov, K.H. Chan, K.M. Sturm- Predicting the global spread of H5N1 avian infl u- Ramirez, C.L. Cheung, Y.H. Leung, K.Y. Yuen, enza. Proceedings of the National Academy of R.G. Webster, and J.S. Peiris. 2004. H5N1 infl u- Sciences U S A 103:19368–19373. enza: a protean pandemic threat. Proceedings of 51. Kou, Z., F.M. Lei, J. Yu, Z.J. Fan, Z.H. Yin, C.X. the National Academy of Sciences U S A Jia, K.J. Xiong, Y.H. Sun, X.W. Zhang, X.M. 101:8156–8161. Wu, X.B. Gao, and T.X. Li. 2005. New genotype 44. Guan, Y., K.F. Shortridge, S. Krauss, and R.G. of avian infl uenza H5N1 viruses isolated from Webster. 1999. Molecular characterization of tree sparrows in China. Journal of Virology H9N2 infl uenza viruses: were they the donors of 79:15460–15466. the “internal” genes of H5N1 viruses in Hong 52. Kuiken, T., G. Rimmelzwaan, D. van Riel, G. van Kong? Proceedings of the National Academy of Amerongen, M. Baars, R. Fouchier, and A. Oster- Sciences U S A 96:9363–9367. haus. 2004. Avian H5N1 infl uenza in cats. Science 45. He, G.M., J. Qiao, L.H. Zhao, and C. Dong. 2005. 306:241. A/chicken/Hebei/718/2001(H5N1) Residues 1 to 53. Kung, N.Y., Y. Guan, N.R. Perkins, L. Bissett, T. 568. In GenBank accession number ABC69149. Ellis, L. Sims, R.S. Morris, K.F. Shortridge, and Available at http://www.ncbi.nlm.nih.gov/entrez/ J.S.M. Peiris. 2003. The impact of a monthly rest viewer.fcgi?val=ABC69149. National Center for day on avian infl uenza virus isolation rates in Biotechnology Information: Bethesda, MD. retail markets in Hong Kong. Avian Diseases Accessed November 12, 2006. 47:1037–1041. 46. Hulse-Post, D.J., K.M. Sturm-Ramirez, J. 54. Kwon, Y.K., S.J. Joh, M.C. Kim, Y.J. Lee, J.G. Humberd, P. Seiler, E.A. Govorkova, S. Krauss, Choi, E.K. Lee, S.H. Wee, H.W. Sung, J.H. C. Scholtissek, P. Puthavathana, C. Buranathai, Kwon, M.I. Kang, and J.H. Kim. 2005. Highly T.D. Nguyen, H.T. Long, T.S. Naipospos, H. pathogenic avian infl uenza in Magpies (Pica pica Chen, T.M. Ellis, Y. Guan, J.S. Peiris, and R.G. sericea) in South Korea. Journal of Wildlife Dis- Webster. 2005. Role of domestic ducks in the eases 41:618–623. propagation and biological evolution of highly 55. Lee, M.S., M.C. Deng, Y.J. Lin, C.Y. Chang, pathogenic H5N1 infl uenza viruses in Asia. Pro- H.K. Shieh, J.Z. Shiau, and C.C. Huang. 2007. ceedings of the National Academy of Sciences Characterization of an H5N1 avian infl uenza U S A 102:10682–10687. virus from Taiwan. Veterinary Microbiology 47. Imai, K., K. Nakamura, M. Mase, K. Tsukamoto, April 2007. T. Imada, and S. Yamaguchi. 2007. Partial 56. Lee, C.W., D.L. Suarez, T.M. Tumpey, H.W. protection against challenge with the highly Sung, Y.K. Kwon, Y.J. Lee, J.G. Choi, S.J. Joh, pathogenic H5N1 infl uenza virus isolated in M.C. Kim, E.K. Lee, J.M. Park, X. Lu, J.M. Katz, Japan in chickens infected with the H9N2 infl u- E. Spackman, D.E. Swayne, and J.H. Kim. 2005. enza virus. Archives of Virology 152(7):1395– Characterization of highly pathogenic H5N1 1400. avian infl uenza A viruses isolated from South 48. Irza, V.N. 2006. Avian infl uenza in Russia. Korea. Journal of Virology 79:3692–3702. Current situation and control strategies Presenta- 57. Leschnik, M., J. Weikel, K. Möstl, S. Revilla- tion to the Twelth Annual Meeting of the Avian Fernández, E. Wodak, Z. Bagó, E. Vanek, V. Infl uenza and Newcastle Disease Community Benetka, M. Hess, and J.G. Thalhammer. 2007. Reference Laboratories. October 2006. Available Subclinical infection with avian infl uenza A at http://ec.europa.eu/food/animal/diseases/con- (H5N1) virus in cats. Emerging Infectious Dis- trolmeasures/avian/docs/pres2_jam2006.pdf. eases 13(2):243–247. Accessed January 12, 2007. 58. Liao, X., X. Zhang, J. Wang, J. Yu, and N. Jin. 49. Keawcharoen, J., K. Oraveerakul, T. Kuiken, 2006. A/chicken/Jilin/hd/2002(H5N1). Residues R.A. Fouchier, A. Amonsin, S. Payungporn, S. 1 to 568. In GenBank accession number Noppornpanth, S. Wattanodorn, A. Theamboo- ABI98919. Available at http://www.ncbi.nlm. niers, R. Tantilertcharoen, R. Pattanarangsan, N. nih.gov/entrez/viewer.fcgi?val=ABI98919. Arya, P. Ratanakorn, D.M. Osterhaus, and Y. National Center for Biotechnology Information: Poovorawan. 2004. Avian infl uenza H5N1 in Bethesda, Maryland. Accessed March 31, 2007. tigers and leopards. Emerging Infectious Diseases 59. Liao, X., X. Zhang, J. Wang, J. Yu, and T. Li. 10:2189–2191. 2006. A/chicken/Hubei/wh/1997(H5N1) Resi- 50. Kilpatrick, A.M., A.A. Chmura, D.W. Gibbons, dues 1 to 568. In GenBank accession number 282 Avian Influenza

ABI94754. Available at http://www.ncbi.nlm. maff.go.jp/tori/20040630e_report.pdf. Accessed nih.gov/entrez/viewer.fcgi?val=ABI94754. October 7, 2006. National Center for Biotechnology Information: 67. Mase, M., K. Tsukamoto, T. Imada, K. Imai, N. Bethesda, Maryland. Accessed March 31, 2007. Tanimura, K. Nakamura, Y. Yamamoto, T. 60. Liao, X., X. Zhang, J. Wang, J. Yu, and Z. Chen. Hitomi, T. Kira, T. Nakai, M. Kiso, T. Horimoto, 2006. A/chicken/Jiangsu/cz1/2002(H5N1). Resi- Y. Kawaoka, and S. Yamaguchi. 2005. Charac- dues 1 to 568. In GenBank accession number terization of H5N1 infl uenza A viruses isolated ABI96767. Available at http://www.ncbi.nlm. during the 2003–2004 infl uenza outbreaks in nih.gov/entrez/viewer.fcgi?val=ABI96767. Japan. Virology 332:167–176. National Center for Biotechnology Information: 68. Melville, D.S., and Shortridge, K.F. 2004. Infl u- Bethesda, Maryland. Accessed March 31, 2007. enza: time to come to grips with the avian dimen- 61. Li, K.S., Y. Guan, J. Wang, G.J. Smith, K.M. Xu, sion. Lancet Infectious Diseases 4:261–262. L. Duan, A.P. Rahardjo, P. Puthavathana, C. 69. Ministry of Agriculture, Peoples’ Republic of Buranathai, T.D. Nguyen, A.T. Estoepangestie, China. 2006 Surveillance on highly pathogenic A. Chaisingh, P. Auewarakul, H.T. Long, N.T. avian infl uenza (HPAI) in the mainland of the Hanh, R.J. Webby, L.L. Poon, H. Chen, K.F. P. R. China from January to October 2006. Shortridge, K.Y. Yuen, R.G. Webster, and J.S. Available at http://www.agri.gov.cn/cwgk/xgzl/ Peiris. 2004. Genesis of a highly pathogenic and P020061227356059896189.pdf. Accessed potentially pandemic H5N1 infl uenza virus in February 1, 2007. eastern Asia. Nature 430:209–213. 70. Minta, Z., K. Smietanka, K. Domanska-Blicharz, 62. Li, Y., Z. Lin, J. Shi, Q. Qi, G. Deng, Z. Li, X. G. Tomczyk and T. Wijaszka. 2006. HPAI H5N1 Wang, G. Tian, and H. Chen. 2006. Detection of infection of a mute swan fl ock in the city of Torun Hong Kong 97-like H5N1 infl uenza viruses from in Poland. In: Joint Twelfth Annual Meetings of eggs of Vietnamese waterfowl. Archives of the National Laboratories for Newcastle Disease Virology 151:1615–1624. and Avian Infl uenza of European Union Member 63. Li, H.Y., K.Z. Yu, H.L. Yang, X.G. Xin, J.Y. States 2006. Available at http://ec.europa.eu/ Chen, P. Zhao, Y.Z. Bi, and H.L. Chen. 2004. food/animal/diseases/controlmeasures/avian/ Isolation and characterisisation of H5N1 and docs/pres6_jam2006.pdf. Accessed January 20, H9N2 infl uenza viruses from pigs in China. 2007. Chinese Journal of Veterinary Preventive Medi- 71. Mounts, A.W., H. Kwong, H.S. Izurieta, Y. Ho, cine 26(1):1–6. T. Au, M. Lee, B.C. Buxton, S.W. Williams, K. 64. Lipatov, A.S., V.A. Evseenko, H.L. Yen, A.V. H. Mak, J.M. Katz, W.W. Thompson, N.J. Cox, Zaykovskaya, A.G. Durimanov, S.I. Zolotykh, and K. Fukuda. 1999. Case-control study of risk S.V. Netesov, I.G. Drozdov, G.G. Onishchenko, factors for avian infl uenza A (H5N1) disease, R.G. Webster, and A.M. Shestopalov. 2007. Hong Kong, 1997. Journal of Infectious Diseases Infl uenza (H5N1) viruses in poultry, Russian 180:505–508. Federation, 2005–2006. Emerging Infectious 72. Muramoto, Y., T.Q. Le, L.S. Phuong, T. Nguyen, Diseases 13:539–546. T.H. Nguyen, Y. Sakai-Tagawa, K. Iwatsuki- 65. Lvov, D.K., M.I.U. Schelkanov, P.G. Deriabin, Horimoto, T. Horimoto, H. Kida, and Y. Kawaoka. T.V. Grebennikova, A.G. Prilipov, Y.A. Nepok- 2006. Molecular characterization of the hemag- lonov, G.G. Onishchenko, N.A. Vlasov, T.I. glutinin and neuraminidase genes of H5N1 infl u- Aliper, A.D. Zaberezhnyi, Y.D. Kireyev, O.P. enza A viruses isolated from poultry in Vietnam Krasheninnikov, S.T. Kiryukhin, Y.I. Burtseva, from 2004 to 2005. Journal of Veterinary Medical and A.N. Slepushkin. 2006. Isolation of infl u- Science 68:527–531. enza A/H5N1 virus strains from poultry and 73. Nguyen, D.C., T.M. Uyeki, S. Jadhao, T. Maines, wild birds in west Siberia during epizooty (July M. Shaw, Y. Matsuoka, C. Smith, T. Rowe, X. 2005) and their depositing to the state collection Lu, H. Hall, X. Xu, A. Balish, A. Klimov, T.M. of viruses (August 2005). Voprosy Virusologii Tumpey, D.E. Swayne, L.P. Huynh, H.K. 51:11–14. Nghiem, H.H. Nguyen, L.T. Hoang, N.J. Cox, 66. MAFF, Japan, Highly Pathogenic Avian Infl u- and J.M. Katz. 2005. Isolation and characteriza- enza Infection Route Elucidation Team. 2004. tion of avian infl uenza viruses, including highly Routes of infection of highly pathogenic avian pathogenic H5N1, from poultry in live bird infl uenza in Japan. Food Safety and Consumer markets in Hanoi, Vietnam, in 2001. Journal of Bureau, MAFF, Japan. Available at http://www. Virology 79:4201–4212. 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 283

74. Nishiguchi, A., T. Yamamoto, T. Tsutsui, T. T. Lai, W.L. Lim, K.Y. Yuen, and Y. Guan. 2004. Sugizaki, M. Mase, K. Tsukamoto, T. Ito, and N. Re-emergence of fatal human infl uenza A subtype Terakado. 2005. Control of an outbreak of highly H5N1 disease. Lancet 363:617–619. pathogenic avian infl uenza, caused by the virus 86. Perkins, L.E., and D.E. Swayne. 2001. Pathobiol- subtype H5N1, in Japan in 2004. Scientifi c and ogy of A/chicken/Hong Kong/220/97 (H5N1) Technical Review (OIE) 24:933–944. avian infl uenza virus in seven gallinaceous 75. Offi ce Internationale des Epizooties. 2005. Highly species. Veterinary Pathology 38:149–164. pathogenic avian infl uenza in peninsular Malay- 87. Perkins, L.E., and D.E. Swayne. 2002. Patho- sia: follow up report No. 7 (fi nal report). Disease genicity of a Hong Kong-origin H5N1 highly Information 18:2. pathogenic avian infl uenza virus for emus, geese, 76. Offi ce Internationale des Epizooties. 2006. Highly ducks, and pigeons. Avian Diseases 46:53–63. pathogenic avian infl uenza in China (Peoples 88. Perkins, L.E., and D.E. Swayne. 2003. Compara- Republic of) Follow up report No. 16. Disease tive susceptibility of selected avian and mam- Information 19:25. malian species to a Hong Kong-origin H5N1 77. Offi ce Internationale des Epizooties. 2006. Highly high-pathogenicity avian infl uenza virus. Avian pathogenic avian infl uenza, Romania. Follow up Diseases 47:956–967. report No. 24. Available at http://www.oie.int/ 89. Pittman, M., R. Freigofas, V. Piazza, A. Brouw, wahid-prod/reports/en_fup_0000005244_ A. Laddomada, and I.H. Brown. 2007. Surveil- 20060427_092705.pdf. lance, prevention and disease management of 78. Offi ce Internationale des Epizooties. 2006. Ter- avian infl uenza in the European Union. Journal of restrial Animal Health Code 16th Edition. World Wildlife Diseases. 43:546–570. Organization for Animal Health: Paris, France. 90. Rimmelzwaan, G.F., D. van Riel, M. Baars, T.M. Available at http://www.oie.int/eng/normes/ Bestebroer, G. van Amerongen, R.A. Fouchier, mcode/en_sommaire.htm. Accessed March 5, A.D. Osterhaus, and T. Kuiken. 2006. Infl uenza 2007. A virus (H5N1) infection in cats causes systemic 79. Offi ce Internationale des Epizooties. 2007. disease with potential novel routes of virus spread Update on avian infl uenza in animals (type within and between hosts. American Journal of H5). Available at http://www.oie.int/downld/ Pathology 168:176–183. AVIAN%20INFLUENZA/A_AI-Asia.htm. 91. Roberton, S.I., D.J. Bell, G.J. Smith, J.M. Nich- Accessed May 10, 2007. olls, K.H. Chan, D.T. Nguyen, P.Q. Tran, U. 80. Oner, A.F., A. Bay, S. Arslan, H. Akdeniz, H.A. Streicher, L.L. Poon, H. Chen, P. Horby, M. Sahin, Y. Cesur, S. Epcacan, N. Yilmaz, I. Deger, Guardo, Y. Guan, and J.S. Peiris. 2006. Avian B. Kizilyildiz, H. Karsen, and M. Ceyhan. (2006) infl uenza H5N1 in viverrids: implications for Avian infl uenza A (H5N1) infection in eastern wildlife health and conservation. Proceedings of Turkey in 2006. New England Journal of Medi- the Royal Society B. 273:1729–1732. cine 355:2179–2185. 92. Salzberg, S.L., C. Kingsford, G. Cattoli, D.J. 81. Pantin-Jackwood, M.J., and D.E. Swayne. 2007. Spiro, D.A. Janies, M.M. Aly, I.H. Brown, E. Pathobiology of Asian highly pathogenic avian Couacy-Hymann, G.M. de Mia, D.H. Dung, A. infl uenza H5N1 virus infections in ducks. Avian Guercio, T. Joannis, A.S.M. Ali, A. Osmani, I. Diseases 51:250–259. Padalino, M.D. Saad, V. Savic´, N.A. Sengama- 82. Pereira, H.G., B. Tumova, and V.G. Law. 1965. lay, S. Yingst, J. Zaborsky, O. Zorman-Rojs, E. Avian infl uenza A viruses. Bulletin of the WHO Ghedin, and I. Capua. 2007. Genome analysis 32:855–860. linking recent European and African infl uenza 83. Peiris, J.S.M., M. de Jong, and Y. Guan. 2007. (H5N1) viruses. Emerging Infectious Diseases Avian infl uenza (H5N1): a threat to human health. 13(5):713–718. Clinical Microbiology Reviews 20:243–267. 93. Seo, S.H., and R.G. Webster. 2001. Cross-reac- 84. Peiris, J.S., Y. Guan, D. Markwell, P. Ghose, tive, cell-mediated immunity and protection of R.G. Webster, and K.F. Shortridge. 2001. Cocir- chickens from lethal H5N1 infl uenza virus infec- culation of avian H9N2 and contemporary tion in Hong Kong poultry markets. Journal of “human” H3N2 infl uenza A viruses in pigs in Virology 75:2516–2525. southeastern China: potential for genetic reassort- 94. Shortridge, K.F. 1999. Poultry and the infl uenza ment? Journal of Virology 75:9679–9686. H5N1 outbreak in Hong Kong, 1997: abridged 85. Peiris, J.S., W.C. Yu, C.W. Leung, C.Y. Cheung, chronology and virus isolation. Vaccine 17:S26– W.F. Ng, J.M. Nicholls, T.K. Ng, K.H. Chan, S. S29. 284 Avian Influenza

95. Shortridge, K.F., P. Gao, Y. Guan, T. Ito, Y. 104. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- Kawaoka, D. Markwell, A. Takada, and R.G. Heng, N. Meemak, N. Pariyothorn, S. Payung- Webster. 2000. Interspecies transmission of infl u- porn, A. Theamboonlers, and Y. Poovorawan. enza viruses: H5N1 virus and a Hong Kong SAR 2006. Avian infl uenza H5N1 in naturally infected perspective. Veterinary Microbiology 74:141– domestic cat. Emerging Infectious Diseases 147. 12:681–683. 96. Sims, L.D. 2006. Surveillance data and control of 105. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- H5N1 avian infl uenza in Asia. In: Proceedings of Heng, N. Pariyothorn, S. Payungporn, A. Theam- ISVEE XI Conference. Available at http://www. boonlers, S. Chutinimitkul, R. Thanawongnuwech, sciquest.org.nz/crusher_download. and Y. Poovorawan. 2006. Fatal avian infl uenza asp?article=10002911. Accessed October 10, A H5N1 in a dog. Emerging Infectious Diseases 2006. 12:1744–1747. 97. Sims, L.D. 2007. Lessons learned from Asian 106. Spackman, E., D.A. Senne, L.L. Bulaga, T.J. H5N1 outbreak control. Avian Diseases 51:174– Myers, M.L. Perdue, L.P. Garber, K. Lohman, 181. L.T. Daum, and D.L. Suarez. 2003. Develop- 98. Sims, L.D., J. Domenech, C. Benigno, S. Kahn, ment of real-time RT-PCR for the detection of A. Kamata, J. Lubroth, V. Martin, and P. Roeder. avian infl uenza virus. Avian Diseases 47:1079– 2005. Origin and evolution of highly pathogenic 1082. H5N1 avian infl uenza in Asia. Veterinary Record 107. Sturm-Ramirez, K.M., T. Ellis, B. Bousfi eld, L. 157:159–164. Bissett, K. Dyrting, J.E. Rehg, L. Poon, Y. Guan, 99. Sims, L.D., T.M. Ellis, K.K. Liu, K. Dyrting, H. M. Peiris, and R.G. Webster. 2004. Reemerging Wong, M. Peiris, Y. Guan, and K.F. Shortridge. H5N1 infl uenza viruses in Hong Kong in 2002 2003. Avian infl uenza in Hong Kong 1997–2002. are highly pathogenic to ducks. Journal of Virol- Avian Diseases 47:832–838. ogy 78:4892–4901. 100. Slomka, M.J., T. Pavlidis, J. Banks, W. Shell, A. 108. Sturm-Ramirez, K.M., D.J. Hulse-Post, E.A. McNally, S. Essen, and I.H. Brown. 2007 Vali- Govorkova, J. Humberd, P. Seiler, P. Putha- dated H5 Eurasian real-time reverse transcrip- vathana, C. Buranathai, T.D. Nguyen, A. Chais- tase-polymerase chain reaction and its application ingh, H.T. Long, T.S. Naipospos, H. Chen, T.M. in H5N1 outbreaks in 2005–2006. Avian Dis- Ellis, Y. Guan, J.S. Peiris, and R.G. Webster. eases 51:373–377. 2005. Are ducks contributing to the endemicity 101. Smith, C., and S. Jadhao. 2007. A/duck/Viet of highly pathogenic H5N1 infl uenza virus in Nam/342/2001(H5N1) Residues 1 to 557. Asia? Journal of Virology 79:11269–11279. GenBank accession number ABP51972. Avail- 109. Suetens, C., R. Snacken, G. Hanquet, B. Broch- able at http://www.ncbi.nlm.nih.gov/entrez/ ier, B. Maes, I. Thomas, F. Yane, T. van den viewer.fcgi?val=ABP51972. National Center for Berg, B. Lambrecht, and S. Van Borm. 2004. Biotechnology Information: Bethesda, MD. Eagles testing positive for H5N1 imported ille- Accessed March 31, 2007. gally into Europe from Thailand. Eurosurveil- 102. Smith, G.J., X.H. Fan, J. Wang, K.S. Li, K. Qin, lance 8:44. J.X. Zhang, D. Vijaykrishna, C.L. Cheung, K. 110. Swayne, D.E. (2007) Understanding the complex Huang, J.M. Rayner, J.S. Peiris, H. Chen, R.G. pathobiology of high pathogenicity avian infl u- Webster, and Y. Guan. 2006a. Emergence and enza viruses in birds. Avian Diseases 51:242– predominance of an H5N1 infl uenza variant in 249. China. Proceedings of the National Academy of 111. Thanawongnuwech, R., A. Amonsin, R. Tantil- Sciences U S A 103:16936–16941. ertcharoen, S. Damrongwatanapokin, A. Theam- 103. Smith, G.J., T.S. Naipospos, T.D. Nguyen, M.D. boonlers, S. Payungporn, K. Nanthapornphiphat, de Jong, D. Vijaykrishna, T.B. Usman, S.S. S. Ratanamungklanon, E. Tunak, T. Songserm, Hassan, T.V. Nguyen, T.V. Dao, N.A. Bui, Y.H. V. Vivatthanavanich, T. Lekdumrongsak, S. Kes- Leung, C.L. Cheung, J.M. Rayner, J.X. Zhang, dangsakonwut, S. Tunhikorn, and Y. Poovorawan. L.J. Zhang, L.L. Poon, K.S. Li, V.C. Nguyen, T. 2005. Probable tiger-to-tiger transmission of T. Hien, J. Farrar, R.G. Webster, H. Chen, J.S. avian infl uenza H5N1. Emerging Infectious Peiris, and Y. Guan. 2006 Evolution and adapta- Diseases 11:699–701. tion of H5N1 infl uenza virus in avian and human 112. Thiry, E., A. Zicola, D. Addie, H. Egberink, K. hosts in Indonesia and Vietnam. Virology Hartmann, H. Lutz, H. Poulet, and M.C. Horzinek. 350:258–268. 2007. Highly pathogenic avian infl uenza H5N1 11 / Multicontinental Epidemic of H5N1 HPAI Virus (1996–2007) 285

virus in cats and other carnivores. Veterinary viruses that continue to circulate in geese in Microbiology 122(1–2):25–31. southeastern China. Journal of Virology 76:118– 113. Tiensin, T., P. Chaitaweesub, T. Songserm, A. 126. Chaisingh, W. Hoonsuwan, C. Buranathai, T. 122. Wee, S.H., C.K. Park, H.M. Nam, C.H. Kim, H. Parakamawongsa, S. Premashthira, A. Amonsin, Yoon, S.J. Kim, E.S. Lee, B.Y. Lee, J.H. Kim, M. Gilbert, M. Nielen, and A. Stegeman. 2005. J.H. Lee, and C.S. Kim. 2006. Outbreaks of Highly pathogenic avian infl uenza H5N1, Thai- highly pathogenic avian infl uenza (H5N1) in the land, 2004. Emerging Infectious Diseases Republic of Korea in 2003/04. Veterinary Record 11:1664–1672. 158:341–344. 114. Tiensin, T., M. Nielen, T. Songserm, W. 123. Winker, K., K.G. McCracken, D.D. Gibson, C.L. Kalpravidh, P. Chaitaweesub, A. Amonsin, S. Pruett, R. Meier, F. Huettmann, M. Wege, I.V. Chotiprasatintara, A. Chaisingh, S. Damrong- Kulikova, Y.N. Zhuravlev, M.L. Perdue, E. watanapokin, S. Wongkasemjit, C. Antarasena, Spackman, D.L. Suarez, and D.E. Swayne. 2007. V. Songkitti, K. Chanachai, W. Thanapongtham, Avian infl uenza migration from Asia to Alaska. and J.A. Stegeman. 2007. Geographic and tempo- Emerging Infectious Diseases 13(4):547–552. ral distribution of highly pathogenic avian infl u- 124. World Health Organization. 2005. Evolution of enza A virus (H5N1) in Thailand, 2004–2005: an H5N1 avian infl uenza viruses in Asia. Emerging overview. Avian Diseases 51:182–188. Infectious Diseases 11:1515–1521. 115. Tanaka,Y., H. Kida, and Y. Sakoda. 2006. A/ 125. World Health Organization. 2006. Antigenic and duck/Hokkaido/55/96(H1N1). Bases 1 to 1422. genetic characteristics of H5N1 viruses and can- GenBank accession number AB271116. Avail- didate H5N1 vaccine viruses developed for able at http://www.ncbi.nlm.nih.gov/entrez/ potential use as pre-pandemic vaccines. Available viewer.fcgi?val=AB271116. National Center for at http://www.who.int/csr/disease/avian_infl u- Biotechnology Information: Bethesda, MD. enza/guidelines/recommendationvaccine.pdf. Accessed March 3, 2007. Accessed October 12, 2006. 116. Tumpey, T.M., D.L. Suarez, L.E. Perkins, D.A. 126. World Health Organization. 2006. Avian infl u- Senne, J.G. Lee, Y.J. Lee, I.P. Mo, H.W. Sung, enza: H5N1 infection found in a stone marten and D.E. Swayne. 2002. Characterization of a in Germany. Available at http://www.who.int/ highly pathogenic H5N1 avian infl uenza A virus csr/don/2006_03_09a/en/index.html. Accessed isolated from duck meat. Journal of Virology October 10, 2006. 76:6344–6355. 127. World Health Organization. 2007. Cumulative 117. Vahlenkamp, T.W., and T.C. Harder. 2006. Infl u- total of confi rmed human cases of infl uenza A enza virus infections in mammals. Berliner und (H5N1) reported to the WHO as at 4 April 2007. Munchener tierarztliche Wochenschrift 119:123– Available at http://www.who.int/csr/disease/ 131. avian_infl uenza/country/cases_table_2007_04_ 118. Van Borm, S., I. Thomas, G. Hanquet, B. Lam- 11/en/index.html. Accessed April 10, 2007. brecht, M. Boschmans, G. Dupont, M. Decaestek- 128. World Health Organization. 2006. Weekly Epide- ker, R. Snacken, and T. van den Berg. 2005. miology Report 81:183–188. Available at http:// Highly pathogenic H5N1 infl uenza virus in www.who.int/wer/2006/wer8118.pdf. Accessed smuggled Thai eagles, Belgium. Emerging Infec- April 10, 2007. tious Diseases 11:702–705. 129. World Health Organization. 2007. Antigenic and 119. Wan, X.F., X. Wu, G. Lin, S.B. Holton, R.A. genetic characteristics of H5N1 viruses and Desmone, C.R. Shyu, Y. Guan, and M.E. Emch. candidate H5N1 vaccine viruses developed for 2007. Computational identifi cation of reassort- potential use as pre-pandemic vaccines, March ments in avian infl uenza viruses. Avian Diseases 2007. Weekly Epidemiology Report 82(19):164– 51:434–439. 167. 120. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. 130. WHO, OIE, and FAO (H5N1 Evolution Working Chambers, and Y. Kawaoka. 1992. Evolution and Group). 2007. Towards a unifi ed nomenclature ecology of infl uenza A viruses. Microbiology system for the highly pathogenic H5N1 avian Reviews 56:152–179. infl uenza viruses. Poster presentation at Options 121. Webster, R.G., Y. Guan, M. Peiris, D. Walker, S. for Control of Infl uenza VI. June 17–23 2007, Krauss, N.N. Zhou, E.A. Govorkova, T.M. Ellis, Toronto Canada. Available at http://www.who. K.C. Dyrting, T. Sit, D.R. Perez, and K.F. Short- int/csr/disease/avian_infl uenza/guidelines/ ridge. 2002. Characterization of H5N1 infl uenza nomenclature/en/index.html. 286 Avian Influenza

131. Xu, X., Subbarao, N.J. Cox, and Y. Guo. 1999. 133. Yu, Z., Y. Song, H. Zhou, X. Xu, Q. Hu, H. Wu, A. Genetic characterization of the pathogenic Zhang, Y.J. Zhou, J.F. Chen, H.B. Dan, Q. Luo, X.M. infl uenza A/goose/Guangdong/1/96 (H5N1) virus: Li, H. Chen, and M. Jin. 2007. Avian infl uenza similarity of its hemagglutinin gene to those of (H5N1) virus in waterfowl and chickens, central H5N1 viruses from the 1997 outbreaks in Hong China. Emerging Infectious Diseases 13:5. Kong. Virology 261:15–19. 134. Zhou, N.N., K.F. Shortridge, E.C. Claas, S.L. 132. Yang, S.T., H.L. Wang, N. Feng, X.Z. Xia, Y.W. Krauss, and R.G. Webster. 1999. Rapid evolution Gao, C.Y. Wang, X.H. Zou, J.Q. Su, H.T. Sun, of H5N1 infl uenza viruses in chickens in Hong S. Chang, X.Y. Zheng, and T.C. Wang. 2006. Kong. Journal of Virology 73:3366–3374. Mice as a model for studying the avian H5N1 135. Zhu, Q.Y., E.D. Qin, W. Wang, J. Yu, B.H. Liu, infl uenza virus in mammals. Virologica Sinica Y. Hu, J.F. Hu, and W.C. Cao. 2006. Fatal infec- 2006:4. Abstract available at http://www.ilib.cn/ tion with infl uenza A (H5N1) virus in China. New A-zgbdx200604010.html. England Journal of Medicine 354:2731–2732. 12 Avian Influenza Control Strategies

David E. Swayne

INTRODUCTION LPAI control programs have been more diverse than There is no “one” control strategy for avian infl u- for HPAI and have ranged from no action to stamp- enza (AI) to fi t every country and all bird species ing-out strategies (47). (49). The strategies developed and used have depended on a variety of factors including presence Low Pathogenicity Avian Infl uenza or absence of AI virus in the country, pathogenicity The existence of LPAI viruses in a variety of wild of the virus (low pathogenicity [LP] versus high aquatic birds, causing mostly asymptomatic infec- pathogenicity [HP]), the hemagglutinin (HA) tions and as part of ecosystems on all seven conti- subtype of the AI virus (H5 and H7 HA subtypes nents suggests the presence of LPAI viruses is versus the other 14 HA subtypes), species of natural, has existed for eons, and is of minimal con- birds at risk or infected (wild birds, captive birds, sequence in its natural setting. Therefore, humans or domesticated species), the type of ecosystem have had and will continue to have minimal impact (natural, zoological exhibits/nature preserves, house- on control of LPAI viruses in wild bird populations. hold pets, or agricultural systems), demonstration of However, humankind’s primary focus for control of regionalization or compartmentalization, fi nancial LPAI should emphasize preventing the introduction resources available (government verses private), of wild bird–origin AI viruses into domestic poultry veterinary medical infrastructure, political will and and other birds when raised under a variety of agri- authority, and desired goal or outcome. cultural and nonagricultural conditions, including In most developed countries, HPAI has been zoos and exhibits, village or rural small holder, handled by variations of stamping-out programs for small commercial farms, and large commercial pro- the past 125 years, using concepts originating with duction systems. In the United States, Canada, Euro- Giovanni Mario Lancisi in Italy during the early pean Union (EU), Australia, and other developed 18th century to control exotic livestock diseases countries, serological and virological active and such as Rinderpest in cattle. However, in past 10 passive surveillance programs in commercial poultry years with H7N3 and H5N1 HPAI in many develop- have demonstrated most commercial farms are free ing countries, the poultry industries and govern- of AI infections. In these countries, the primary ments have not been able to achieve eradication control strategy has been preventing the introduction through traditional stamping-out programs alone but of LPAI virus into agricultural systems. However, have managed HPAI through the use of combina- the village/rural poultry sectors, live poultry market tions of stamping-out and reduction of clinical (LPM) systems and in certain locations during spe- disease. Compared with HPAI, control of LPAI has cifi c times of year in outdoor-reared specialty not become a poultry health issue until recently. The poultry, such as organics, are at risk for introduction identifi cation and need for control emerged most of LPAI viruses from wild bird reservoirs, followed prominently in the 1960s in association with clinical by secondary spread and transmission to other syndromes of respiratory disease and drops in egg poultry including the outdoor and indoor commer- production primarily within turkeys but also within cial systems. The LPAI viruses are not maintained pheasants, quails, and partridges (13). Globally, in wild gallinaceous species of birds (31, 42).

In the United States, control of LPAI falls to the designation LPNAI has increased the use of stamp- state governments and/or private industry except for ing-out programs in dealing with these two AI sub- H5 and H7 LPAI, which is handled jointly by federal types as a means to prevent emergence of HPAI and state government authorities. Notably success- viruses, although stamping out is not required by ful state LPAI control programs have been devel- OIE for LNPAI. oped in Minnesota (15, 32), Pennsylvania (6), and A more thorough discussion on control of LPAI the Delmarva region (11) through partnerships is presented in Chapter 23 (Control of Low Patho- between the poultry industries and state govern- genicity Avian Infl uenza). ments. These programs have been used to eradicate a variety of LPAI viruses on multiple occasions over High Pathogenicity Avian Infl uenza the past 30 years (9, 16, 17, 39, 47). The Minnesota The HPAI viruses have not arisen nor are they main- plan, initially developed in the early 1980s, has been tained in a wild bird reservoir (34). Instead, H5 and the model for many other state and national LPAI H7 LPAI viruses are introduced from wild aquatic control plans, and has fi ve specifi c components: edu- birds into village or commercial poultry and HPAI cation, preventing exposure, monitoring, reporting, virus strains arise through specifi c mutations in the and a “responsible response” (32). The Minnesota HA gene following circulation for days to years LPAI control program is presented in Chapter 23 (31). In most HPAI epizootics, wild aquatic birds (Control of Low Pathogenicity Avian Infl uenza). have not been infected by HPAI viruses and thus Since 2000, Italy has developed and implemented were not biological vectors of HPAI viruses. In successful control programs for H5 and H7 LPAI, some outbreaks, wild birds have been mechanical including the use of emergency or prophylactic vac- vectors, by spreading the virus through fomites on cination with inactivated AI vaccines containing a feet and feathers, but this has been dependent on the neuraminidase (NA) subtype different from the fi eld presence or absence of biosecurity measures to virus (i.e., heterologous NA), monitoring for infec- prevent wild bird access to premises. There are two tion in vaccinated poultry by NA differentiating exceptions to this rule: (1) an H5N3 HPAI virus serological tests (differentiating infected from vac- infected common terns in South Africa during 1961 cinated animals [DIVA]), use of sentinel birds, and (2) the Asian lineage of H5N1 HPAI virus that elimination of infected fl ocks by stamping-out or initially did not involve wild birds, but in 2002, controlled marketing, and strict restriction measures strains emerged with the capacity to infect a variety (7, 8, 26, 27). of wild and captive birds, and some outbreaks In the past two decades, some H5 and H7 LPAI in 2005–2007 involved wild bird infections and viruses have mutated and become HPAI viruses (see transmission. However, including wild birds in Chapters 2 and 6, Molecular Determinants of Patho- stamping-out programs is not condoned for ecolog- genicity for Avian Infl uenza Viruses and The Global ical reasons, and such measures may be counterpro- Nature of Avian Infl uenza), but determining which ductive by encouraging wild birds to disperse, thus LPAI viruses have the potential to mutate to HP has moving the virus to new premises and geographic not always been predictive in laboratory models regions (38). (48). In mid-1990s, molecular criteria based on H5 Because some wild aquatic birds can be reservoirs and H7 HA proteolytic cleavage site were added to of LPAI viruses, some individuals have the miscon- classify AI viruses as HP or potential to become HP ception that all species of wild birds are infected and irrespective of in vivo lethality for chickens (28, 54). are spreading HPAI viruses, especially the H5N1 of However, some H5 and H7 AI viruses without the recent years. In epidemiological terms, the move- molecular criteria have mutated and become HPAI ment of infected poultry, contaminated products viruses, which necessitated changes in national and such as manure and contaminated equipment and international guidelines for AI control, to include clothing/shoes are the major ways HPAI virus is not only HPAI viruses but all H5 and H7 LPAI spread from farm to farm. Such movement of viruses (30). In the World Organization for Animal infected poultry or derived materials may be through Health (Offi ce Internationale des Epizooties [OIE]) legal or illegal movement, including across porous Code, such H5 and H7 LPAI viruses are now called borders between countries. In some outbreaks, H5 and H7 LP notifi able AI (LPNAI) viruses. The aerosol generation during depopulation or cleaning 12 / Avian Influenza Control Strategies 289 has been implicated in farm-to-farm transmission. It binations of fi ve specifi c components or features is quite easy to blame wild birds for spreading HPAI (45, 47): (1) education, i.e., providing knowledge on because such a mechanism is beyond control of how AI is transmitted and each individual’s role in humankind. However, to admit to illegal movement prevention, management or eradication; (2) biosecu- of poultry including fi ghting cocks, poultry prod- rity, i.e., management practices and procedures to ucts, or contaminated equipment is seen by some as prevent virus introduction or, if present, from leaving admitting failure of a government’s ability to safe- a premise; (3) diagnostics and surveillance, i.e., guard its people and agricultural production. The ability to detect the virus or evidence of infections recommendations and responsibilities for containing in bird populations or their environment, or a means HPAI outbreaks have been described previously to verify freedom from such infections; (4) elimina- (14). tion of infected poultry, i.e., removal of the infection From 1959 to 1992, most developed countries source or susceptible sources to prevent continued eradicated HPAI epizootics or outbreaks within a environmental contamination and dissemination; few weeks to a year by traditional stamping-out pro- and (5) decreasing host susceptibility; i.e., increas- grams. However, since 1992, some developing ing host resistance so if exposure occurs, infection countries have been unable to achieve immediate is prevented or the negative consequences of infec- eradication through traditional stamping-out pro- tion are minimal. How effective the comprehensive grams because of the lack of fi nancial resources and strategy is at controlling AI is dependent upon how indemnities, diffi culty in developing and enforcing many of the fi ve components are used and how thor- movement controls, inadequate veterinary infra- oughly they are practiced in the fi eld. The goals for structure and high level of poultry production at the individual LPAI and HPAI control strategies may be village or rural level. In such outbreaks, manage- different depending on the country, subtype of the ment of the disease to a low infection rate has been virus, economic situation, and risk to public health. a realistic immediate option with the long-term goal The following sections concentrate on components of eradication. of control programs focused on agricultural and allied industry production systems. GOALS OF AVIAN INFLUENZA CONTROL AND COMPONENTS USED IN A Education CONTROL STRATEGY One critical aspect in control is the education of all There are three different goals or outcomes in the poultry and allied industry personnel, government control of AI: (1) prevention, (2) management, and personnel, and others involved in the control process (3) eradication (45, 47). Which goal to pursue greatly regarding how AI viruses are introduced, how they depends on the infection status of the country, zone, are spread, and how such events can be prevented. or compartment (CZC). If the CZC is free from AI, The control of risky behaviors and actions greatly the goal should be preventing either introduction reduces the spread of AI virus by controlling fomite from a wild bird reservoir (LPAI virus) or from and aerosol dispersion of virus, thus preventing AI infected poultry (LPAI or HPAI virus) within a virus movement onto the farm and between farms. neighboring affected CZC. If the CZC has infected Also, the general public should be included in the fl ocks, the goal could be to manage the disease to education process by communicating information on reduce economic losses followed by eradication or risks and dispelling incorrect information and immediate eradication through stamping out. Man- rumors. agement is a process of decreasing the clinical man- Detailed information on the educational compo- ifestations of the disease by reducing infection rates nents in a control strategy are covered in Chapter 21 and the negative economic consequences of infec- (The Role of Educational Programs in Control of tion by allowing production to continue. Eradica- Avian Infl uenza). Chapter 21 provides detailed tion, the ultimate goal of any AI control plan, is information on methods of education and communi- complete elimination of the AI virus from the cation to all individuals in the poultry production CZC. chain on what AI is and how they can prevent its These goals or outcomes are accomplished based introduction to their premises, or CZC. Of particular on comprehensive strategies developed using com- importance is training of producers and on farm 290 Avian Influenza personnel in biosecurity measures to prevent intro- were reared outdoors on range and experienced out- duction of AI on a premises and how to prevent breaks of LPAI following exposure to infected wild spread once AI virus has been introduced. ducks, but moving commercial turkeys production indoors in late 1990s, has nearly eliminated LPAI Biosecurity infections in the Minnesota turkey industry (18). In Biosecurity is the fi rst line of defense against both some countries, the village/rural poultry sector and LPAI and HPAI and involves management practices associated LPM systems have become an important to prevent or reduce AI virus spread by preventing entry point for LPAI viruses from wild aquatic birds contamination, controlling the movement of birds or into agricultural systems and have served as the their products, people and equipment, or reducing major reservoir for LPAI viruses in agricultural the amount of the virus (e.g., cleaning and disinfec- systems of many developed countries (23, 41, 43). tion) (15, 49). Conceptually, biosecurity falls into If biosecurity is lax and the poultry density is high, two broad categories (45, 47): (1) inclusion biosecu- AI viruses can spread to the commercial industry rity, such as quarantine and other measures, which and rapidly move within the integrated commercial are designed to keep the AI virus on an affected system resulting in epidemics of HPAI or LPAI. premises or in an affected CZC, and (2) exclusion Biosecurity is covered in more detail in Chapter biosecurity, which is practiced to keep the AI virus 16 (Farm and Regional Biosecurity Practices). Issues out of an AI-free premises or CZC. The highest risk covered include movement controls, controlling source of AI virus for naïve birds is direct exposure fomites by controlling people and equipment move- to infected birds which excrete high levels of virus ment, cleaning and disinfection, and quarantine. from the respiratory and/or alimentary systems into In addition, Chapter 17 (Farm Biosecurity Risk their immediate environments. Typically, transmis- Assessment and Audits) presents the principles for sion occurs when naive birds come into close direct assessing biosecurity risk on a farm and develop- contact with infected birds or, indirectly, when ment of mitigation strategies through use of a survey exposed to contaminated materials from the envi- instrument to assess on farm risk for introduction ronment of infected birds especially poultry manure of AI. or equipment contaminated by poultry manure. Exposure and infection usually result from inhala- Diagnostics and Surveillance tion, contact with mucus membranes or ingestion of Early and successful control of AI requires an accu- AI virus–contaminated dust, water droplets, or other rate and rapid diagnosis (49). The speed with which forms of contaminated materials (49). Cleaning and AI is controlled and the cost for such control are disinfection of equipment and clothing/footware of greatly dependent on how fast the fi rst case or cases personnel are critical to prevent introduction and are diagnosed, the level of biosecurity practiced in farm-to-farm spread of AI virus. Chapter 18 the area, and how quickly control strategies can be (Methods for Inactivation of Avian Infl uenza Virus) implemented, especially if eradication is the goal. provides information on commonly used detergents Passive surveillance or diagnostic work-ups are and disinfectants, and their application to inactiva- critical to identify LPAI virus as the etiology of tion of AI viruses. Impact of thermal application, respiratory disease or drops in egg production, or different pH values, and physical inactivation HPAI virus as the cause of high mortality events. methods are discussed in the context of contribution More broadly, active surveillance through planned to inactivation process. Proper methods for cleaning statistical sampling is critical in identifying where and disinfection of equipment, buildings, and prem- the AI virus or AI viral infection is located within a ises are discussed. CZC, or for certifying a CZC as AI free. Such testing In preventing primary introduction of LPAI is typically accomplished through serological detec- viruses from wild aquatic birds, poultry should be tion of AI-specifi c antibodies and/or detection of AI raised in confi nement or, if raised with outdoor virus by real-time reverse transcriptase–polymerase access, confi ned or separated during specifi c periods chain reaction (RRT-PCR) or antigen-capture that correspond to migrations periods of potentially ELISA tests. The tests used for diagnosis and sur- infected wild aquatic birds. During the late 1970s to veillance of AI are presented in Chapter 13 (Avian mid-1990s, some commercial turkeys in Minnesota Infl uenza Diagnostics and Surveillance Methods). 12 / Avian Influenza Control Strategies 291

This chapter provides a synopsis of the classic fi nancial compensation or the ability to market methods used to diagnose AI, including virus isola- recovered poultry. Stamping-out and disposal pro- tion in cell culture and embryonating chicken eggs, grams will only be successful if indemnities are immunological methods for identifying and subtyp- paid by federal or state governments in a timely ing AI viruses, and newer biotechnology methods manner. for virus detection including antigen and nucleic acid detection methods. Decreasing Host Susceptibility Finally, surveillance is critical for evaluating the If poultry are at risk for exposure to infected birds success of control strategies and for use in decision or a contaminated environment, decreasing suscep- making as a prelude to improving control strategies. tibility of the birds to infection may be necessary to Special virological and serological strategies to break the infection cycle (49). Such decreased sus- identify infected birds within vaccinated populations ceptibility can be achieved either through using a (i.e., DIVA) are discussed in Chapter 19 (Vaccines, host strain or breed that is genetically resistant to AI Vaccination, and Immunology). virus infections or by producing active or passive immunity against the AI virus in a susceptible host Elimination of Infected Poultry species, breed, or strain. Elimination of the source of AI virus in CZC is At this time, human anti-infl uenza drugs are not critical for stopping an outbreak and eradicating the recommended for treatment of food-producing disease. Once an affected fl ock is identifi ed, the animals. Use of such medications has been shown high-risk materials should be eliminated, including to favor rapid development of AI strains resistant to the infected birds, eggs, and manure (49). However, the antivirals and compromise the effectiveness of the safest and most economical method for elimina- the specifi c antiviral therapies for humans infected tion varies with the virus strain, local conditions, with AI viruses (2, 3, 10, 12, 22, 55). For LPAI virus biosecurity level practiced on the farm and in the infections, supportive care and antibiotic treatment area, and available fi nancial and personnel resources. have been used to reduce the negative effects of For HPAI, elimination has typically meant humane concurrent bacterial infections. depopulation and disposal of carcasses, eggs and manure in an environmentally friendly method such Genetic resistance as composting, incineration, rendering or landfi ll Concerning genetic resistance, very little research burial. Humane euthanasia is covered in Chapter 14 has been done to identify the natural occurrence or (Humane Mass Depopulation as an Effective for classic selection of resistance to AI viruses in Measure), and disposal methods are covered in poultry. Some commercial chicken strains have Chapter 15 (Methods for Disposal of Poultry resistance to renal pathology following intravenous Carcasses). challenge with LPAI viruses (50). Recently, a popu- However, for LPAI, traditional stamping-out and lation survey of Leung-Hahng-Kow and Pradoo- disposal methods have been used less commonly Hahng-Dam native chicken breeds in Thailand and instead alternative control methods including identifi ed the A9, B14, and B21 major histocompat- controlled marketing of birds 2 to 3 weeks following ibility class (MHC) I and II haplotypes as being recovery from infection and washing of eggs before more frequently present in survivors after H5N1 marketed have been used successfully (49). The HPAI village outbreaks, while A1, B12, B13, and alternatives are plausible because most AI virus B19 haplotypes were predominant in the chickens shedding occurs during the fi rst 2 weeks after initia- that died and B2, B4, and B5 haplotypes were tion of infection and usually by 4 weeks, virus equally present in survivors and fatalities (5). cannot be detected. Therefore, antibody positive Although these are promising empirical data, this fl ocks have a low risk of transmission if maintained study did not verify that the survivors had actually under strict biosecurity. Because the economic been challenged or infected by H5N1 HPAI virus; losses due to AI may be severe, any AI control thus, any conclusions as to defi nitive resistance program should not unnecessarily penalize the resulting from specifi c MHC haplotypes are very growers, especially the small producers and farmers tentative. Studies in mice have identifi ed a func- who cannot withstand the economic losses without tional Mx1+ gene as conferring some resistance to 292 Avian Influenza laboratory-adapted human infl uenza A viruses (19). with gene inserts of AI HA have been licensed and An Mx homolog has been demonstrated in ducks used in some countries. and chickens and has shown variable in vitro antivi- Experimental studies have demonstrated that ral properties against infl uenza A viruses and other high-quality and properly used AI vaccines can viruses (1, 4, 21, 24), but whether it can confer provide protection against mortality, morbidity, and resistance to AI virus infection in the bird is declines in egg production (49). Furthermore, vac- unknown. Finally, a new technology that inserts cines increase resistance to AI virus infection, reduce small interfering RNAs (siRNA) has silenced the number of birds shedding virus, greatly reduce expression of some AI viral genes in avian and the titer of challenge virus shed, prevent contact mammalian cells, thus conferring resistance to AI transmission, and reduce environmental contamina- virus replication (25). Use of siRNA in the respiration. Therefore, vaccines can be a single tool for use tory tract produced protection in a mouse model in a comprehensive AI control program when used system following lethal infl uenza A virus challenge in concert with other disease control components. (53). Combining siRNA with transgenic technology The topic of vaccines and their use are covered in holds the potential for the development of AI more detail in Chapter 19 (Vaccines, Vaccination, virus–resistant birds. and Immunology).

Immunity ECONOMIC COSTS The established and practiced method to produce Economic costs from AI can result from direct and resistance to AI viruses is through active or passive indirect losses from morbidity and mortality in immunity, principally against the AI virus HA, and, affected fl ocks, loss of consumer confi dence in to a lesser extent, the NA, but such protection is poultry products from nonaffected CZC, and down- subtype specifi c. In practice, immunity has been time in farming operations, as well as costs to done mainly through vaccination and, to a lesser prevent, manage, or eradicate the disease or infec- extent, through maternal antibodies passed to tion. The costs are variable and dependent on virus progeny via the egg yolk. Maternal antibodies only strain, host species, type of agricultural system provided protection for the fi rst 1 to 3 weeks post affected, number of premises involved, the control hatching, while active immunity was effective for strategies used, and the speed with which the control longer periods of time. AI vaccines have been used program is implemented (49). In most developed in a variety of poultry species but predominantly in countries, neither HP nor LPAI has been an endemic chickens and turkeys. disease in the commercial poultry industries, but A variety of vaccine technologies have been LPAI has been identifi ed as causing sporadic to developed and shown to be effi cacious in the labora- endemic infections in some backyard premises and tory setting against LPAI and HPAI viruses (45). in LPM systems that serve ethnic populations of Field use has been dependent on licensing by national large metropolitan areas. Most outbreaks, and the veterinary authorities following demonstration of resulting economic losses, have been from HPAI purity, safety, effi cacy, and potency (35) and a dem- epidemics in commercial as well as noncommercial onstrated need in the control of AI. The majority of chickens and turkeys. By contrast, in many develop- vaccine used in the fi eld has been inactivated whole ing countries, LPAI viruses have become endemic AI virus vaccines, typically made using LPAI fi eld in commercial and noncommercial poultry, espe- outbreak strains and, more recently, reverse geneti- cially viruses of the H9N2 subtype during the 1990s, cally generated AI vaccine strains, followed by and caused ongoing increased cost for poultry pro- chemical inactivation and oil emulsifi cation (46). duction. Since 1996, the H5N1 HPAI virus caused Use of HPAI viruses as inactivated AI vaccine epidemics in various Asian, African, and European strains has occurred but requires manufacturing in countries. Beginning in 2003, H5N1 HPAI became special high biocontainment facilities to prevent endemic in village poultry, especially domestic accidental escape and infection of susceptible poultry ducks, in some Asian and African countries. in the community. Since the late 1990s, live fowl Infections by LPAI viruses in poultry have caused poxvirus and avian paramyxovirus type 1 (lento- signifi cant economic losses from the illness and genic Newcastle disease viruses) vectored vaccines mortality in infected birds, especially when accom- 12 / Avian Influenza Control Strategies 293 panied by secondary bacterial or viral pathogens but such infections have been uncommon consider- (49). However, accurate fi gures for economic losses ing the number of exposures to H5N1 HPAI and are generally not documented or are unavailable. In H9N2 LPAI that have occurred in Asia and Africa general, the losses from LPAI outbreaks have been over the past 10 years and the number of human less than those with HPAI outbreaks because of infections with endemic H1N1 and H3N2 human lower morbidity and mortality rates with LPAI; in infl uenza A viruses that occur each year around the some situations, recovered LPAI-infected fl ocks globe. Although rare, AI viruses have caused indi- have been eliminated through a controlled market- vidual sporadic infections or AI virus genes have ing program that provides some fi nancial recupera- appeared in infl uenza A viruses that infected humans tion to farmers; federal or state eradication costs (i.e., reassortment of gene segments). have been less commonly incurred with LPAI and, Between 1959 and 1997, only 6 incidences involv- typically, LPAI has caused minimal disruption in ing 15 nonfatal cases were reported, but from 1997 national and international trade in poultry and through 2006, two AI virus strains were responsible poultry products. Endemic H9N2 LPAI infections in for 356 cases, the 2003 Netherlands H7N7 and poultry of Asia and the Middle East and H5N2 LPAI 1997–2006 H5N1 HPAI (reviewed in Ref. 50). In infections of poultry in Mexico and Central America these latter two incidents, 151 deaths occurred in have caused a signifi cant fi nancial burden on poultry association with H5N1 HPAI viruses and 1 death production and are now controlled by routine vac- was associated with H7N7 HPAI virus. In most cination and management programs to control sec- human cases, the H5N1 HPAI virus–infected indi- ondary bacterial and viral pathogens. However, in vidual had close contact with live or dead infected some developed countries, H5 and H7 LPAI viruses poultry in the village (sector 4) or LPM system have been handled with traditional stamping-out (sector 3), while H7N7 HPAI virus infections were programs at a higher fi nancial cost. For example, in farmers, poultry veterinarians, and depopulation the stamping-out program undertaken in Virginia crews in the commercial production sector (sectors during 2002 for H7N2 LPAI had a federal eradica- 1 and 2). Exposure to live or dead infected birds was tion cost of $461,000 per farm, which was slightly determined as the primary risk factor (31, 40). This more than the $275,000 per farm for H5N2 HPAI emphasizes the need for precautions in working with eradication in the United States when adjusted for birds infected by some strains of HPAI viruses. At infl ation to 2006 funds (49). Typically, the economic this point, neither the H7N7 nor H5N1 HPAI viruses losses have been greater for HPAI than LPAI with have acquired biological features of a human pan- costs being proportional to the number of birds that demic virus. died plus those that were preemptively culled. AI viruses have contributed genes to previous However, the projected cost for not implementing human pandemic infl uenza viruses through reassort- an HPAI eradication program would be even more ment events. The 1957 (H2N2) and 1968 (H3N2) costly in terms of animal health losses and loss of human pandemic infl uenza viruses arose following export markets (49). More detailed coverage of the reassortment of three (HA, NA, and PB1) and two costs of AI, especially the H5N1 HPAI epidemic, is (HA and PB1) AI viral genes with fi ve and six presented in Chapter 24 (Economics of Avian human infl uenza internal viral genes, respectively Infl uenza) (49). (20, 33, 36, 37). It is unclear if this reassortment occurred in humans or a “mixing vessel” species Public Health Aspects such as swine, but with the discovery of multiple In general, infl uenza A viruses express host adapta- human infections by AI viruses in the past 10 years, tion with transmission and infection occurring most a mixing vessel may not be necessary to produce frequently and with ease between individuals of the human pandemic infl uenza A viruses. Some recent same or closely related species (e.g., chicken to evidence suggests that the 1918 pandemic virus may turkey), occasionally causing infection in unrelated not have been derived by reassortment but might species but within the same class (e.g., pig to human have arisen by direct adaptation of a complete AI or wild duck to turkey) and, rarely, interspecies and virus to humans (52). interclass infections (e.g., chicken to human) (44). Chapter 20 (Public Health Implications of Avian Infections by AI viruses have occurred in humans, Infl uenza Viruses) discusses in more detail the 294 Avian Influenza zoonotic aspects of AI viruses and the infections control strategy must be tailored to the AI virus and they have caused. the local situation and needs. In most developed countries, HPAI has been handled by variations of ROLE OF INTERNATIONAL ANIMAL stamping-out programs, but with the emergence of HEALTH ORGANIZATIONS H5N1 HPAI in Asia 10 years ago, the strategies to Assessment of pathogenicity, pathobiology, epide- control HPAI as well as LPAI have broadened, espe- miology, and molecular features of AI viruses is cially for developing countries, to incorporate a critical to develop appropriate control strategies and variety of options. to assess the impact of AI viruses on international There are three different goals or outcomes in the trade (51). Previously, HPAI was categorized as a control of AI: (1) prevention, (2) management, and List A disease by the World Organization for Animal (3) eradication. These goals are accomplished based Health (Offi ce International des Epizooties, OIE), on comprehensive strategies developed using combi- and as an exotic disease, HPAI has been used as a nations of fi ve specifi c components or features: (1) legitimate trade barrier to protect countries or regions education, i.e., providing knowledge on how AI is from introduction of this devastating animal health transmitted and each individual’s role in prevention, disease. Although Lists A and B have been elimi- management or eradication; (2) biosecurity, i.e., nated, OIE still recognizes the seriousness of HPAI management practices and procedures to prevent and has developed, through a panel of experts from virus introduction or, if present, from leaving a member countries, a code that lists animal health premises or CZC; (3) diagnostics and surveillance, measures applicable to imports and exports of i.e., ability to detect the virus or evidence of infec- animals and animal products (29). Since 2004, OIE tions in bird populations or their environment, or has codifi ed animal health measures for HP notifi - methods to verify freedom from such infections; (4) able AI and the less serious LPNAI (i.e., H5 and H7 elimination of infected poultry, i.e., removal of the LPAI). Chapter 22 (Trade and Food Safety Issues infection source or susceptible sources to prevent for Avian Infl uenza) discusses in more detail the continued environmental contamination and dissem- measures taken to reduce risk in trade of birds and ination; and (5) decreasing host susceptibility; i.e., their products. Topics covered include risk of trans- increasing host resistance to prevent infections or if mission of AI viruses through meat or other products infection occurs minimize the negative conse- to poultry and humans, distribution of LPAI and quences. How effective the comprehensive strategy HPAI virus in poultry meat and tissues of infected is at controlling AI is dependent upon how many of birds, and methods to inactivate AI virus in foods. the fi ve components are used and how thoroughly While OIE has a major role in setting interna- they are practiced in the fi eld. The immediate goals tional animal health standards for trade in animals for individual LPAI and HPAI control strategies may and animal products, the Food and Agricultural be different depending on the country, subtype of the Organization (FAO) of the United Nations plays a virus, economic situation and risk to public health, major role in the developing world for livestock but the long term goal should be elimination of AI. production and animal health programs. The FAO Economic costs associated with AI can result facilitates countries in development of programs that from direct and indirect losses from illness and mor- enable the production of clean and safe animal prod- tality in affected fl ocks, loss of consumer confi dence ucts for consumers, particularly in assisting to build in poultry products from nonaffected CZC and and strengthen opportunities for smallholder live- downtime in farming operations, as well as costs to stock farmers in developing countries. Chapter 25 prevent, manage, or eradicate the disease or infec- (Global Strategy for Highly Pathogenic Avian tion. The costs are variable and dependent on virus Infl uenza: Progressive Control and Eradication, and strain, host species, type of agricultural system Postoutbreak Recovery) covers the general topic of affected, number of premises involved, the control international control strategies for AI including the strategies used, and the speed with which the control recovery process. program is implemented. Infections by AI viruses have occurred in humans, CONCLUSIONS but such infections have been uncommon consider- There is no “one” control strategy to fi t all types of ing the number of exposures to H5N1 HPAI and AI for every country and in all bird species. Each H9N2 LPAI that have occurred in Asia and Africa 12 / Avian Influenza Control Strategies 295 over the past 10 years, and the number of human 11. Delmarva Poultry Industry. 2004. Proceedings of infections with endemic H1N1 and H3N2 human the 39th National Meeting on Poultry Health and infl uenza A viruses that occur each year around the Processing. Delmarva Poultry Industry: George- globe. town, DE, pp. 1–96. 12. Easterday, B.C., V.S. Hinshaw, and D.A. Halvor- son. 1997. Infl uenza. In: B.W. Calnek, H.J. Barnes, REFERENCES C.W. Beard, L.R. McDougald, and Y.M. Saif 1. Bazzigher, L., A. Schwarz, and P. Staeheli. 1993. (eds.). Diseases of Poultry, 10th ed. Iowa State No enhanced infl uenza virus resistance of murine University Press: Ames, IA, pp. 583–605. and avian cells expressing cloned duck Mx protein. 13. Easterday, B.C., and B. Tumova. 1972. Avian Virology 195:100–112. infl uenza. In: M.S. Hofstad, B.W. Calnek, C.F. 2. Beard, C.W., M. Brugh, and D.C. Johnson. 1984. Helmbolt, W.M. Reid, and H.W. Yoder, Jr. (eds.). Laboratory studies with the Pennsylvania avian Diseases of Poultry, 6th ed. Iowa State University infl uenza viruses (H5N2). Proceedings of the Press: Ames, IA, pp. 670–700. United States Animal Health Association 88:462– 14. Fichtner, G.J. 1987. The Pennsylvania/Virginia 473. experience in eradication of avian infl uenza 3. Beard, C.W., M. Brugh, and R.G. Webster. 1987. (H5N2). In: B.C. Easterday and C.W. Beard (eds.). Emergence of amantadine-resistant H5N2 avian Proceedings of the Second International Sympo- infl uenza virus during a simulated layer fl ock sium on Avian Infl uenza, Georgia Center for Con- treatment program. Avian Diseases 31:533– tinuing Education, the University of Georgia, 537. Athens, Georgia, USA, September 3–5, 1986. 4. Bernasconi, D., U. Schultz, and P. Staeheli. 1995. Symposium on Avian Infl uenza, US Animal Health The interferon-induced Mx protein of chickens Association: Richmond, VA, pp. 33–38. lacks antiviral activity. Journal of Interferon Cyto- 15. Halvorson, D.A. 1987. A Minnesota cooperative kine Research 15:47–53. control program. In: B.C. Easterday and C.W. 5. Boonyanuwat, K., S. Thummabutra, N. Sookma- Beard (eds.). Proceedings of the Second Interna- nee, V. Vatchavalkhu, V. Siripholvat, and T. Mit- tional Symposium on Avian Infl uenza, Georgia suhashi. 2006. Infl uences of MHC class II Center for Continuing Education, the University of haplotypes on avian infl uenza traits in Thai indig- Georgia, Athens, Georgia, USA, September 3–5, enous chicken. The Journal of Poultry Science 1986. Symposium on Avian Infl uenza, US Animal 43:120–125. Health Association: Richmond, VA, pp. 327–336. 6. Brugh, M., and D.C. Johnson. 1987. Epidemiology 16. Halvorson, D.A. 1995. Avian infl uenza control in of avian infl uenza in domestic poultry. In: B.C. Minnesota. Poultry Digest 54(9):12–19. Easterday and C.W. Beard (eds.). Proceedings of 17. Halvorson, D.A. 1998. Epidemiology and control the Second International Symposium on Avian of avian infl uenza in Minnesota. In: Proceedings Infl uenza, Georgia Center for Continuing Educa- of the 47th New England Poultry Health Confer- tion, the University of Georgia, Athens, Georgia, ence. New England Poultry Association: Ports- USA, September 3–5, 1986. Symposium on Avian mouth, NH, pp. 5–11. Infl uenza, US Animal Health Association: Rich- 18. Halvorson, D.A. 2002. Twenty-fi ve years of avian mond, VA, pp. 177–186. infl uenza in Minnesota. In: Proceedings of the 53rd 7. Capua, I., and D.J. Alexander. 2004. Avian infl u- North Central Avian Disease Conference. NCADC: enza: recent developments. Avian Pathology Minneapolis, MN, pp. 65–69. 33:393–404. 19. Jin, H.K., T. Yamasihita, K. Ochiai, O. Haller, and 8. Capua, I., and D.J. Alexander. 2006. The challenge T. Watanabe. 1998. Characterization and expres- of avian infl uenza to the veterinary community. sion of the Mx1 gene in wild mouse species. Avian Pathology 35(3):189–205. Biochemical Genetics 36(9/10):311–322. 9. Davison, S., R.J. Eckroade, and A.F. Ziegler. 2003. 20. Kawaoka, Y., S. Krauss, and R.G. Webster. 1989. A review of the 1996–1998 nonpathogenic H7N2 Avian-to-human transmission of the PB1 gene of avian infl uenza outbreak in Pennsylvania. Avian infl uenza A viruses in the 1957 and 1968 pandem- Diseases 47(Suppl.):823–827. ics. Journal of Virology 63:4603–4608. 10. Dolin, R., R.C. Reichman, H.P. Madore, R. 21. Ko, J.H., A. Takada, T. Mitsuhashi, T. Agui, and Maynard, P.N. Linton, and J. Webber Jones. 1982. T. Watanabe. 2004. Native antiviral specifi city of A controlled trial of amantadine and rimantadine chicken Mx protein depends on amino acid varia- in the prophylaxis of infl uenza A infection. New tion at position 631. Animal Genetics 35(2):119– England Journal of Medicine 307:580–584. 122. 296 Avian Influenza

22. Lang, G., O. Narayan, and B.T. Rouse. 1970. Pre- 33. Reid, A.H., and J.K. Taubenberger. 1999. The vention of malignant avian infl uenza by 1-adaman- 1918 fl u and other infl uenza pandemics: “over tanamine hydrochloride. Archiv Gesamte there” and back again. Lab Invest 79(2):95–101. Virusforschung 32:171–184. 34. Rohm, C., T. Horimoto, Y. Kawaoka, J. Suss, and 23. Lee, C.W., D.A. Senne, J.A. Linares, P.R. Wool- R.G. Webster. 1995. Do hemagglutinin genes of cock, D.E. Stallknecht, E. Spackman, D.E. Swayne, highly pathogenic avian infl uenza viruses consti- and D.L. Suarez. 2004. Characterization of recent tute unique phylogenetic lineages? Virology H5 subtype avian infl uenza viruses from US 209:664–670. poultry. Avian Pathology 33(3):288–297. 35. Roth, J.A., and L.M. Henderson. 2001. New tech- 24. Li, X.Y., L.J. Qu, J.F. Yao, and N. Yang. 2006. nology for improving vaccine safety and effi cacy. Skewed allele frequencies of an Mx gene mutation Veterinary Clinics of North America: Food Animal with potential resistance to avian infl uenza virus in Practice 17:585–597. different chicken populations. Poultry Science 36. Schafer, W. 1955. Vergleichende sero-immunolo- 85(7):1327–1329. gische untersuchungen uber die viren der infl uenza 25. Li, Y.C., L.H. Kong, B.Z. Cheng, and K.S. Li. unf klassichen gefl ugelpest. Zeitschrift fur Natur- 2005. Construction of infl uenza virus siRNA forschung 10B:81–91. expression vectors and their inhibitory effects on 37. Scholtissek, C., I. Koennecke, and R. Rott. 1978. multiplication of infl uenza virus. Avian Diseases Host range recombinants of fowl plague (infl uenza 49(4):562–573. A) virus. Virology 91:79–85. 26. Marangon, S., and I. Capua. Control of avian infl u- 38. Schoustra, W., J. Lubroth, C. Bruschke, W.B. enza in Italy: from stamping out to emergency Karesh, S. Marangon, R. Fouchier, T. Mundkur, N. and prophylactic vaccination. Developments in Marano, H. Kida, and S. Newman. 2006. Conclu- Biologicals 124:109–115. sions and recommendations. FAO and OIE Inter- 27. Marangon, S., I. Capua, G. Pozza, and U. Santucci. national Scientifi c Conference on Avian Infl uenza 2004. Field experiences in the control of avian and Wild Birds. Available at http://www.fao.org/ infl uenza outbreaks in densely populated poultry ag/againfo/subjects/en/health/diseases-cards/ areas. Developments in Biologicals 119:155– conference/documents/FinalRecommendations. 164. pdf. Accessed March 23, 2007. 28. Offi ce International des Epizooties. 1996. Highly 39. Senne, D.A. 2004. Avian infl uenza. Proceedings of pathogenic avian infl uenza (fowl plague). In: the United States Animal Health Association Manual of Standards for Diagnostic Tests and Vac- 108:545–547. cines, 3rd ed. Offi ce International des Epizooties: 40. Shortridge, K.F. 1999. Poultry and the infl uenza Paris, pp. 155–160. H5N1 outbreak in Hong Kong, 1997: Abridged 29. Offi ce International des Epizooties. 2007. Terres- chronology and virus isolation. Vaccine 17:S26– trial Animal Health Code, 2006. Available at S29. http://www.oie.int/eng/normes/en_mcode.htm- 41. Spackman, E., D.A. Senne, S. Davison, and D.L. Paris: Offi ce International des Epizooties. Accessed Suarez. 2003. Sequence analysis of recent H7 March 28, 2007. avian infl uenza viruses associated with three dif- 30. OIE. 2006. Avian infl uenza. International Animal ferent outbreaks in commercial poultry in the Health Code—2006. Available at http://www.oie. United States. Journal of Virology 77(24):13399– int/eng/normes/mcode/en_chapitre_2.7.12.htm. 13402. OIE: Paris. Accessed September 8, 2006. 42. Suarez, D.L., and C.S. Schultz. 2000. Immunology 31. Perdue, M.L., D.L. Suarez, and D.E. Swayne. of avian infl uenza virus: a review. Developmental 2000. Avian infl uenza in the 1990s. Avian and and Comparative Immunology 24(2–3):269–283. Poultry Biology Reviews 11(1):1–20. 43. Suarez, D.L., E. Spackman, and D.A. Senne. 2003. 32. Poss, P.E., K.A. Friendshuh, and L.T. Ausherman. Update on molecular epidemiology of H1, H5, and 1987. The control of avian infl uenza. In: B.C. East- H7 infl uenza virus infections in poultry in North erday and C.W. Beard (eds.). Proceedings of the America. Avian Diseases 47:888–897. Second International Symposium on Avian Infl u- 44. Swayne, D.E. 2000. Understanding the ecology enza, Georgia Center for Continuing Education, and epidemiology of avian infl uenza viruses: the University of Georgia, Athens, Georgia, USA, implications for zoonotic potential. In: C.C. Brown September 3–5, 1986. Symposium on Avian Infl u- and C.A. Bolin (eds.). Emerging Diseases of enza, US Animal Health Association: Richmond, Animals. ASM Press: Washington, DC, pp. 101– VA, pp. 318–326. 130. 12 / Avian Influenza Control Strategies 297

45. Swayne, D.E. 2004. Application of new vaccine 50. Swayne, D.E., M.J. Radin, T.M. Hoepf, and R.D. technologies for the control of transboundary Slemons. 1994. Acute renal failure as the cause of diseases. Developments in Biologicals 119:219– death in chickens following intravenous inocula- 228. tion with avian infl uenza virus A/chicken/Alabama/ 46. Swayne, D.E. 2006. Avian infl uenza vaccine tech- 7395/75 (H4N8). Avian Diseases 38:151–157. nologies and laboratory methods for assessing 51. Swayne, D.E., and D.L. Suarez. 2000. Highly protection. In: Proceedings for the Requirements pathogenic avian infl uenza. Revue Scientifi que et for Production and Control of Avian Infl uenza Technique Offi ce International des Epizooties Vaccines. European Directorate for the Quality of 19(2):463–482. Medicines: Strasbourg, France pp. 15–25. 52. Taubenberger, J.K. 2005. Characterization of the 47. Swayne, D.E., and B. Akey. 2005. Avian infl uenza 1918 infl uenza virus polymerase genes. Nature control strategies in the United States of America. (London) 437(7060):889–893. In: R.S. Schrijver and G. Koch (eds.). Avian Infl u- 53. Tompkins, S.M., C.Y. Lo, T.M. Tumpey, and S.L. enza. Prevention and Control. Springer: Dordrecht, Epstein. 2004. Protection against lethal infl uenza pp. 113–130. virus challenge by RNA interference in vivo. Pro- 48. Swayne, D.E., J.R. Beck, M. Garcia, M.L. Perdue, ceedings of the National Academy of Sciences and M. Brugh. 1998. Pathogenicity shifts in exper- U S A 101(23):8682–8686. imental avian infl uenza virus infections in chick- 54. United States Animal Health Association. 1994. ens. In: D.E. Swayne and R.D. Slemons (eds.). Report of the Committee on Transmissible Dis- Proceedings of the Fourth International Sympo- eases of Poultry and Other Avian Species. Criteria sium on Avian Infl uenza, May 29–31, 1997, for determining that an AI virus isolation causing Athens, Georgia. Symposium on Avian Infl uenza, an outbreak must be considered for eradication. US Animal Health Association: Richmond, VA, Proceedings of the United States Animal Health pp. 171–181. Association 98:522. 49. Swayne, D.E., and D.A. Halvorson. 2008. Infl u- 55. Webster, R.G., Y. Kawaoka, W.J. Bean, C.W. enza. In: Y.M. Saif, J.R. Glisson, A.M. Fadly, Beard, and M. Brugh. 1985. Chemotherapy and L.R. McDougald, and L. Nolan, (eds.). Diseases vaccination: a possible strategy for the control of of Poultry, 12th ed. Blackwell: Ames, IA, in highly virulent infl uenza virus. Journal of Virology press. 55:173–176. 13 Avian Influenza Diagnostics and Surveillance Methods

Erica Spackman, David L. Suarez, and Dennis A. Senne

INTRODUCTION VIRUS DETECTION TESTS The clinical presentation of avian infl uenza (AI) varies by virus strain and host species. The clini- Virus Isolation cal disease and lesions the virus produces in poultry The reference standard for the diagnosis of AI virus are not pathognomonic for avian infl uenza; there- is virus isolation (VI), and although other methods fore, diagnosis of AI virus infection requires a may be used to make a presumptive diagnosis, VI is laboratory test. Detection of AI virus infection or necessary to confi rm the presence of virus in an index exposure may be accomplished by isolating the virus case and to perform further characterization of the in eggs or cell cultures or through the detection of virus. Embryonating chicken eggs (at 9 to 11 days of viral protein, viral nucleic acid, or antibody to AI incubation) is the most widely used system for the virus. isolation of AI virus, although cell cultures, often Diagnostic tests for AI virus can be classifi ed with avian origin cell lines, Madin-Darby canine as type A specifi c, meaning they can identify any kidney cells, or other cell lines, may also be used. type A infl uenza virus of avian and mammalian However, when cell cultures are used for isolation or origin, or the test can be subtype specifi c and will propagation of low pathogenic AI (LPAI) viruses, only detect a specifi c subtype (or antibodies to trypsin must be added to the medium to facilitate a specifi c subtype). Subtype-specifi c tests most hemagglutination (HA) protein cleavage, a require- often target the H5 and H7 hemagglutinin (H) ment for productive replication in these systems. subtypes because of the potential for these subtypes The embryonating chicken egg is considered to to be highly pathogenic (HP) in gallinaceous be the most sensitive host system for the isolation poultry. of poultry adapted AI virus and can be used with all For poultry, numerous tests have been described sample types: tissue homogenates, cloacal swabs, for both virus and antibody detection (Table 13.1). tracheal swabs, oropharyngeal swabs, and environ- Selection and use of diagnostic tests depend on mental samples. However, to achieve optimal sensi- application as well as factors such as cost, sensitiv- tivity, it may be necessary to serially passage a ity, specifi city, speed, complexity, and the avail- specimen, often referred to as a “blind passage,” in ability of human resources, materials, and test embryonating eggs a second or third time, but this reagents. This chapter outlines the most widely substantially increases the time it takes to complete available and frequently used tests for detecting AI the test. virus and antibody to AI virus. Additional tests rou- Because of the high sensitivity of VI, this method tinely used for in-depth characterization of a virus may be used to detect AI virus during any stage isolate are also briefl y discussed. of an active infection. Depending on numerous

Table 13.1. Characteristics of selected avian inﬂ uenza virus diagnostic assays. Relative Relative Relative cost Time to Assay Target sensitivity speciﬁ city per sample result

Virus isolation Viable virus Very high Moderate High 1 to 2 weeks Antigen detection AIV protein Low High Moderate 15 minutes immunoassays (commercial kits) Real-time RT-PCR AIV RNA Very high Very high Moderate 3 hours Agar gel 1. Type A infl uenza virus Moderate High Moderate 48 hours immunodiffusion nucleoprotein and matrix protein 2. Antibody to type A infl uenza nucleoprotein and matrix protein ELISA (commercial Antibody to type A Moderate Moderate Low 2 to 3 hours kits) infl uenza Hemagglutination 1. Identifi cation of AIV High Moderate Moderate to 2 hours (HA)-inhibition HA subtype to high high 2. Antibody to a specifi c HA subtype Neuraminidase 1. Identifi cation of AIV Moderate Moderate Moderate 3 hours (NA)-inhibition NA subtype to high 2. Antibody to a specifi c NA subtype AIV = avian infl uenza virus. host- and virus-related factors, AI virus may be says, agar gel immunodiffusion (AGID) assay, and detected within 24 hours of infection in an individ- real-time reverse transcriptase–polymerase chain ual bird and for several weeks postexposure in a reaction (RRT-PCR) tests on undiluted egg or cell fl ock (31). culture fl uids. Alternatively, the HA positive samples Although VI is very sensitive, it is not highly can be directly subtyped using AI virus–specifi c specifi c or selective because other agents that may subtyping serums in the HI or neuraminidase-inhibi- be present in a poultry-origin specimen will readily tion (NI) assays. grow in chicken embryos or cell cultures (e.g., avian Despite the high sensitivity of embryonating eggs paramyxoviruses). For this reason, additional tests for detecting AI virus by isolation, there are some on fl uids from eggs or cell cultures are required to practical considerations that should be taken into confi rm presence of AI virus. To accomplish this, account. First, virus isolation is relatively expensive fl uids from eggs or cell cultures inoculated with the and is not easily scaled-up because procurement and test material are usually tested for HA by a standard incubation of eggs have to be scheduled well in HA assay (reviewed in Ref. 32). A sample positive advance. Second, when performing virus isolation, for HA is then tested by the hemagglutination-inhi- the infectious virus is amplifi ed to a high level, bition (HI) assay to differentiate AI virus from other signifi cantly increasing the potential for cross- hemagglutinating viruses, most commonly avian contamination among samples and exposure of paramyxoviruses (e.g., Newcastle disease virus). laboratory personnel to infectious virus. For this Once avian paramyxoviruses have been ruled out, reason, VI is generally performed in laboratories the presence of AI virus can be confi rmed by type- with enhanced biosecurity (e.g., BSL-3, BSL-3Ag, specifi c tests such as antigen detection immunoas- or P3), especially if the specimen is suspected to 13 / Avian Influenza Diagnostics and Surveillance Methods 301 contain HPAI (or virulent Newcastle disease virus). poultry or wild birds. Finally, the sample types that Virus isolation also requires a high level of technical can be used with antigen detection tests are limited; skill to perform and interpret results because embry- some can accommodate only tracheal or oropharyn- onating eggs can support the growth of many differ- geal swab specimens. ent avian viruses as well as bacteria, which can Antigen detection kits also have some major complicate diagnosis. Finally, VI has the longest advantages: they are very rapid, producing results time to result of any AI virus detection test. The VI within 15 to 20 minutes, and are highly specifi c. procedure may take from 1 to 2 weeks to complete Additionally, commercial antigen detection tests are depending on the number of passages used and how convenient, self-contained, and easy to use and quickly the virus grows to a suffi ciently high titer to interpret. Therefore, they are ideal for use on the be detected by HA or other methods. farm as a “pen-side” test. Antigen detection tests are commonly used in state diagnostic laboratories as a Antigen Detection Imunnoassays rapid screening test for AI virus in clinical speci- Numerous commercial type A infl uenza antigen mens and for identifying suspect AI virus isolates. detection immunoassay kits are available (38), but The cost per sample of running the commercial most are only licensed for human use. At least one antigen detection tests varies among manufacturers test has been conditionally licensed in the U.S. for but is less expensive than VI and is similar to RRT- veterinary use (FluDetect, Synbiotics Inc., San PCR. Antigen detection kits are best suited for rapid Diego, CA), while other test kit licensed for human screening of sick or recently dead chickens or diagnostic use (Directigen Flu A test, Becton-Dick- turkeys on the farm or for use in the laboratory enson, Franklin Lakes, NJ), have been used in during an outbreak. poultry and other species as an unlicensed test for several years with good results (1, 11, 40). These Agar Gel Immunodiffusion Assay kits use a monoclonal antibody directed against the The AGID assay (5) is also referred to as the agar highly conserved type A infl uenza nucleoprotein to gel precipitin test, which is the application of the bind viral antigen on a fi lter strip or membrane. Ouchterlony immuno-double diffusion to AI virus Results can be visualized by the appearance of a (Fig. 13.1). The principle of AGID is to visualize band or pattern on the test strip or membrane following a chromatographic immunochemical reaction. Due to the immense interest in the recent Asian H5N1 HPAI virus, a commercial H5-specifi c test has been developed (Animal Genetics, Inc., Gyeonggi-do, Korea), and other similar kits may become available. However, development of subtype-specifi c antigen detection tests are more challenging because the monoclonal antibodies used in these tests must be directed to the highly variable HA antigen, making the test less reliable than type A–specifi c assays. The greatest limitation of antigen detection kits is their low sensitivity. Most kits have an analytical 4 5 sensitivity of approximately 10 to 10 EID50 (38, unpublished data). However, birds presenting with clinical disease or that die from AI virus infection generally shed adequate virus titers for detection Figure 13.1. Seven-well pattern used to test with the antigen detection kits. In contrast, if the for antibodies to avian infl uenza by the agar disease is subclinical, the level of virus being shed gel immunodiffusion (AGID) test. In this by the animal is often below the detection threshold scheme, antigen (AG) is placed in the center well and test samples (1, 2, 3) and antiserum of the test. Therefore, antigen detection assays are (AS) are placed in alternate peripheral wells. not recommended for screening apparently healthy 302 Avian Influenza the immunoprecipitation reaction of AI virus anti- available, where results can be obtained in less than body and antigen after diffusion in an agar matrix. 3 hours; (2) it is more specifi c than conventional Although AGID is most widely used in a diagnostic RT-PCR when used with a hybridization probe; and setting to detect antibody using a reference antigen, (3) the potential for cross-contamination is reduced it can also be used to detect type A infl uenza antigen because samples are not manipulated after amp- by using a reference antibody, such as to confi rm AI lifi cation. The major disadvantage of RRT-PCR is virus in allantoic fl uid from embryonating chicken the high start-up cost for equipment, which has hin- eggs in virus isolation. dered some smaller laboratories from using this AGID is inexpensive and simple to run and does technology. not require unusual supplies or expensive equip- Because AI virus nucleic acid–based detection ment. However, preparation of the reagents with tests can detect both live and inactivated virus proper quality assurance is expensive and time con- (30), these methods may not be appropriate for envi- suming; therefore, many laboratories use reagents ronmental testing where the goal is to certify a produced by reference laboratories. Additionally premises free of live virus. Care must also be taken AGID requires moderate skill to interpret test results. to ensure that cross-contamination is prevented, Results may be read in 24 hours, but the test may because molecular methods are highly sensitive take up to 48 hours to detect some weak positive (37). Most laboratories doing molecular diagnostic reactions. work separate the RNA extraction, RT-PCR setup, and product analysis steps into different rooms to Molecular/Nucleic Acid–Based Tests reduce the risk of cross-contamination. Finally, In recent years, the application of molecular methods molecular methods require a moderate skill level for for the detection of viral nucleic acid has become an sample processing, performing the test, and interpre- important tool for the detection of AI virus and iden- tation of results. tifi cation of HA and NA subtypes. RT-PCR–based RRT-PCR and other molecular methods are ideal tests are the most widely used molecular method. for rapid, high through-put, relatively inexpensive Alternative amplifi cation methods are also avail- screening of specimens during routine surveillance able, such as the nucleic acid sequence–based ampli- or during an outbreak. Although a positive result fi cation (NASBA) test, an isothermic method for should be confi rmed by VI, RRT-PCR provides amplifying nucleic acids (8–10, 16). Although NASBA initial information that can be used as the basis for is sensitive, the test has not had widespread use. an immediate response. Real-time RT-PCR (28) and conventional RT- PCR tests (13, 21, 29) have both been reported for SUBTYPE-SPECIFIC TESTS the detection of type A infl uenza virus or AI virus The HI assay and NI assay can be used to identify and subsequent identifi cation of certain HA sub- the HA and NA subtypes of either an AI virus isolate types, often H5 and H7 (17, 19, 21, 28) and certain or the HA and NA antibody specifi city from AI NA subtypes (33). The recent Asian H5N1 HPAI virus–exposed animals. Because of the number of viruses have probably been the most targeted, with HA and NA subtypes, it is not practical to use these numerous reports of HA and NA subtype–specifi c tests for blind screening of diagnostic samples; tests (12, 18–20, 36), although few of the reported therefore, they are routinely used only after a posi- tests have been fi eld validated. tive result with a type-specifi c test. In some cases, Molecular methods offer numerous advantages however, because of the importance of H5 and H7 for AI virus detection: high sensitivity, which is subtype viruses, the HI assay may be used to screen similar to VI (3, 7, 22, 28); high specifi city; scal- for just these two subtypes. ability, the ability to accommodate any sample type with proper sample processing; minimization of Hemagglutination-Inhibition Assay contact with infectious materials, because the virus The HI assay (reviewed in Ref. 34) is a simple but is inactivated early during sample processing; and useful test that can be used several ways. The HI test reasonable cost. RRT-PCR, which is more widely can be used as a confi rmatory test for the presence used than conventional RT-PCR, offers additional of subtype-specifi c AI virus in hemagglutinating egg advantages: (1) it is the most rapid molecular test fl uids, to further characterize AI virus isolates by 13 / Avian Influenza Diagnostics and Surveillance Methods 303 identifying the HA subtype, or to identify subtype- From a practical standpoint, the HI assay is rela- specifi c antibodies to AI virus in serum, plasma, or tively expensive when used to identify isolates or egg yolk. when used as a screening test for detecting antibod- Confi rmation of AI virus presence in a specimen ies because of the number of antigens or antiserums is usually done by fi rst excluding the presence of required to test for all 16 HA subtypes. However, avian paramyxovirus type-1 (APMV-1, or Newcas- the HI assay is rapid (results are available within a tle disease virus) with APMV-1–specifi c antibody. couple of hours) and simple to perform and requires A negative HI assay result with APMV-1 antiserum low to moderate skill to interpret test results. A indicates that APMV-1 is not present and that the major advantage of the HI assay is that inactiva- specimen is suspect for AI virus. ted antigens can be used, eliminating the need for Suspect isolates are identifi ed by HI with a panel special biosecurity or biosafety measures in the of subtype-specifi c antisera representing each HA laboratory. subtype. Because false-positive reactions, a phenomenon referred to as steric inhibition, can be Neuraminidase Inhibition Assay caused by presence of a homologous NA (23), more The NI assay can be used to detect NA subtype–spe- than one reference serum per HA subtype is often cifi c antibodies or antigen, but is more frequently necessary to ensure adequate specifi city. The used as a method for identifying the NA subtype of problem of steric inhibition can be overcome by the a newly isolated infl uenza virus. use of antisera prepared by DNA vaccines contain- The principle of the NI assay is to inhibit the ing only the HA gene (14). Additionally, some enzymatic activity of the NA with subtype-specifi c cross-reaction can occur between HA subtypes, antibodies (4). For characterization of new isolates, making results more diffi cult to interpret; therefore, a panel of reference antibodies corresponding to all the HI assay specifi city is highly dependent on the nine NA subtypes is needed to perform the NI assay. quality of the antibody panel. The test format uses a colorimetric reaction that does Conversely, HI assay may be used to identify the not occur when the NA activity is blocked, indicat- HA subtype of AI virus antibodies in a serum, ing a match between the antibody and test virus plasma, or egg yolk specimen by using viruses of subtype. known subtype as the antigen in the assay. Again, a From a practical standpoint the NI assay is similar panel of all 16 HA subtypes is needed to evaluate to HI assay in numerous ways. First, sensitivity is all of the different possible subtypes. not a critical characteristic of NI assay since virus Sensitivity is not a major concern when the HI isolates are used. Also like HI assay, the specifi city assay is used to identify AI virus isolates because is good, but it also depends on the quality of the the test is used with amplifi ed virus as opposed to reference sera used (35). The NI assay is a more clinical specimens where the concentration of virus complicated procedure than the HI assay, and may be low and unknown isolates are also standard- although it can be completed within a few hours, it ized to a specifi c HA titer before identifi cation by is typically performed in reference laboratories. The the HI assay. However, sensitivity of the HI assay NI assay is more expensive than the HI assay because for antibody detection is a concern. Reduced sensi- the substrate used in the test is relatively expensive. tivity can occur when signifi cant antigenic drift The assay can be performed in a 96-well microtiter occurs within a subtype resulting in low reactivity format or in tubes, but the microtiter assay requires between the antigens used in the HI assay and anti- special white-colored plates to make interpretation bodies found in test sera. Despite these concerns, the of color differences easier. HI assay is still considered to be more sensitive than AGID (15), and HI will detect AI virus antibody for METHODS FOR FURTHER a longer period post exposure than AGID. Although CHARACTERIZATION OF AI not routinely used for species other than chickens VIRUS ISOLATES and turkeys, the HI assay is not species specifi c and Once AI virus is isolated from a diagnostic speci- may be considered for use in other species, such men, further characterization by methods that as ducks, for which there are no other consistent are not primary diagnostic or detection tests is serologic tests available. often necessary. Such tests include the chicken 304 Avian Influenza pathogenicity test to differentiate LP from HP Sequence data can also be used to confi rm or even viruses, sequencing the HA gene protelytic cleavage be the primary method for determining the HA and site to assess virulence characteristics, and phyloge- NA subtypes of an AI virus isolate. netic analysis, which can be used for molecular epi- Because AI virus has a segmented genome, all demiologic purposes. Characterization of AI virus eight genome segments need to be considered for isolates is not routinely done at veterinary diagnostic complete sequence analysis. Sequencing of the full- laboratories because it requires higher biosafety and length coding region of the entire genome of newly biosecurity conditions, specialized knowledge, and isolated AI virus can take several weeks to com- facilities (i.e., animal facilities for pathotyping) par- plete. However, individual genes and portions of all ticular for a suspect HPAI virus. eight gene segments that are adequate for analysis Because advanced analysis is expensive and labor can be sequenced faster. intensive, it is not feasible to perform extensive Constraints for sequence analysis are the cost and testing for all isolates. Therefore, the H5 and H7 HA time to both produce and analyze the data, which subtypes are targeted because of their potential high limits the number of isolates that can be evaluated. impact. During an outbreak, generally only the index Finally, sequence analysis for either molecular epi- case is fully characterized, although continued mon- demiology or assessment of genetic features requires itoring of viruses isolated during an outbreak is advanced knowledge and skill, which limits its needed to detect any changes in the virus character- application. istics to ensure that other diagnostic tests used are, or remain, sensitive to the outbreak virus. Less com- In Vivo Pathotyping prehensive genetic analysis may be used to evaluate In vivo Pathotyping is used to classify H5 or H7 specifi c genetic features, such as the HA protein subtype AI virus isolates as HP or LP for chickens cleavage site to evaluate virulence for a larger based on the defi nition of the OIE. Briefl y, a virus number of isolates. is classifi ed as HP if it kills 75% or more of eight 4- to 8-week-old susceptible chickens within 10 Sequence Analysis days of inoculation with the test isolate administered The generation of sequence data from AI virus iso- by the intravenous route. If the virus kills fewer lates is important for two reasons. First, the sequence than six chickens (75%), it is considered to be LP. of the HA proteolytic cleavage site of the H5 and Although the in vivo test and genetic analysis do not H7 subtype AI virus HA protein is critical for correlate in every case, the proteolytic cleavage site quickly predicting the pathotype of the virus. One sequence of the HA gene can give an indication of criteria of the World Organization of Animal Health whether a virus is HP or LP. (Offi ce Internationale des Epizooties [OIE]) for Pathotyping in chickens provides important infor- reporting an AI virus as HP notifi able AI is a cleav- mation; however, by the nature of the test, it is age site sequence compatible with previously char- expensive and requires BSL-3Ag animal facilities acterized HPAI viruses (2, 24). Advancements in and trained personnel. automated sequencing technology has made it possible to determine the HA gene proteolytic cleavage ANTIBODY DETECTION site sequence in less than 24 hours. This has impor- Antibody detection is a common and relatively inex- tant implications for developing appropriate control pensive method of surveillance for detecting prior strategies. exposure of poultry fl ocks to AI virus. One of the The second objective of AI virus sequence analy- primary applications of antibody testing is in the sis is to perform molecular epidemiological analysis support of trade to certify fl ocks or poultry products to identify the likely geographic and species origin as free of exposure to AI virus. For this reason, of an AI virus isolate and to determine if the isolate antibody tests are performed on millions of samples is related to previously characterized AI virus iso- yearly in the United States alone. Numerous test lates. As genome sequencing has become relatively formats are used for AI virus antibody detection: less expensive and as more and more AI virus AGID assay, HI assay (previously described), and sequence data have become available in recent years, commercial enzyme-linked immunosorbent assay molecular epidemiology has become more practical. (ELISA). 13 / Avian Influenza Diagnostics and Surveillance Methods 305

Agar Gel Immunodiffusion Assay types for AI virus, although in some cases tissue will The AGID assay previously described for antigen also be collected. Tissues are not optimal for detec- detection is most widely used for AI virus antibody tion of LPAI virus, except for lung. Numerous detection. For antibody detection, AGID has moder- tissues may be collected for HPAI virus, including ate sensitivity and can detect antibody earlier post lung, heart, spleen, and pancreas. The type of sample infection than other antibody detection tests because collected and processing methods are dependent on it detects IgM instead of IgG. Antibody may be numerous interrelated factors such as the purpose of detected as early as 5 days post infection and may testing, type of tests used, and the target species. be detected for many weeks or months post infection First, the purpose of testing is to detect prior expo- (32), although the response and duration of antibody sure to AI virus, antibody testing is optimal, but are affected by both the host and virus strain. The when detection of active infection is the objective, AGID test is suitable for testing serum, plasma, and sampling tissue or organs where the virus replicates egg yolk (6) from any species. Importantly, however, is necessary. Sometimes only certain subtypes, such AGID does not produce consistent results with as H5 and H7, will be the primary target; therefore, serum from some species, such as ducks (26, 31); subtype-specifi c tests may be the primary test used therefore, other tests may be more appropriate for to make a diagnosis. samples from species other than chickens and Second, the test itself dictates the appropriate turkeys. specimen type. It is not uncommon for a single sample to be tested by more than one assay, particu- ELISA Antibody Testing larly when the results of a screening test, such as an Several commercial ELISA kits are available for the antigen detection kit, must be confi rmed by a second, type-specifi c detection of AI virus antibodies in more-sensitive test such as VI. Because VI requires serum, plasma, and egg yolk from chickens. ELISA live virus and the AI virus antigen detection and tests can be more sensitive than AGID tests but may RRT-PCR assays do not, it is important that the cold give false-positive results due to poorer specifi city chain for a specimen be maintained to ensure optimal (31). ELISA-positive tests are routinely confi rmed conditions for all tests. with the AGID test. Third, the target species must be taken into The primary advantage of ELISA is that it is account. The tissue tropism of AI virus is often faster than AGID (results may be available in a few species specifi c, although there are some strain- hours), it is amenable to high-throughput testing, dependent exceptions. Cloacal swab specimens are and interpretation of results is less subjective than more appropriate when testing waterfowl because that for AGID. The current commercially available LPAI virus replication is primarily in the intestinal ELISA kits are species specifi c for chicken and tract (2, 27, 32). However, in chickens, infections turkey specimens, which limits how the test can be tend to be upper respiratory, so tracheal or oropha- applied. These tests do not provide accurate assess- ryngeal swabs are more appropriate. To achieve the ment of sera from ducks, geese, or wild and captive highest level of sensitivity, both types of samples birds. Some laboratories have developed in-house should be collected from poultry (32), but this competitive or blocking ELISA tests that can detect approach also increases the cost of testing. antibody from any species (25, 39); however, these tests are not commercially available. Several com- CONCLUSION mercial companies have developed prototype block- One of the most critical aspects of implementing ing ELISA tests, which should be available in the diagnostic and detection tests for any disease is near future. fi tness-for-purpose. The practical aspects of the test are as important as its analytical performance. A SAMPLE COLLECTION, PROCESSING, given test may have superior sensitivity and specifi c- AND HANDLING ity, like RRT-PCR, but the rapid and portable nature Successful detection of AI virus infection or expo- of antigen detection kits make them ideal for on- sure relies on proper sample collection, processing, farm testing, whereas RRT-PCR must be done in a and handling. Tracheal or oropharyngeal swabs and laboratory. Test goals are important to defi ne as cloacal swabs are the most widely used specimen well; for example, active surveillance will have 306 Avian Influenza different diagnostic needs than virus detection for antibody testing as a method to determine infl u- containment purposes during an outbreak. Finally, enza status in white leghorn hens. Avian Diseases unlike many poultry disease agents, regulatory 47:1196–1199. aspects, such as OIE guidelines, need to be consid- 7. Cattoli, G., A. Drago, S. Maniero, A. Toffan, E. ered when implementing AI virus diagnostics, as Bertoli, S. Fassina, C. Terregino, C. Robbi, G. Vicenzoni, and I. Capua. 2004. Comparison of these may dictate which tests can be used and how three rapid detection systems for type A infl uenza an outbreak is handled. virus on tracheal swabs of experimentally and As technology advances and new AI virus detec- naturally infected birds. Avian Pathology 33:432– tion tests are developed, there is one fi nal con- 437. sideration that must be taken into account regarding 8. Collins, R.A., L.S. Ko, K.Y. Fung, K.Y. Chan, J. the suitability of new tests—validation of perfor- Xing, L.T. Lau, and A.C. Yu. 2003. Rapid and mance data and fi tness-for-purpose. Numerous in- sensitive detection of avian infl uenza virus subtype house tests have been developed and published. H7 using NASBA. Biochemistry Biophysics However, only a few of these tests have been Research Communications 300:507–515. evaluated to the point where they can be considered 9. Collins, R.A., L.S. Ko, K.L. So, T. Ellis, L.T. Lau, to be validated for a particular purpose. Validation and A.C. Yu. 2002. Detection of highly pathogenic and low pathogenic avian infl uenza subtype H5 should be considered for both the type of sample (Eurasian lineage) using NASBA. Journal of Viro- and the species involved. One additional aspect logical Methods 103:213–225. of validation is the ability to transfer the test proce- 10. Collins, R.A., L.S. Ko, K.L. So, T. Ellis, L.T. Lau, dure to other laboratories and to maintain sensitivity and A.C. Yu. 2003. A NASBA method to detect and accuracy. The availability of validated tests high- and low-pathogenicity H5 avian infl uenza is crucial to implementing sensitive and specifi c viruses. Avian Diseases 47:1069–1074. testing that will readily be accepted nationally and 11. Davison, S., A.F. Ziegler, and R.J. Eckroade. 1998. internationally. Comparison of an antigen-capture enzyme immunoassay with virus isolation for avian infl uenza REFERENCES from fi eld samples. Avian Diseases 42:791–795. 1. Akey, B.L. 2003. Low-pathogenicity H7N2 avian 12. Fedorko, D.P., N.A. Nelson, J.M. McAuliffe, and infl uenza outbreak in Virginia during 2002. Avian K. Subbarao. 2006. Performance of rapid tests for Diseases 47:1099–1103. detection of avian infl uenza A virus types H5N1 2. Alexander, D.J. 2005. Avian Infl uenza, In: Manual and H9N2. Journal of Clinical Microbiology of Diagnostic Tests and Vaccines for Terrestrial 44:1596–1597. Animals. World Organization for Animal Health: 13. Fouchier, R.A., T.M. Bestebroer, S. Herfst, L. Van Paris, France. Available at http://www.oie.int/eng/ Der Kemp, G.F. Rimmelzwaan, and A.D. Oster- normes/mmanual/A_00037.htm. Accessed January haus. 2000. Detection of infl uenza A viruses from 15, 2007. different species by PCR amplifi cation of con- 3. Atmar, R.L., B.D. Baxter, E.A. Dominguez, and served sequences in the matrix gene. Journal of L.H. Taber. 1996. Comparison of reverse tran- Clinincal Microbiology 38:4096–4101. scription-PCR with tissue culture and other rapid 14. Lee, C.W., D.A. Senne, and D.L. Suarez. 2006. diagnostic assays for detection of type A infl uenza Development and application of reference antisera virus. Journal of Clinical Microbiology 34:2604– against 15 hemagglutinin subtypes of infl uenza 2606. virus by DNA vaccination of chickens. Clinical 4. Aymard-Henry, M., M.T. Coleman, W.R. Dowdle, and Vaccine Immunology 13:395–402. W.G. Laver, G.C. Schild, and R.G. Webster. 1973. 15. Meulemans, G., M.C. Carlier, M. Gonze, and P. Infl uenza virus neuraminidase and neuraminidase- Petit. 1987. Comparison of hemagglutination- inhibition test procedures. Bulletin of the World inhibition, agar gel precipitin, and enzyme-linked Health Organisation 48:199–202. immunosorbent assay for measuring antibodies 5. Beard, C.W. 1970. Demonstration of type-specifi c against infl uenza viruses in chickens. Avian Dis- infl uenza antibody in mammalian and avian sera eases 31:560–563. by immunodiffusion. Bulletin of the World Health 16. Moore, C., S. Hibbitts, N. Owen, S. Corden, G. Organization 42:779–785. Harrison, J. Fox, C. Gelder, and D. Westmoreland. 6. Beck, J.R., D.E. Swayne, S. Davison, S. Casavant, 2004. Development and evaluation of a real-time and C. Gutierrez. 2003. Validation of egg yolk nucleic acid sequence based amplifi cation assay 13 / Avian Influenza Diagnostics and Surveillance Methods 307

for rapid detection of infl uenza A. Journal of 26. Slemons, R.D., and B.C. Easterday. 1972. Host Medical Virology 74:619–628. response differences among 5 avian species to an 17. Munch, M., L.P. Nielsen, K.J. Handberg, and P.H. influenzavirus-A-turkey-Ontario-7732–66 Jorgensen. 2001. Detection and subtyping (H5 and (Hav5N?). Bulletin of the World Health Organiza- H7) of avian type A infl uenza virus by reverse tion 47:521–555. transcription-PCR and PCR-ELISA. Archives of 27. Slemons, R.D., and D.E. Swayne. 1995. Tissue Virology 146:87–97. tropism and replicative properties of waterfowl- 18. Ng, L.F., I. Barr, T. Nguyen, S.M. Noor, R.S. Tan, origin infl uenza viruses in chickens. Avian Dis- L.V. Agathe, S. Gupta, H. Khalil, T.L. To, S.S. eases 39:521–527. Hassan, and E.C. Ren. 2006. Specifi c detection of 28. Spackman, E., D.A. Senne, T.J. Myers, L.L. H5N1 avian infl uenza A virus in fi eld specimens Bulaga, L.P. Garber, M.L. Perdue, K. Lohman, by a one-step RT-PCR assay. BMC Infectious L.T. Daum, and D.L. Suarez. 2002. Development Diseases 6:40. of a real-time reverse transcriptase PCR assay for 19. Payungporn, S., S. Chutinimitkul, A. Chaisingh, S. type A infl uenza virus and the avian H5 and H7 Damrongwantanapok, C. Buranathai, A. Amonsin, hemagglutinin subtypes. Journal of Clinical Micro- A. Theamboonlers, and Y. Poovorawan. 2006. biology 40:3256–3260. Single step multiplex real-time RT-PCR for H5N1 29. Starick, E., A. Romer-Oberdorfer, and O. Werner. infl uenza A virus detection. Journal of Virological 2000. Type- and subtype-specifi c RT-PCR assays Methods 131:143–147. for avian infl uenza A viruses (AI virus). Journal 20. Payungporn, S., P. Phakdeewirot, S. Chutinimit- of Veterinary Medicine B Infectious Diseases in kul, A. Theamboonlers, J. Keawcharoen, K. Veterinary and Public Health 47:295–301. Oraveerakul, A. Amonsin, and Y. Poovorawan. 30. Suarez, D.L., E. Spackman, D.A. Senne, L. Bulaga, 2004. Single-step multiplex reverse transcription- A.C. Welsch, and K. Froberg. 2003. The effect of polymerase chain reaction (RT-PCR) for infl uenza various disinfectants on detection of avian infl u- A virus subtype H5N1 detection. Viral Immunol- enza virus by real time RT-PCR. Avian Diseases ogy 17:588–593. 47:1091–1095. 21. Poddar, S.K. 2002. Infl uenza virus types and sub- 31. Swayne, D.E., and D.A. Halvorson. 2003. Infl u- types detection by single step single tube multiplex enza. In: Y.M. Saif, H.J. Barnes, J.R. Glisson, A. reverse transcription-polymerase chain reaction M. Fadly, L.R. McDougald, and D.E. Swayne (RT-PCR) and agarose gel electrophoresis. Journal (eds.). Diseases of Poultry, 11th ed. Iowa State of Virological Methods 99:63–70. Press: Ames, IA, pp. 135–160. 22. Pregliasco, F., C. Mensi, L. Camorali, and G. 32. Swayne, D.E., D.A. Senne, and C.W. Beard. 1998. Anselmi. 1998. Comparison of RT-PCR with Avian infl uenza. In: D.E. Swayne, J.R. Glisson, other diagnostic assays for rapid detection of M.W. Jackwood, J.E. Pearson, and W.M. Reed infl uenza viruses. Journal of Medical Virology (eds.). A Laboratory Manual for the Isolation and 56:168–173. Identifi cation of Avian Pathogens, 4th ed. Ameri- 23. Schulman, J.L., and E.D. Kilbourne. 1969. Inde- can Association of Avian Pathologists: Kennett pendent variation in nature of hemagglutinin and Square, PA, pp. 150–155. neuraminidase antigens of infl uenza virus: distinc- 33. Takao, S., Y. Shimazu, S. Fukuda, M. Kuwayama, tiveness of hemagglutinin antigen of Hong Kong- and K. Miyazaki. 2002. Neuraminidase subtyping 68 virus. Proceedings of the National Academy of of human infl uenza A viruses by RT-PCR and its Sciences U S A 63:326–333. application to clinical isolates. Japanese Journal of 24. Senne, D.A., B. Panigrahy, Y. Kawaoka, J. E. Infectious Disease 55:204–205. Pearson, J. Suss, M. Lipkind, H. Kida, and R.G. 34. Thayer, S.G., and C.W. Beard. 1998. Serologic Webster. 1996. Survey of the hemagglutinin (HA) Procedures. In: D.E. Swayne, J.R. Glisson, M.W. cleavage site sequence of H5 and H7 avian infl u- Jackwood, J.E. Pearson, and W.M. Reed (eds.). A enza viruses: amino acid sequence at the HA cleav- Laboratory Manual for the Isolation and Identifi ca- age site as a marker of pathogenicity potential. tion of Avian Pathogens, 4th ed. American Asso- Avian Diseases 40:425–437. ciation of Avian Pathologists: Kennett Square, PA, 25. Shafer, A.L., J.B. Katz, and K.A. Eernisse. 1998. pp. 255–266. Development and validation of a competitive 35. Webster, R.G., and C.H. Campbell. 1972. An inhi- enzyme-linked immunosorbent assay for detection bition test for identifying the neuraminidase antigen of type A infl uenza antibodies in avian sera. Avian on infl uenza viruses. Avian Diseases 16:1057– Diseases 42:28–34. 1066. 308 Avian Influenza

36. Wei, H.L., G.R. Bai, A.S. Mweene, Y.C. Zhou, tion of infl uenza virus type A: evaluation of Y.L. Cong, J. Pu, S. Wang, H. Kida, and J.H. Liu. their use with poultry. Avian Diseases 49:477– 2006. Rapid detection of avian infl uenza virus a 481. and subtype H5N1 by single step multiplex reverse 39. Zhou, E.M., M. Chan, R.A. Heckert, J. Riva, and transcription-polymerase chain reaction. Virus M.F. Cantin. 1998. Evaluation of a competitive Genes 32:261–267. ELISA for detection of antibodies against avian 37. Wiedbrauk, D.L., and J. Stoerker. 1995. Quality infl uenza virus nucleoprotein. Avian Diseases Assurance in the Molecular Virology Laboratory. 42:517–522. In: D.L. Wiedbrauk and D.H. Farkas (eds.). Molec- 40. Ziegler, A.F., S. Davdison, H. Acland, and R.J. ular Methods for Virus Detection. Academic Press: Eckroade. 1999. Characterization of H7N2 (non- San Diego, CA, pp. 25–38. pathogenic) avian infl uenza virus infections in 38. Woolcock, P.R., and C.J. Cardona. 2005. commercial layer, in Pennsylvania, 1997–98. Commercial immunoassay kits for the detec- Avian Diseases 43:142–149. 14 Mass Depopulation as an Effective Measure for Disease Control Purposes

Elizabeth A. Krushinskie, Martin Smeltzer, Patrice Klein, and Harm Kiezebrink

INTRODUCTION control zone. Use of vaccination is an option in both Avian infl uenza (AI) continues to be an important HPAI and H5/H7 LPAI outbreaks as a means to disease threat affecting the poultry industry world- contain the spread of viral infection. In H5/H7 LPAI wide because of its recent implications to human outbreaks, vaccination may be used, in particular, health risk, the potential economic and international for long-lived birds and those of high genetic value trade impacts, and the animal welfare consequences to sustain the productive life of the fl ock. This to the poultry industry. The AI virus is known to chapter, however, focuses on the use of mass depop- circulate in wild bird populations, especially wild ulation as the most rapid measure to minimize the waterfowl and shorebirds, and presents a natural spread of NAI and other highly contagious diseases reservoir for possible transmission to and infection of domesticated birds. of domestic poultry fl ocks with exposure to wild Several national and international animal health birds. Thus, it is essential to maintain strict on-farm organizations have developed world-recognized and biosecurity practices to prevent the spread of AI accepted authoritative reports on euthanasia of disease from wild birds to domestic poultry. Should animals. These include the World Organization of either high pathogenicity AI (HPAI) or low patho- Animal Health (Offi ce Internationale des Epizooties genicity AI (LPAI) infections of the H5 or H7 [OIE])’s Guidelines for the Killing of Animals for subtype occur in domestic fl ocks, defi ned by the Disease Control Purposes (Article 3.7.6.1), the World Organization of Animal Health (OIE) as noti- American Veterinary Medical Association’s 2000 fi able avian infl uenza (NAI), further spread of the Report of the American Veterinary Medical Asso- NAI virus must be prevented. This may be accom- ciation (AVMA) Panel on Euthanasia, the European plished by common response practices of (1) estab- Council Directive (1993), the European Food Safety lishing a control zone around the index case Authority (ESFA) Scientifi c Panel on Animal Health (premises) upon notifi cation of an NAI infection, (2) and Welfare (2004), and the AustVetPlan’s Opera- implementing quarantine and stop movement restric- tional Procedures Manual for the Destruction of tions within the NAI control zone, (3) making deci- Animals (1, 2, 6, 15, 17). The authors of this chapter sions to depopulate the index case (premises) and acknowledge and concur with the general principles any epidemiologically linked premises within 48 to and euthanasia methods described in the aforemen- 72 hours of identifi cation to include preemptive tioned references and encourage general adoption of slaughter of presumptive positive birds in the region, these universally accepted approaches. and/or (4) making decisions to vaccinate the infected It is important to distinguish between euthanasia fl ock and any non–NAI-infected birds within the and mass depopulation. Euthanasia (“good death”)

Avian Influenza Edited by David E. Swayne 309 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 310 Avian Influenza is the transitioning of an animal to death as pain- Destruction of domestic species of animals lessly and stress-free as possible, giving all possible infected with certain highly contagious diseases has consideration to its experience. Mass depopulation been an accepted tool in disease eradication efforts is a method by which large numbers of animals must worldwide. Many synonyms have been used to be destroyed quickly and effi ciently with as much describe this process such as depopulation, euthana- consideration given to the welfare of the animals as sia, culling, or stamping out. In farm production practicable, but where the circumstances and tasks systems, the removal of animals with limited eco- facing those doing the depopulation are understood nomic potential or hopes of recovery from illness or to be extenuating (18). injury is an integral component of modern animal Principally, the mass depopulation method chosen husbandry. The destruction of entire herds of cattle must be safe, humane, and effi cient. Specifi cally, with foot and mouth disease (FMD), swine with there are factors of critical importance that must be classic swine fever (CSF), or poultry with exotic considered. Foremost is human health and safety, Newcastle disease (END) or HPAI is not a part of which includes emotional and physical worker normal production practices but is consistent with fatigue, worker morale, non–English- or foreign international “stamping-out” policies for countries language–speaking worker communication, and or areas seeking to become or maintain disease free personal protective equipment (PPE) for worker bio- status. This control strategy removes the reservoir of safety and biosecurity (9, 17). Of particular interest disease at its source by eliminating the virus load in is the development of specifi c worker protection rec- affected animals and the local premises, reducing ommendations and guidance for those workers the likelihood of spread to other nearby or contact entering the highly contaminated environment to farms, and decreasing the possibility of transmission accomplish these labor-intensive and often physi- by fomites or other indirect contact. cally demanding procedures (4, 11). To ensure worker Similarly, the destruction of large, non-infected, safety and minimize distress to the animals, all per- populations of animals has been used to address sonnel involved in the depopulation process should catastrophic situations such as fl oods or indivi- have relevant training, skill, and competencies. dual farm damage from storms when movement to Furthermore, the plan for depopulation should be alternative production facilities is not available or site specifi c to the individual farm or premises and not feasible. Mass depopulation also has been used should address the species, number, age, and size of to prevent the introduction of meat into the human the birds; the housing type; the availability of equip- food chain from a herd or fl ock of animals that ment and facilities; the cost; and animal welfare has been contaminated with chemical or pesticide concerns to include minimized handling of the birds residues. to reduce stress, irreversibility of the method, rapid Once the decision to depopulate a fl ock has been loss of consciousness, rapid death, continuous mon- made, the specifi c method selected becomes sce- itoring of the procedure to ensure effi cacy and nario and time driven. The methods used primarily humaneness, and maintenance of biosecurity (2, 6, will be based upon the number and type of birds to 7, 17). be destroyed as well as the specifi c reason for the A well-planned depopulation operation will opti- depopulation. Nearly all methods currently described mize each of these critical factors. An assessment of for euthanasia of birds and poultry were developed the risk to the affected birds of distress, injury, and for individual or small numbers of birds as compan- prolonged suffering during the culling process and ion animals or in research settings as part of Institu- risk to human safety compared to the benefi t obtained tional Animal Care and Use Committee (IACUC) by culling for disease eradication to protect suscep- requirements. The diffi culty faced by veterinarians tible, healthy birds and people should be completed and others charged with directing the depopulation to determine the consequences of the measures of large numbers of birds at the farm level is in the undertaken. Mass depopulation for disease control attempt to extrapolate these individual animal guide- purposes can result in large-scale improvement in lines into workable, economical, and acceptable overall bird welfare because other fl ocks of at-risk methods for large scale emergency situations, gener- birds do not become infected and do not develop the ally in a short amount of time and under much scru- disease. tiny. This chapter will attempt to give those 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 311 individuals with the responsibility of directing the birds by fatal depression of the CNS respiratory on-farm depopulation activities during emergency center. Examples include halothane, isofl urane, and responses reasonable guidance and practical appli- sevofl urane. However, not all are reliable to guaran- cation of acceptable methods successfully used in tee an irreversible death in birds. The newer inhalant the past. In this chapter, the principal mechanisms anesthetics such as isofl urane and sevofl urane have for causing death are explained; recognized and a wide margin of safety, even in birds, and may not accepted methods of mass depopulation including have a lethal effect despite the highest concentration newer, conditional methods, are described; practical used. Some inhalant anesthetics (halothane and experiences to guide in the selection of the most methoxyfl urane), which were used in high concen- appropriate method are summarized; and concerns trations to euthanize birds in the past, are no longer about animal welfare are discussed. marketed in the United States and therefore, are unavailable. Inhalant gaseous anesthetics are costly MECHANISMS FOR CAUSING DEATH and more appropriately used in controlled institu- The three principal mechanisms for causing death of tional or clinical settings rather than performed at a animals are as follows: farm site. The use of compressed carbon monoxide (CO) gas in cylinders is not recommended for fi eld 1. Hypoxia—resulting from methods that produce use due to human safety issues and the diffi culty in low oxygen levels causing unconsciousness and obtaining suffi cient quantities of gas. Nitrogen and depression of the respiratory center in the central argon are available in compressed gas cylinders and nervous system (CNS), followed by complete have been used in some commercial poultry slaugh-

loss of brain function and death. terhouses alone or in combination with CO2, oxygen

2. Depression of the neurons in the CNS—resulting (O2), or air (10, 19). These agents cause death by from methods that cause direct depression of the hypoxia, but only are effective when oxygen levels CNS followed by respiratory and cardiac arrest in the sealed chamber are reduced rapidly to less and death. than 2.0% (6). Availability and cost of nitrogen and 3. Physical disruption of the brain—resulting from argon gases also are limiting factors. A physical methods that cause direct, extensive physical method such as cervical dislocation is an acceptable injury or destruction of brain tissue leading to method when properly conducted by trained and cessation of brain function and death (1, 15, 17). skilled persons. However, its use is limited because it is very labor intensive and, therefore, reserved ACCEPTED METHODS OF EUTHANASIA primarily for use in small sized birds (less than OF POULTRY 1.0 kg [2.2 lb]) and for relatively small numbers of Euthanasia is the act of inducing humane death in birds. Electrocution is used in many poultry slaugh- an animal with minimal pain and distress (1, 17). terhouses by water bath stunning systems to stun Current acceptable methods for euthanasia of poultry birds prior to severing the major blood vessels in as described by the OIE, AVMA, and other interna- their necks leading to death by exsanguination. To tionally recognized authorities are either chemical be used as the sole method of euthanasia or mass methods such as lethal injection of barbiturates, depopulation, suffi cient and constant electrical inhalant gaseous anesthetics, carbon dioxide (CO2) current must be ensured to cause instant, simultane- gas, or carbon monoxide gas or physical methods ous destruction of the CNS, cardiac arrest, and death. such as cervical dislocation and electrocution. These This method may be hazardous to personnel so the methods generally are designed to be applied to indi- equipment and technique must be assessed for vidual or small numbers of birds. Barbiturate injec- human health and safety (2, 6, 7, 17). tions typically are limited to use in individual or pet Because of the various limiting factors associated bird euthanasia because this method requires appli- with these aforementioned methods of euthanasia, cation by intravenous injection by a licensed veteri- CO2 gas introduced into some type of chamber has narian and uses legally controlled substances. become a commonly recommended and accepted Inhalant gaseous anesthetics can rapidly induce method of euthanasia and mass depopulation (1, 2, anesthesia and unconsciousness, and when applied 6, 9, 14, 16, 17). This decision is based upon relative in overdose levels, generally will effectively kill ease of obtaining CO2 in needed quantities, wide 312 Avian Influenza worker safety factors, humaneness of the technique Second, the method must be practical, economi- for the animals, and the relatively inexpensive cost. cal, and capable of being implemented and com- Electrocution using specialized equipment similar to pleted as rapidly as possible in the shortest time that used in processing plants also may be an option frame possible. It is preferable to select depopula- for poultry facilities, such as caged layer operations, tion methods that can be conducted inside the poultry where individual birds must be handled (removed house to reduce airborne transmission of contami- from cages) prior to euthanasia. nated feathers or dust and dirt particles. Other Thus, the preferred methods for mass depopula- important factors to consider are the type of birds tion of poultry fl ocks have been the use of either and the husbandry style such as free-range or compressed CO2 gas in some type of chamber, elec- enclosed housing, fl oor-reared, pen-reared, or con- trocution, or cervical dislocation based, in part, on fi ned in cages. The facility’s condition, age, and reasons of practicality, worker safety, and availabil- physical structures such as post placement will also ity of needed materials and equipment. These dictate the practicality of the actual method chosen. methods are discussed in more detail later in the The number of birds to be depopulated will dictate chapter. However, recent reports on the risks of the most reasonable method. A small backyard fl ock exposure to poultry workers of potential zoonotic of less than 100 birds, a multihouse broiler farm with diseases and other safety hazards have raised con- a 200,000-bird capacity, or a table egg complex with cerns for the use of methods that require intimate 1 million or more animals will require different bird handling or involve spillage of body fl uids such equipment, supplies, and number of workers and as blood. Such methods that may pose a greater risk will directly affect the choice of depopulation to the handler are considered to be less desirable. method. Thus, for reasons of human health and safety, newer In a commercial setting, facility design may limit mass depopulation methods are being evaluated. the actual types of euthanasia chambers that can be One of these is the use of water-based foam as mod- constructed and used in a house. The ability of a ifi ed from its original use as a fi refi ghting agent to house to be sealed for whole house CO2 gassing, for suppress fi res. This method minimizes both the example, will be infl uenced by how air-tight the number of workers needed and the direct handling house is, the type of ventilation system present, and of the birds. The conditional use of water-based its physical overall condition. Certain building foam for mass depopulation of domestic poultry is designs such as the caged layer facility not only discussed in more detail later in the chapter but is limit, but effectively determine, the method used. mentioned here relative to identifi cation of methods Caged layer facilities require birds to be individually to address increasing concerns for worker safety. removed from the cages, prior to euthanasia, whether

the bird is placed in a CO2 cart by the cage, taken to

MASS DEPOPULATION METHOD a CO2 chamber at the end of or outside of the house, SELECTION CRITERIA or put on an electrocution line. In commercial poultry production, the number, age, Bird age and size also will affect the method size, and species of birds to be culled, the type of chosen. Depopulating a fl ock of 2-week-old broilers production facility, and the number of available is much less labor intensive than depopulating the laborers will determine the method used. The most same fl ock when it is 8 weeks old. Similarly, a fl ock important criterion for selecting the depopulation of young turkey poults will need to be handled much method is to attempt to minimize the amount of bird differently than a 23-week-old fl ock of turkey handling and bird movement to minimize any dis- toms. tress or possible injury to the birds and any adverse The type/species of bird also infl uences the human safety and animal welfare consequences. method chosen due to inherent physical characteris- Furthermore, although local perceptions may vary tics of different kinds of poultry such meat-type on what may be considered to be acceptable depop- chickens, leghorn-type birds, turkeys, or ducks. This ulation methods, it is paramount that the methods affects the ability to catch, drive, or move each type selected should be recognized as acceptable by the of the bird within the facility they are housed in and OIE, AVMA, and other international and national may require individual bird handling. established authorities on animal euthanasia and Availability of labor, equipment, supplies, water, animal welfare guidance. and/or electricity also will dictate the type of depop- 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 313 ulation method that can be done quickly and effi - up. Experience has shown that most valves will ciently. In addition, because depopulation and freeze up after approximately 7 to 8 minutes. disposal often are tightly linked and coordinated, the Optimum delivery of the calculated volume should speed at which the resulting carcasses can be be delivered into the chamber to displace 20% of the removed from the facility or premises, if needed, will chamber volume per minute so that CO2 has replaced affect the speed at which depopulation can occur. the ambient air within the chamber in 5 to 10 minutes. Therefore, a signifi cant amount of excess

CARBON DIOXIDE USE IN liquid CO2 is necessary to ensure the delivery of

MASS DEPOPULATION adequate volumes of CO2 gas. Carbon dioxide is a colorless, odorless gas that is Typically, birds are moved or placed into some 50% heavier than air with a molecular weight of type of chamber or container, the container sealed

44.01. Therefore, when released to the environment, or closed, and large amounts of CO2 gas are intro-

CO2 will settle to the ground and displace the sur- duced rapidly to reach a targeted level of 70%. The rounding air. CO2 also is nonﬂ ammable and nonex- inhalation of CO2 gas initially produces an irritating plosive with a relatively low safety hazard for effect in the conscious animal due to the formation personnel (1, 9). Liquid CO2 is commercially sold of carbonic acid on mucous membranes in the eyes, by weight and is available in most localities. It is nasal and oral cavities. The noxious sensation elicits available in amounts of 20 lb (9 kg), 50 lb (23 kg), sneezing, coughing, and agitation (1, 9, 10, 19). and 387 lb (175 kg) in pressurized metal cylinders. Once the CO2 blood levels become elevated in the Larger amounts in the thousands of pounds are bird, usually within 1 to 2 minutes, a secondary available for delivery by tanker trucks. Frozen CO2 graduating anesthetic state is produced. Deep anes- is available as dry ice. This can be purchased either thesia is followed by death from cardiac and respira- in a pellet or as shaved or crushed ice. tory arrest. Death is achieved at CO2 concentrations

The physical properties of liquid CO2 are such of 70% of the container’s volume. This percentage that one pound of liquid CO2 will produce a volume compensates, in part, for the fact that most chambers 3 of 8.75 ft of CO2 gas at 70º F (21.1º C) and at 1 are not 100% air-tight and there will be CO2 leakage, atmosphere of pressure (7). CO2 concentrations of some air inﬁ ltration, and variability in the amount

50% to 55% for euthanasia of adult chickens have of CO2 actually delivered. This provides a wide been recommended (13, 14, 16). Slightly higher margin of error to assure the levels delivered into concentrations at 65% CO2 are needed for loss of the chamber are sufﬁ cient for the desired lethal pain perception and brain function in turkeys (12). effect to be achieved.

To reliably ensure death in all birds exposed to CO2 If CO2 gas cylinders are not available, dry ice gas, an estimated ﬁ nal concentration of 70% CO2 in (frozen CO2) may be used as an alternate method to the chamber or enclosure is recommended at an deliver CO2 gas in lethal concentrations by sublima- optimum ﬂ ow rate that will displace 20% of the tion of the dry ice. The dry ice is placed in the chamber volume per min (2, 7, 9). Appendix 14.1 bottom of a deep container and is physically sepa- provides guidance on calculating the amount of CO2 rated from direct contact by the birds. The addition needed for euthanasia. of a small volume of warm water will accelerate the

Liquid CO2 is held under extremely high pressure sublimation process and result in high levels of CO2 in the cylinders, up to 1200 PSI. When the cylinders within minutes. Direct contact with dry ice will are opened and large amounts of CO2 are released cause painful thermal burns to the skin. The birds rapidly, the pressure within the tank will fall. When remain in the container until unconsciousness and the pressure falls below 70 PSI in the storage tank, death are achieved (2, 8). the liquid CO2 begins to solidify before turning to the gaseous state. The problem most commonly PRINCIPLES OF ELECTROCUTION associated with rapid release of CO2 gas from cyl- Electrocution is used in many poultry slaughter- inders is freezing of the cylinder valves which can houses to stun birds in a water bath stunning system prevent the complete release of all the contents. It is prior to severing major blood vessels in the neck important that an adequate volume of liquid CO2 is resulting in death by exsanguination. provided to deliver the desired quantities of CO2 gas Shackled poultry are conveyed through an electri- in suffi cient time before the cylinders’ valves freeze fi ed water bath stunner with their heads fully 314 Avian Influenza immersed. When suffi cient current is applied, the proper preplanning and pretraining of culling per- poultry will be stunned and killed. Required sonnel. minimum currents to dry birds are quail (100 mA/ Some types of birds, such as ducks, turkeys, and bird), chickens (160 mA/bird), ducks and geese broiler breeders, can be walked or driven into the (200 mA/bird), and turkeys (250 mA/bird). Higher euthanasia chambers. Others, such as broilers, must currents are required for wetted birds (6, 7, 17). be individually caught and hand carried. Birds It is important to specify that the electric stunner that can be effectively migrated should be moved in should generate a low-frequency current (30 to at least three or four separate waves to allow birds 60 Hz) sinusoidal waveform applied for a minimum to move and migrate as desired without becoming of 3 seconds and that the currents specifi ed are stressed, sitting down, running back around the han- related to this frequency and waveform (17). It has dlers, or requiring the use of force to push them been shown that as frequency increases, the inci- along the desired route. Additionally, it is necessary dence of ventricular fi brillation decreases. Many to move birds slowly and quietly to prevent over- modern water bath electrical stunners use a high- crowding of those in front and the excessive frequency waveform to reduce carcass damage agitation of fl ighty birds with subsequent piling, (broken bones, red wingtips, breast hemorrhages, crowding, and overheating. The use of exper- etc.). There also are many electrical stunners in the ienced poultry workers in a calm and systematic United States that use a pulsed direct current (DC) fashion will minimize these adverse animal welfare waveform that is unlikely to induce ventricular consequences. fi brillation. Voltage levels must be between 220 and 400 V alternating current (AC) and applied for 10 WORKER SAFETY AND HEALTH ISSUES seconds to ensure lethal effect (Jules Sperry, per- In the past, few concerns were raised over the poten- sonal communication). tial zoonotic risks of certain strains of AI viruses. During the HPAI outbreak in the Netherlands Recently, however, public health experts at the (2003), specially designed electrocution equipment Centers for Disease Control and Prevention (CDC) was developed. This equipment was effi cient and have recommended that any H5 and H7 AI subtype effective for depopulating 2,500 to 10,000 birds per viruses, of both HP and LP, has the potential to be hour. These are mobile units suitable for use for a zoonotic pathogen (4). This characterization has large numbers of birds of different species and sizes dramatically elevated the concerns related to worker and may be powered in rural areas by generators. safety when depopulating large number of AI- infected birds is involved and has greatly infl uenced ANIMAL WELFARE ISSUES who can and should be permitted to accomplish this

The use of CO2 is an internationally accepted method task (4, 11). for euthanasia of individual small mammals and At a minimum, worker safety programs particu- birds except diving animals and neonates that are larly targeted at reducing respiratory and ocular known to be hypoxia tolerant (1, 17). Carbon dioxide exposure should be emphasized. This usually entails also is considered to be acceptable for mass depop- the use of PPE that provides effective protection of ulation of domestic poultry. Uniform delivery of the wearer from exposure to disease agents and also suffi cient quantities in a timely manner addresses prevents the contamination of underlying clothing most of the concerns related to humane killing of and shoes, which could contribute to the spread of large numbers of birds. Animal welfare issues such disease from one premise to another. PPE typically as increased bird stress, piling, excitement, and consist of some type of impervious coveralls, head overheating are of concern, particularly with fl oor cover, face mask, goggles, gloves, and boots birds, and are primarily related to attempting to designed to prevent exposure of the wearer to the euthanize large numbers of birds too rapidly, in too disease agent. There are various models and manu- small of an enclosure, or with insuffi ciently trained facturers of this equipment, but all must meet personnel resulting in ineffective bird movement, minimal safety requirements and be tolerable by improper equipment placement, or packing birds too the wearer. Decreased mobility, the inadequate tightly or penning them up too long before euthana- removal of body heat, and the need to maintain sia procedures begin. These can be addressed by the protective barrier while accomplishing normal 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 315 body functions make using PPE very problematic should be available for staff to change into and out during the depopulation exercise. Current guidance of PPE before proceeding to and from the infected on the appropriate use of PPE must be obtained from premises. Only essential staff and equipment are public health offi cials prior to attempting a mass permitted access to the infected premises for the depopulation effort involving zoonotic organisms depopulation process. (4, 11). It is currently recommended by public health offi - Emergencies cials that workers assigned culling tasks involving A procedure must be established to address any AI-infected fl ocks be vaccinated with the current emergencies that could occur during the depopula- seasonal human infl uenza A strains, preferably at tion process to include fi res, injuries, and other least 7 days in advance of the culling operation, to medical emergencies on site. Ambulances and emer- reduce the chance of infl uenza virus recombination gency medical personnel will be permitted immedi- in vivo. In addition, follow up monitoring of workers’ ate access to the site and should take all necessary health should be done for at least 7 days post- precautions to protect themselves from contact with culling. infected animals and associated materials. Every Therapeutic agents, such as oseltamivir (Tamifl u; effort should be made to disinfect any equipment Hoffmann-La Roche Inc., Nutley, NJ, USA), may and personnel leaving the site without hindering also be recommended for prophylactic treatment of the emergency medical care and response. Local workers potentially exposed to zoonotic viruses (4, hospital authorities should be informed of the desti- 11). nation of the ambulance and to determine the risk and need for appropriate disinfection that may be STANDARD OPERATIONAL PROCEDURES required of the medical personnel and equipment FOR MASS DEPOPULATION OF POULTRY upon arrival. Fire service vehicles and offi cers should be General Principles allowed immediate access to the site. Any vehicles, Standard operational procedures (SOPs) for the equipment, clothing, and personnel who have entered depopulation of poultry should be established prior the site must be cleaned and disinfected before to any response. departure. A register of personnel attending the inci- SOPs should address: dent should be maintained and appropriate health protection measures taken. Fire service orders for 1. Appropriate and adequate training of personnel incidents on infected premises should be discussed to perform the depopulation method in advance with the appropriate authorities. 2. Availability and maintenance of equipment and PPE MASS DEPOPULATION METHODS FOR 3. Availability and health status of personnel DIFFERENT TYPES OF POULTRY 4. Staging areas for depopulation equipment and MANAGEMENT PRACTICES personnel 5. Contingency plans for medical emergencies Floor-Reared Poultry in Enclosed Housing If the birds to be depopulated are fl oor-reared birds A designated staging area on the perimeter of the in enclosed housing, the choice of depopulation infected premises should be established to allow methods may include: controlled access for the depopulation equipment and personnel. There should be adequate space for 1. Carbon dioxide compressed gas using: vehicle parking, equipment delivery, PPE applica- a. Free standing panel enclosure within a tion, rest area, hand washing and toilet facilities, and confi ned area of the poultry house that is facilities for decontamination of personnel, equip- covered and sealed with plastic or tarps ment, and vehicles prior to leaving the premises. b. Plastic tent-type wrap that envelopes the Appropriate cleaning and disinfecting facilities for birds under plastic on the fl oor equipment and personnel should be set up at the c. The entire house or isolated section sealed to entrance/exit of the staging area. Suitable facilities conduct “whole house” CO2 gassing 316 Avian Influenza

d. Live haul cages placed inside a CO2 The height of the panels should be sufﬁ cient so euthanasia chamber or “containerized that birds will not be able to escape and there is gassing unit” enough head room to ensure an adequate volume of 2. Water-based foam application covering the birds gas can be added to achieve euthanasia, typically 4 ft (conditionally accepted procedure) (1.2 m) in height. The size of the enclosure is directly related to the number and size of the birds to be

With all fl oor methods that use CO2 and do not euthanized (see Appendix 14.1). involve the catching of the birds, the birds should be Panels may be constructed in any confi guration of moved to a smaller designated area and confi ned to width and length as long as the CO2 gas can be an appropriate stocking density for the type, age, and delivered quickly and uniformly. Experience has size of the birds. Stocking densities of 34 to 38 kg/m2 shown that widths greater than 16 ft (5.0 m) and (6.8 to 7.6 lb/ft2) are used in the United Kingdom for lengths greater than 75 ft (23 m) can become cum- slaughter weight broilers, for example. Stocking bersome and diffi cult to manage. The size of the densities for other bird types should be known by internal volume of the enclosure will determine the the poultry farmer. With such high stocking densi- amount of CO2 gas needed. Once the panels are ties, it is essential that proper preplanning, preposi- placed, extra litter from the fl oor is piled against the tioning, and worker instruction be done ahead of inside and outside of the panels to seal the enclosure time to allow the euthanasia process to begin imme- at the fl oor level. One or more panels at the short diately, proceed quickly, and to prevent unnecessary end(s) should be left open allowing birds to be overheating, piling, and suffocation of the birds. directed inside the enclosure before it is closed and

Introduction of CO2 gas should begin immediately sealed. Proper placement of the enclosed chamber after the birds are placed inside the prepared enclo- and entry openings will greatly assist in the efﬁ cient sure and it is closed or sealed. Optimal delivery of movement of birds into the enclosure (Fig. 14.1).

CO2 should result in rapid and uniform gas distribu- A ceiling can be constructed of plastic by drawing tion within the enclosure and to achieve rapid eutha- either clear or black plastic sheeting over the top of nasia of the birds. Cessation of most bird movement the paneled enclosure effectively sealing it. This should be achieved within 5 minutes and all move- may be constructed from plastic polyethylene sheet- ment should stop within 10 minutes. It is recom- ing of 5- to 6-mil thickness. Clear or opaque plastic mended to maintain a sealed enclosure for an is advantageous because it will permit viewing additional 20 minutes before the seal is removed to inside the enclosure to assess the condition of the ensure all the birds are dead (2, 9). A properly con- birds during the euthanasia process. The plastic ducted CO2 gas euthanasia procedure should dem- sheeting should be prepared outside the poultry onstrate uniform distribution of the bird carcasses house, if weather conditions permit, to facilitate over the fl oor area in a single layer and not crowded measuring and cutting. The plastic is cut to the or piled in the corners of the enclosure or house. length and width of the paneled enclosure plus a minimum additional 2 ft (0.6 m) for each side and Co2 Compressed Gas Using Specifi c Floor end. This will provide area for placement of pulling Method Procedures and weighting devices or ropes to position the plastic and hold it in place. Free standing panel enclosure Once the plastic is cut to the needed length and With this method, a solid-walled frame is con- width, it should be opened and refolded in an accor- structed and plastic sheeting is drawn over the top dion-style folding of pleats approximately 4 ft to seal the chamber. Metal or plywood side and end (1.2 m) wide. The plastic should be folded in such a panels are fashioned into a square or rectangular manner that it can be secured along the longest side shaped enclosure on the house fl oor. The panel (length) of the paneling and pulled over the shortest enclosure should be constructed in a level area inside side (width) once the birds are inside. This will the house. Suffi cient space on each side wall facilitate the unfolding and pulling of the plastic (approximately 4 ft or 1.2 m) should be left to permit over the top of the enclosure. Once this initial folding adequate work space and access to all sides of the pattern is completed, the plastic may be additionally chamber. folded or rolled, lengthwise, to facilitate bringing it 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 317

Figure 14.1. Free-standing panel enclosures for use with compressed gas fl oor method. (A) Erection of fl oor panels Source: M. Smeltzer, USDA. (B) Birds confi ned within panel enclosure Source: M. Smeltzer, USDA.

into the house. The plastic sheeting should be placed door and working his way forward. All persons on one long side of the panels. One end of the plastic should leave the building and the house fans should should be secured to the side of the panels to keep be turned off. The CO2 gas tanks generally will fl ow it from being pulled over too far as the opposite side for 8 to 10 minutes before either emptying or freez- is drawn or pulled across the width of the panels to ing up. Most bird sounds and activity will cease seal the enclosure. If the enclosed chamber is over within 5 minutes. All sounds and movement should 30 ft (9 m) in length, a rope should be placed midway cease within 10 minutes. Wait an additional 20 or every 30 ft (9 m) to assist in drawing the middle minutes (total of 30 minutes) before designated per- areas of the plastic across the paneled enclosure and sonnel enter the house to turn off the tanks valves, preventing the plastic “ceiling” from collapsing into remove the plastic ceiling to one side, and confi rm the enclosure at bird head level. that all birds have been euthanized. Carbon dioxide cylinders should be distributed Subsequently, the house fans should be turned on evenly inside the perimeter of the enclosed chamber to ventilate the building interior for approximately and secured to the inside of the panels. Once the CO2 20 to 30 minutes. Once the Safety Offi cer declares cylinders are secured and the plastic is in place on the house is safe to enter, the workers may proceed one lengthwise side of the panels, the birds may be to dismantle the paneled enclosure. They will detach moved into the enclosure. Birds are driven into the and remove the plastic ceiling, remove the empty enclosed chamber to a stocking density appropriate CO2 tanks, and break down the panels to allow for to the type, age, and size of the birds, resulting in a the disposal of the carcasses to proceed by the closely packed, uniform layer of birds without appropriate method selected for this site. piling. Once the birds are in the enclosed chamber, the Plastic tenting plastic sheeting is unfolded, drawn over the top of This method can be used in broiler houses con- the panels, and attached to the opposite side to create structed without fl oor posts or other immovable a ceiling over the enclosed chamber using one fl oor obstructions and in breeder houses with central person on each corner and at each “throw” rope. scratch areas. Equipment such as feeders and water- Once the enclosure is sealed, the euthanasia process lines must be raised so as to not obstruct the process. should begin immediately. The person(s) responsi- The birds should be herded into an area limited to ble for the CO2 gas tanks should turn on the gas tank one fourth to one half of the total fl oor area for the valves beginning with the tank farthest from the exit depopulation procedures. 318 Avian Influenza

Two sheets of 6-mil clear plastic measuring the width of the house and one fourth to one half the house length to enclose the birds on the fl oor space are prepared outside of the house. The plastic is unrolled, unfolded, and refolded lengthwise before entering the house. One sheet of plastic sheeting is placed along one of the sidewalls of the house and its outer edge is secured by covering it with approximately 2 to 3 ft (0.6 to 1.0 m) of litter. The embedding of this plastic edge in the litter should secure the plastic against pulling as the other edge is drawn across the fl oor space to cover the birds in that confi ned area of the house. Temporarily roll the remainder of the free edge of plastic sheeting toward the house sidewall to maintain open fl oor space during Figure 14.2. Plastic tenting procedure for setup. This process should be repeated on the oppo- use with compressed gas method. Plastic site side of the house with the other sheet of plastic. sheets are drawn across the fl oor to cover Each sidewall of the house will have a folded the birds and the horizontally placed sheet of plastic anchored by litter against the side compressed gas cylinders Source: D.A. wall and a free edge to be drawn across the fl oor Bautista, University of Delaware. space to cover the birds. The CO2 gas cylinders should be evenly distributed along the sides of the house. They should be placed along the inside length pleted, all workers immediately leave the house. A of the house, perpendicular to the sidewalls, alternat- designated person remains who is trained in the use ing against the opposing sheets of plastic, with the of and wearing a self-contained breathing equipment nozzles pointed horizontally toward the center of the (SCBA), and proceeds to gradually open all the CO2 house. Gas cylinders locations should be marked by compressed gas cylinders valves to fully release the hanging plastic ribbons from the ceiling over the CO2 gas under the plastic. This person proceeds location of the tanks in order to easily identify the from the farthest end of the house toward the exit location of the tank valves. Once covered with and leaves immediately once all cylinder valves are the plastic, the gas cylinders cannot be readily opened (Fig. 14.2). located without prior marking of each location. After the CO2 gas has been dispersed under the Once the plastic sheeting and cylinders are in plastic for a minimum of 20 minutes, the designated place, the birds should be herded to the prepared person wearing SCBA protective equipment reen- house section. Birds should be moved slowly to ters the house to evaluate the process and determine prevent piling, sitting down, and running back if there is any remaining bird movement or activity. around the handlers. Workers will be evenly spaced If the depopulation procedure is completed, the the length of the house and will grasp the free edge house fans should be turned on and the curtains or of plastic on one sidewall of the house. This free vents opened to ventilate the house interior. The edge will be drawn over the birds to the opposite Safety Offi cer will determine when it is safe for the side of the house and dropped inside the opposite work crew to reenter the house, remove the plastic plastic sheet. Similarly, the second sheet of plastic sheeting, and begin carcass disposal. The plastic will be grasped by its free edge and pulled over the sheeting should be pulled to the side walls to expose

ﬁ rst sheet to the contralateral side of the house effec- the dead birds. The CO2 gas cylinder valves should tively covering the birds in two layers of plastic be closed and the cylinders moved outside of the sheeting. The ﬁ nal step to secure the plastic sheeting house. Carcass disposal should proceed as planned is to fold over the front and rear ends of the plastic for this site. The use of in-house composting as the and immediately cover these with 4 ft (1.2 m) of disposal method will greatly reduce the amount of litter. House fans should be turned off. Once com- carcass handling needed for disposal. 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 319

Whole house (or an isolated portion of the house) The advantage to whole-house gassing is that it CO2 gassing can be used on large fl ocks of birds (meat-type This method uses the entire house or a portion of it chickens and turkeys) within a relatively short period as the mass depopulation chamber. Floor equipment of time and with minimal training of personnel. such as feeders, watering devices, and nest boxes However, there are several signifi cant disadvan- must be raised if present. The ventilation system will tages. Once the process has been initiated, it cannot be shut down during the gassing procedure. The be stopped and observation of the birds is logisti- entire house (or designated portion) is completely cally limited or impractical. Furthermore, the intro- sealed off to permit the CO2 to fi ll up to 4 to 6 ft duction of CO2 gas into the sealed house is gradual (2 m) from the ground (4). Plastic sheeting may be over a longer period of time than in a paneled enclo- used to cover the side walls and air intakes to provide sure or small chamber, thereby delaying lethal CO2 more air tightness depending upon the construction exposure and time to unconsciousness of the birds and integrity of the house. Calculation of the volume (6). This may be cause for possible animal welfare of CO2 gas needed is based upon the length and concerns. width of this defi ned area and determined as described earlier. Delivery of suffi cient concentra- Live haul cage enclosures tion of CO2 gas to displace ambient air with a fi nal This method incorporates the standard capture target level of 70% CO2 will involve larger total method used to collect birds for transport to process- volumes to compensate for the larger house ing facilities. A catching crew is employed to catch (“chamber”) volume and gas leakage from the house. the birds and place them in live haul cages or rolling The most effi cient approach to achieve this large racks per standard catching procedures. The fi lled volume of CO2 is to have direct delivery from a CO2 cages or racks then are placed at the designated area tanker truck with high volume and fl ow rate, rather at the end of the house where they are covered with than from multiple individual metal cylinders. It is or rolled into a preconstructed CO2 euthanasia important to adequately secure the tanker truck’s chamber. release nozzle or vaporizer because the high pres- The CO2 euthanasia chamber is constructed of sure released from this nozzle can easily break loose, sheet metal or plywood to fi t over a desired number causing a dangerous whipping action of the hose of live haul cages. Units that are designed to be lifted apparatus. Once the building preparation is com- over the cages should have a “U” channel on the top pleted and the tanker hose is secured, the birds of the chamber so that these can be moved with a should be herded to and confi ned within the desig- fork lift. A manifold directs the CO2 gas to the top nated area of the house. The ventilation fans are of the unit whereby it is evenly distributed within turned off, the curtains closed, and the compressed the chamber and settles down over the birds. There

CO2 is introduced. Experiences in the Netherlands should be a connection to secure the CO2 tank to the HPAI outbreak (2003) and elsewhere have shown unit right below the intake pipe. A similarly con- that lethal effects will begin after 35 minutes and the structed box-type chamber may be constructed to depopulation process completed in 2 to 3 hours (4). roll the carts holding the cages into the chamber with Bird movement and activity should diminish within a door to close and seal the chamber once the carts 10 minutes and cease within 20 to 30 minutes. The are positioned inside. critical factor is uniform and thorough distribution A designated working area or pad for using the of lethal levels of CO2 gas to all the confi ned birds CO2 euthanasia chamber is prepared in a fl at area by to ensure a humane death without complications of placing and smoothing with sand or limestone dust heat stress and suffocation of piled birds. Before to improve bottom seal. Chambers are assembled workers may reenter the house, the Safety Offi cer with CO2 tanks at a designated area at the end of the should assess the situation and give clearance for a house or immediately outside. Racks with full cages designated worker in SCBA to enter the house to are placed on the pad area side by side or rolled into turn on the exhaust fans and ventilate the house. the chambers. The chamber is either closed (for roll- Once the house is adequately ventilated, workers in cages) or placed over the racks of cages with a may enter to begin the disposal process. forklift. 320 Avian Influenza

Carbon dioxide compressed gas is introduced into The units are operated in tandem for optimum the chamber at ﬂ ow rates to achieve euthanasia. This capacity, and throughputs per pair of up to 4000 system may be used in all types of poultry housing chickens per hour are possible—in most cases, the but is especially advantageous for breeder birds and rate-limiting factor being the speed of catching. The caged-layer houses. The primary disadvantage is CGUs are seen by the U.K. veterinary authority as that individual bird handling is necessary. After being a rapid, ﬂ exible, and humane system for mass birds are euthanized, the chambers are removed and depopulation in disease outbreaks (G. Hickman, the carcasses are deposited into disposal containers. DEFRA, personal communication) (Fig. 14.3). Also any dead birds must be individually removed from housing coops or cages. Water-Based Foam

A similar CO2 gas chamber system has been The use of commercially available water-based foam developed for the mass depopulation of poultry in as a method of mass depopulation of chickens and the United Kingdom called Containerised Gassing turkeys offers an alternative to CO2 gassing (3, 5). Units (CGUs; International Patent Application No. Water-based foam induces anoxia in the birds by cre- PCT/GB2007/000943). This novel system consists ating a mechanical obstruction in the upper airway. of a steel chamber with a lockable access door, Water-based foam as a depopulation agent has designed to accommodate an industry standard many advantages over CO2 gassing: personnel (Anglia Autoﬂ ow or Stork) transport module. The requirements are reduced; exposure to infected birds chamber is preﬁ tted with gas lines and diffusers, is limited; entry into poultry housing may not be which in turn are connected via a system of mani- essential; depopulation efforts may be accomplished folds and gas regulators to four gas cylinders. Birds more quickly and therefore allow more effective are caught by experienced catchers and placed in the containment; the method is synergistic with in-house modules. The modules are then loaded by forklift composting; and measurements of animal welfare into the CGU, and a lethal mixture of gas is intro- parameters suggest that the method is at least equiv- duced into the chamber. alent and perhaps in some parameters superior to

Following extensive research into the aversiveness those reported when using CO2 gassing as employed of various gas mixtures, the United Kingdom veteri- under fi eld conditions. nary authorities decided to use 80% argon/20% CO2 The foam and dispensing equipment are adapted gas mixture. This provides rapid depopulation of a wide from fi re retardant technology used by many fi re- range of poultry species, including ducks and geese. fi ghting companies (3, 5). Foam is formed from

Figure 14.3. Examples of live haul cage enclosures for use in compressed gas method. This process is used commonly in caged layer and breeder facilities. (A) Enclosure used in The Netherlands. Source: H. Kiezebrink and J. Sparry: B-F-C Bird Flu Control GmbH. (B) Enclosure used in the United Kingdom. Source: G. Hickman, DEFRA, UK. 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 321 water, air, and concentrated foaming agents that based foam as a method of mass depopulation of combine to form a stable mass of small, air-fi lled fl oor-reared chickens and turkeys to be used under bubbles. The mass depopulation procedure involves any one of the following conditions (18): confi ning poultry to sections of the house, lowering the light intensity to reduce activity, and then creat- 1. Animals infected with a potentially zoonotic ing a cover of dense foam over the birds. Current disease, or discussions have focused on whether this method is 2. Animals infected with a rapidly spreading infec- humane and whether chickens and turkeys and other tious disease that, in the opinion of state or federal poultry subjected to this method suffer more or less regulatory offi cials, cannot be contained by con- distress than with current CO2 euthanasia methods. ventional or currently accepted means of mass The U.S. Department of Agriculture–Animal and depopulation, or Plant Health Inspection Service (USDA/APHIS) 3. Animals housed in structurally unsound build- and the AVMA maintain that destroying chickens ings which are hazardous for human entry (such and turkeys with water-based foam, while not a as those damaged during a natural disaster). method of routine euthanasia, is a conditionally accepted method for mass depopulation (18). Pre- The conditional use of this foam method is contin- liminary measurements of some physiologic param- gent upon further research conducted to better refi ne eters suggest this method is at least equivalent to the method and address humane considerations. parameters associated with the CO2 gas method Water-based foam has been shown to be an effective under fi eld conditions (3, 5). Several formal pub- agent of mass depopulation for fl oor-reared poultry, lished studies recently have been completed on this i.e., meat-type chickens and turkeys. The use of method but have not yet determined unequivocally water-based foam on all types of poultry may not be the cause of death. It is theorized that death is by appropriate at this time due to housing situation, age, asphyxiation within 5 to 7 minutes for the majority or species. Further development of the technology of birds and all birds have succumbed within 10 to may permit expansion of the range of species and 15 minutes. Time to death is comparable to that poultry types for water-based foam depopulation. observed by the CO2 gaseous method used to depop- More comprehensive acceptance also could be ulate chickens and turkeys in poultry houses. expanded as new data on meeting humane criteria The USDA/APHIS and the AVMA have stated and use in other poultry species become available their conditional acceptance for the use of water- (Fig. 14.4).

Figure 14.4. Units constructed for use of the water-based foam method. (A) Unit designed by North Carolina Department of Agriculture and Consumer Services. Source: L. Dufour-Zavala, Georgia Poultry Laboratory Network. (B) Unit designed by University of Delaware and KifCo, Inc. Source: M. Smeltzer, USDA. 322 Avian Influenza

Caged-House Methods which the birds are placed into the CO2 euthanasia The use of battery cages by the table egg industry chamber. One end contains a hinged door that opens presents a different challenge compared to fl oor- from the bottom through which the carcasses are reared management systems when depopulation is removed. The fl oor of the chamber is sloped to to be done in this type of poultry house. One key facilitate the removal of the dead carcasses by tilting factor dramatically changes the available choices: it the cart. A small (20 lb/9 kg) CO2 gas cylinder is is not practical or feasible to depopulate birds while attached to the cart and periodically discharged into in the cages. There are logistical complications that the chamber through a manifold, providing a uniform can delay effective, prompt removal of the bird car- and repetitive charging of the chamber with CO2 gas casses from the cages and subsequent rapid decom- as needed. Incomplete charging with CO2 gas will position if the carcasses are not removed promptly. lead to smothering of the birds rather than humane Another major factor is the extremely large numbers euthanasia. The cart containing dead birds is moved of birds housed in these cage layer operations. to the end of the house where the end door is opened, Average house size today in the table egg industry the cart tilted, and the carcasses dumped out into is well over 75,000 birds in a single house. Most disposal containers. table egg farms contain multiple houses. Some com- The catching crew is instructed to catch and place plexes house well over 1 million birds on a single birds in the carts precharged with CO2 gas. One 20- farm. Depopulating and disposing of this number of lb (9 kg) cylinder commonly will last for 2 hours. birds in a short time is both labor intensive, expen- An experienced crew of 10 catchers with fi ve carts sive, and very site dependent. Birds typically are can catch and euthanize approximately 25,000 to confi ned in batteries of cages that are placed over a 30,000 birds in an 8- to 10-hour period. Under deep manure pit into which the droppings fall or routine circumstances, such as depopulating spent directly over a manure belt that removes the drop- hens in a layer house of 90,000 to 100,000 birds will pings. Another complicating feature is that the avail- take 3 days to complete using an experienced crew able aisle space between rows of cages within a with minimal PPE requirements (Fig. 14.5). layer house is generally no more than 2.0 to 2.5 ft (0.6 to 0.8 m) with aisles often 400 to 500 ft (122 to Caged-Layer Euthanasia Chamber Method 152 m) in length. Both the worker and the culling With this method, a dumpster or large rectangular equipment must maneuver within this space, creat- box with sides high enough to permit placing of ing signifi cant traffi c fl ow problems and interference birds and adequate CO2 levels is used as the chamber. with the process of removing birds from the cages This is lined with 5- to 6-mil plastic. The chamber and out of the house. Workers and equipment must is charged with CO2 prior to bird placement and is travel up the aisle and back again multiple times and also continuously dispensed as birds are placed in often cannot pass by each other coming and going. the chamber, maintaining a minimum of 50% CO2 This also presents a logistical problem simply to concentration level and targeted fi nal concentration catch and carry birds to a CO2 euthanasia chamber of 70% CO2. A catching crew is utilized to remove or for electrocution at the end of the poultry house. birds from cages and manually carry them to the end Caged-house euthanasia methods include (1) of the house where the birds are dropped or slid into euthanasia carts inside the poultry house, (2) eutha- the chamber. With this method, it is important to nasia chambers outside of the poultry house, and (3) place the chamber below the level of the loading electrocution outside of the poultry house. ramp so the workers may drop or place birds down into the chamber rather than have to lift or raise the Caged-House Euthanasia Cart Method birds up into the chamber. Chamber may be used Caged-layers can be euthanized in a container on until approximately 50% fi lled at which time it must wheels (cart) into which CO2 gas is infused. The cart be emptied and reset up (Fig. 14.6). essentially is a metal box with dimensions of 3 ft Another method used for caged birds is similar to length ×3 ft height ×1.5 ft width (0.9 × 0.9 × 0.5 m). the fl oor bird live haul cage method. Birds are caught The cart has wheels on the bottom and is rolled per standard catching procedures and placed in down the aisle in front of the cages fi lled with birds. rolling live haul cages. These cages are then moved The euthanasia cart contains a door on the top into to the end of the house and rolled into a euthanasia 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 323

Figure 14.5. Carts on wheels into which compressed CO2 gas is infused for use in caged- houses. (A) Multiple carts are covered with plastic sheeting as the carts are rolled into a constructed frame and then lay plastic over the top into which CO2 gas is infused. Source: USDA/APHIS. (B) Diagram depicting rolling cart process. Source: USDA/APHIS.

chamber where CO2 gas is introduced. The carcasses are removed from the cages and placed in a disposal container.

Electrocution of Caged-Layers Poultry must be manually removed from their cages, inverted, and shackled onto a line which conveys them through a water bath stunner with their heads fully immersed. Electrical contact is made between the water and the earthed shackle so that when suf- ﬁ cient current (amperage) is applied, poultry will be simultaneously stunned and killed. This method is capable of depopulating large numbers of birds effectively and reliably. The units designed for use in the Netherlands during the HPAI outbreak (2003) are mobile, can process 2,500 to 10,000 birds per Figure 14.6. Caged layer chamber method hour, and require only one operator. The major dis- into which are placed multiple, individual advantage to the use of this procedure is the birds removed from the layer cages. handling of poultry, which cause more stress to the Compressed CO gas charges the chamber. 2 birds and may present risks to human health and Source: S. Gasper, FPM, Inc. safety (6). 324 Avian Influenza

Noncommercial or Backyard Bird tions (barbiturates), (2) cervical dislocation, (3)

Euthanasia Methods liquid CO2 (compressed CO2 gas) euthanasia in a Euthanizing backyard or noncommercial birds will chamber, and (4) culling bag method using frozen be very situation dependent and based upon the CO2 (dry ice). number and types of birds on the premises as well as the on-site facilities available. The ﬂ ock may be Injectable Anesthetics in an urban, suburban, or rural setting. The owner or Lethal injection using high doses of anesthetic and individual may have an emotional attachment to the sedative drugs will cause direct, rapid CNS depres- animals in addition to a business interest in the sion, unconsciousness, and death. Barbiturate solu- operation, such as an avicultural hobby, a small tions such as sodium pentobarbital are most family traditional business, or individual interests. commonly used but are categorized as controlled Therefore, the aesthetics and likely presence of other substances with legal requirements and restricted use interested individuals must be factored into the by veterinarians. Intravenous administration is the method of choice. required route because the solutions will irritate sur- For ﬂ ocks with small numbers of birds involved, rounding tissues if given by intramuscular or subcu- the use of lethal injection of barbiturate solutions taneous routes of administration. Intracardiac would be the most appropriate and humane method administration is not considered to be a humane

(1). The use of CO2 in a small chamber such as method in a conscious animal but may be used if the plastic waste bin or a culling bag also may be used animal fi rst is anesthetized. Intraperitoneal (intracoe- effectively. Species differences in response to CO2 lomic or intra-abdominal) administration may be euthanasia may render one method more advanta- used in small birds without prominent peripheral geous than another. Cervical dislocation may be a vein access but the lethal procedure will take longer reasonable alternative method, but many people due to slower absorption rate. Restraint is required to consider physical methods of euthanasia to be objec- permit proper administration, which may be stressful tionable and perceived as violent. Thus, use of phys- to the birds. Prior sedation may be used to ameliorate ical methods of euthanasia should be determined the stress due to handling and restraint. Lethal injec- based on the overall situation. tion is labor intensive and costly and may cause Noncommercial or backyard fl ock euthanasia signifi cant distress in the birds and therefore should methods may include (1) injectable euthanasia solu- be limited to use in small numbers of birds (Fig. 14.7).

Figure 14.7. Diagram of intravenous injection site for euthanasia solution administration. Shown in the diagram is the anatomic orientation of the medial metatarsal vein of the avian leg. Source: K. Carter, University of Georgia. 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 325

Cervical Dislocation hip, with the bird’s back facing the operator. The Cervical dislocation is accepted by the AVMA as a back of the bird’s head is placed between the fi rst method of euthanasia for poultry and may be a prac- two fi ngers of the other hand, with the back of the tical and economical method when small numbers hand facing the operator. Cervical dislocation is of birds are involved (1, 17). The objective is to accomplished by smoothly and rapidly extending quickly and effectively disarticulate the skull from the operator’s arm that is holding the bird’s head and the spinal column and to completely sever the spinal stretching the bird’s head and neck so that the base cord. If done correctly, this procedure produces of the skull is lifted off the end of the last cervical instantaneous loss of consciousness. Refl exive mus- vertebrae. Once suffi ciently stretched, the head cular contractions will occur, but these do not indi- should be rolled backwards by simultaneous back- cate awareness by the animal. ward fl exion of the operator’s wrist. This stretching of the neck and pulling of the head backwards will Chicks separate the skull from the neck vertebra and rupture The neck of young chicks can be easily dislocated the spinal cord, killing the bird instantly. When done by pressing the head and neck junction fi rmly against correctly, a distinct popping sound and feeling is a sharp edge, or by pinching this region between the achieved and the separation between the cervical thumb and index fi nger. Alternatively, the end of vertebrae and the skull can be palpated through the scissors with the thumb and fi nger holes (as opposed intact neck skin. It is important to perform this task to the cutting end) can be used. The chick’s neck is in a controlled fashion so as not to tear the bird’s placed between the handles of the scissors and the neck or pull off the head causing a signifi cant spill- handles are closed, immediately dislocating the head age of blood (Fig. 14.9). from the neck without breaking the skin or causing bleeding (Fig. 14.8). Larger birds—mechanical method (Burdizzo-method) Larger birds—manual method Bovine castration forceps (e.g., Burdizzo) can be The bird’s legs are held in a fi xed position in one used for killing large birds such as male chickens hand, which is often stabilized against the operator’s and species with strong necks such as geese or ducks. It is diffi cult for one person to perform this operation and hold the bird at the same time, but it

Figure 14.8. Mechanical cervical dislocation is shown using the ﬁ nger-hold ends of scissors placed around the neck of day-old Figure 14.9. Manual cervical dislocation for chicks and small birds to mechanically larger birds is shown by demonstrating the separate the uppermost cervical vertebrae manual restraint and position of the bird’s and spinal cord. Source: K. Carter, University neck, feet, and body. Source: S. Collett, of Georgia. University of Georgia. 326 Avian Influenza

Figure 14.10. Mechanical cervical dislocation for larger birds is shown using ‘Burdizzo’ instrument placed around the bird’s neck to mechanically separate the cervical vertebrae and spinal cord. Source: K. Carter, University of Georgia. Figure 14.11. Simple chamber for

compressed CO2 gas using a plastic barrel with secure lid which should be lined with a plastic bag and into which CO gas is infused. is quite easily done with the aid of an assistant. The 2 Source: M. Smeltzer, USDA. instrument is opened and the bird’s neck is placed in the jaws of the Burdizzo. The jaws are then closed and the cervical vertebrate and spinal cord are effectively separated (Fig. 14.10). can proceed until the can is half full. More than this amount will result in difﬁ culty lifting and removing Compressed CO2 Gas Euthanasia in a Chamber the birds in the plastic bags lining the can. The A 50- to 90-gallon (190- to 340-liter) waste bin or plastic bag should be sealed and the carcasses are barrel should be lined with plastic. A 20- to 50-lb removed for disposal with the plastic bag (Fig.

(9- to 23-kg) cylinder of compressed CO2 can be 14.11). used to introduce CO2 gas into the container through a hose introduced through an opening or hole in the Culling Bag and Frozen CO2 for Euthanasia lid. Birds should have been previously captured and A small CO2 euthanizing bag system has been devel- restrained. In past efforts, long-handled fi sh nets oped for use with dry ice and is available commer- with large net circumference have been helpful in cially (8). This type of system is composed of the capturing birds in various situations. The can is following steps as shown in Figure 14.12: charged with CO2 to a concentration of at least 50% with a targeted fi nal concentration of 70% CO2. A 1. Assemble support boxes. group of four or fi ve birds, depending on species and 2. Insert inner plastic bag inside outer bag. size, are then placed into the can. A cover is replaced 3. Turn on CO2 monitor and check to ensure it is over the can and additional CO2 is introduced. When working. these birds are euthanized, additional birds may be 4. Using leather gloves, pour 2.0 kg (4.4 lb) of dry placed into the can after recharging with CO2. This ice pellets into inner bag. This quantity should 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 327

Figure 14.12. Culling bag system using dry ice (frozen CO2) has been shown to be effective for use with small numbers of birds or small ﬂ ocks in rural areas. The system is very portable and does not require electricity or compressed CO2 gas cylinders Source: H. Kiezebrink and J. Sparry, B-F-C-Bird Flu Control GmbH.

generate a cubic meter of CO2 gas that yields a 6. Wait for monitor to indicate the targeted con-

70% CO2 gas concentration within the bag centration of CO2 gas (70%), this may take 1 to 2 (additional dry ice is later added to maintain the minutes depending on the rate of vaporization. 70% concentration); the addition of 1 to 2 liters 7. Next, the birds are captured and placed in the of warm water can speed up the vaporization bag at a rate of no more than 10/min to allow

rate of the CO2 pellets (Appendix 14.2). birds to become unconscious by inhalation of

5. Place CO2 monitor into neck of bag within CO2; avoid putting too many birds into the bag 20 cm (8 inches) of the top. at once or smothering will result. 328 Avian Influenza

8. If the monitor indicates that the CO2 level falls on noninfected premises. The method chosen must below 70%, more dry ice should be added and be safe, reliable, and efﬁ cient with as much consid- no more birds placed in the bag until the monitor eration given to humaneness as practicable. Critical indicates the required concentration has been factors involving cost, availability of equipment, reached. training of personnel, worker safety, and reliability 9. A maximum of 150 kg (330 lb) of birds are to of the method to minimize pain and distress to the be placed in the bag. animals were discussed. Hopefully these basic prin- 10. Once the bag is full and all birds have died, the ciples and guidelines have provided a template for top of bag should be sealed securely with a conscientious decision-making. plastic cable tie. Nonetheless, several ﬁ nal, pragmatic comments

11. Bottom of outer bag can be opened by pulling may be notable and worthwhile. The use of CO2 on the draw string. The entire outer box can then gas for mass depopulation appears to be the most be lifted up and the inner bag containing the adaptable to various on-farm situations. Suggested dead birds will be left on the ground. improvements to this method include the use of clear 12. The inner bag can be transferred to a disposal plastic tarps or constructing viewing windows so lorry or a burial pit, or stored safely for later birds may be easily observed during the euthanasia

collection. process, positioning the CO2 gas cylinders at equal 13. A new inner bag is inserted into the outer bag distances to promote uniform dispersal of gas, min- and the procedure repeated. imizing valve freezing by opening valves slowly at

14. Up to four outer bags/boxes should be used ﬁ rst to release CO2 gas, and optimizing the CO2 ﬂ ow alternately, it takes a short while for the dry ice rate to displace 20% of chamber volume per minute to sublimate into gas so while one box is being to hasten unconsciousness of the birds and to mini- used, the other can be in preparation. mize heat stress under the tarps or within the cham-

bers. Also, the use of CO2 monitors within the CONCLUSIONS chambers and enclosures is recommended to aid in

Although methods for individual animal euthanasia ensuring adequate lethal levels of CO2 are achieved have been described and reported to be humane and (8, 9). acceptable by recognized leading authorities on Electrocution as a mass depopulation method has animal welfare, adapting these methods for mass limited use due to concerns for proper maintenance depopulation is not a simple matter of scale. Rather, of equipment, access to constant electric current in use of such individual methods for mass depopula- rural areas, welfare of animals if improperly applied, tion still is being developed, discussed, and deter- and human safety. This method does require han- mined to be accepted as humane. The common dling of birds, which may increase the risk of expo- approach by those faced with the depopulation of sure to workers to pathogens with zoonotic disease large numbers of birds has been to extrapolate the potential. Nonetheless, when used by skilled and euthanasia methods used for individual birds to the competent personnel, this method will depopulate larger scale. Unfortunately, success, by humane large numbers of birds in a relatively short time standards, is not always guaranteed despite one’s period. best efforts, and must be evaluated on a situation by Given the few recognized and acceptable methods situation basis. for mass depopulation, researchers have been This chapter has attempted to explain the cur- encouraged to develop newer methods, which mini- rently recognized and accepted methods of mass mize the risk to human health and safety while depopulation including newer, conditional methods; abiding by the criteria established for the humane to describe practical experiences to guide in the death of animals. One such method, water-based selection of appropriate methods for mass depopula- foam, has been shown to cause death in domestic tion; and to address associated animal welfare and chickens and turkeys within a similar time frame to human health and safety concerns. CO2 gas and requires only a few trained workers to Mass depopulation performed on-site is believed conduct the procedure. While this method has to be the best method for preventing further spread received conditional acceptance by the AVMA and of the virus and risk of exposure to healthy animals USDA/APHIS for use in certain mass depopulation 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 329 situations, further research to validate its mechanism Avian Infl uenza—Part III. The EFSA Journal for causing death is needed to determine if it meets 266:1–21. the recognized criteria of animal welfare authorities 7. Galvin, J.W. 2006. Slaughter of Poultry for Disease for a humane death in birds. Control Purposes (Discussion Paper). Available Therefore, to reiterate a primary focus of this at http://www.oie.int/eng/AVIAN_INFLUENZA/ discussion%20paper%20(Galvin).pdf. Accessed chapter, when mass depopulation is the method January 5, 2007. selected for disease control, it is essential that the 8. Kiezebrink, H. 2005. Procedure for use of culling procedure selected be as humane as possible and bags. B-F-C Bird Flu Control GmbH. Available any animal welfare concerns are addressed. This at http://www.bfc-birdfl ucontrol.com. Accessed must begin in the planning stages and be mon- January 5, 2007. itored throughout the process. A fi nal report and 9. Kingston, S.K., C.A. Dussault, R.S. Saidlicz, assessment of the mass depopulation activities N.H. Faltas, M.E. Geib, S. Taylor, T. Holt, and should be submitted by the depopulation team B.A. Porter-Spalding. 2005. Special Report: and on-site safety offi ce to the outbreak res- Evaluation of two methods for mass euthanasia ponse authorities at the incident command post of poultry in disease outbreaks. Journal of the to benefi t those immediately involved and to pro- American Veterinary Medical Association 227(5): 730–738. mote dissemination of knowledge and practical 10. Lambooj, E., M.A. Gerritzen, B. Engel, S.J.W. experience. Hillebrand, J. Lankhaar, and C. Pieterse. 1999. Behavioural responses during exposure of broiler ACKNOWLEDGMENTS chickens to different gas mixtures. Applied Animal The authors wish to thank Mr. Julian Sparrey (B-F- Behaviour Science 62:255–265. C Bird Flu Control GmbH) for his contribution of 11. Occupational Safety and Health Administration. information on the CO culling bag method and for 2006. Guidance Update on Protecting Employees 2 from Avian Infl uenza Viruses. Available at http:// reviewing this document. www.osha.gov/OshDoc/data_AvianFlu/avian_ fl u_guidance_english.pdf. Accessed January 5, REFERENCES 2007. 1. American Veterinary Medical Association. 2001. 12. Raj, A.B.M., and N.G. Gregory. 1994. An evalua- The 2000 Report of the AVMA Panel on Euthana- tion of humane gas stunning methods for turkeys. sia. Journal of the American Veterinary Medical Veterinary Record 135:222–223. Association 218(5):669–696. 13. Raj, A.B.M., N.G. Gregory, and S.B. Wooton. 2. AustVetPlan. 2001. Operational Procedures 1990. Effect of carbon dioxide stunning on somato- Manual, Edition 3.0—Destruction of Animals. sensory evoked potentials in hens. Research in 3. Benson, E., G.W. Malone, R.L. Alphin, M.D. Veterinary Science 49:355–359. Dawson, I. Estevez, and G.L. Van Wicklen. 2007. 14. The European Commission. Scientifi c Committee The use of fi re fi ghting foam for mass emergency on Animal Health and Animal Welfare. 1998. The

euthanasia of fl oor-reared meat-type poultry oper- use of mixtures of the gases CO2, O2, and N2 for ations. Poultry Science 86:219–224. stunning and killing of poultry. Available at www. 4. Centers for Disease Control and Prevention (CDC). europa.eu.int/comm/food/fs/sc/scah/out08_en. 2006. Interim guidance on protection of persons html. Accessed January 5, 2007. involved in U.S. outbreaks of avian infl uenza 15. The European Council Directive. 1993. Protection disease control and eradication activities. Avail- of animals at the time of slaughter or killing. able at http://www.cdc.gov/fl u/avian/professional/ Offi cial Journal of the European Communities pdf/protectionguid.pdf. Accessed January 5, 340(21):21–33. 2007. 16. The Poultry Industry Council for Research and 5. Dawson, M.D., P.L. Reyes, E.R. Benson, G.W. Education (Canada). 1995. Controlled gas stun- Malone, R.L. Alphin, I. Estevez, and G.L. Van ning and killing (European versus North Amer- Wicklen. 2006. Evaluating the use of fi re fi ghting ican perspective). Available at http://www. foam in mass poultry euthanasia. Applied Agricul- poultryindustrycouncil.ca/factsheets/fs_62.pdf. tural Engineering 22(5):787–794. Accessed January 5, 2007. 6. European Food Safety Authority. 2005. Scientifi c 17. World Organization of Animal Health (OIE). 2006. Report on Animal Health and Welfare Aspects of International Animal Health Code. Article 3.7.6.1: 330 Avian Influenza

Guidelines for the Killing of Animals for Disease avma.org/issues/policy/poultry_depopulation. Control Purposes. Available at http://www.oie.int/ asp#Attachment%20A. Accessed April 8, 2007. eng/normes/mcode/code2006_back/en_chapitre_ 19. Webster, A.B., and D.L. Fletcher. 2001. Reactions 3.7.6.htm. Accessed January 5, 2007. of laying hens and broilers to different gases used 18. USDA/APHIS Water-based Foam Euthanasia Per- for stunning poultry. Poultry Science 80:1371– formance Standards. 2006. Available at http://www. 1377. 14 / Mass Depopulation as an Effective Measure for Disease Control Purposes 331

Appendix 14.1 Determining the Amount of Liquid CO2 Needed for Poultry Euthanasia

1. Determine the number and type of birds to be euth- Length ______anized: Width ______a. N = ______Height ______2. Determine size of euthanasia enclosure needed: A = length * height = _____ ft2 (m2) a. This should be calculated based on the number 3. Determine the volume of the empty enclosure: and size of birds to be held in the enclosure a. V = length × width × height = _____ ft3 (m3)

while considering space limitations within the 4. Determine the pounds of CO2 needed to achieve a house. Birds should be closely packed in one 70% concentration of gas in the enclosure: 3 layer. They should not be so tightly packed a. Number (#) of liquid CO2 tanks = 8.75 ft 3 that they try to climb on each other or (0.25 m ) gas CO2

pile up. b. V × (lb CO2) × 0.70 = _____ lb liquid CO2 b. The following estimates may be useful for needed 8.75 ft3 (0.25 m3)

calculating the amount of fl oor space needed: 5. Calculate number of liquid CO2 tanks needed 2 i. Three 8- to 10-lb turkeys/ft (40 to 50 kg/ a. Liquid CO2 is typically available in 50-lb (23- m2) of fl oor space kg) or 387-lb (175-kg) tanks. The larger tanks ii. Four 4- to 6-lb chickens/ft2 (20 to 30 kg/m2) are harder to move, are more likely to freeze, of fl oor space and are more diffi cult to operate. The 50-lb c. The height of the chamber should be calculated (23-kg) tanks are recommended.

to ensure that the gas layer will exceed the b. (lb liquid CO2)/50 = _____ number of 50-lb height the birds can reach with their head and (23-kg) tanks needed neck. This is typically around 4 ft (1.2 m) for chickens and turkeys. Adapted from Kingston et al., JAVMA 227(5):730–738. d. Enclosure size (area) needed in ft2 (m2):

i. Rectangular enclosures are easier to work *EXTRA CO2 TANKS MUST ALWAYS BE AVAILABLE with than square enclosures. AT THE SITE. 332 Avian Influenza

Appendix 14.2 Determining the Amount of Dry Ice (Frozen CO2) Needed for Poultry Euthanasia

Dry ice is generally available for refrigeration and amount of CO2 gas to create >70% CO2 atmosphere cooling purposes in block, pelleted, or shaved form. in a chamber 0.9 m × 0.9 m × 1.2 m (approximately 3 This may be used as a source of CO2 for small-volume 1 cubic meter [m ]/3 ft × 3 ft × 4 ft). During the chambers used for euthanasia. Dry ice will dissipate euthanasia process, an additional 3 kg (6.6 lb) of dry into CO2 gas if left at air temperature over a period of ice should be added periodically to maintain the time. This process is rapidly accelerated by mixing the >70% CO2 gas level as indicated by CO2 monitor. dry ice with warm water, thus giving desired levels of 2. One kilogram (2.2 lb) of pelleted dry ice mixed with

CO2 gas within a small chamber in 1 to 2 minutes. a quart of warm water will deliver a sufﬁ cient

The following gives general amounts that have been amount of CO2 gas to create a >70% CO2 atmo- used for these type of chambers or volumes in ﬁ eld sphere in a chamber 1 yard × 1 yard × 1 yard situations to achieve desired levels: (approximately 1 cubic yard or 1 cubic meter). During the process, an additional 3 kg (6 to 7 lb) of

1. 1 kg (2.2 lb) of pelleted dry ice mixed with 1 liter dry ice should be added to maintain the >70% CO2

(0.26 gal) of warm water will deliver sufﬁ cient gas level as indicated by the CO2 monitor. 3. Chambers of this volume will be sufﬁ cient to euth-

* EXTRA CO2 TANKS MUST ALWAYS BE AVAIL- anize up to 150 kg (approximately 330 lb) of birds ABLE AT THE SITE. or around 50 to 70 adult chickens. 15 Methods for Disposal of Poultry Carcasses

Boris Brglez and John Hahn

INTRODUCTION and federal government personnel to deal with the From December 2003 to September 2006, H5N1 outbreak. The AITFs were confronted with a poten- highly pathogenic avian infl uenza (HPAI) virus tial economic disaster with only limited strategic spread from China to over 50 previously unaffected plans in place to combat it. The purpose of this countries located in Europe, Africa, the Middle East, chapter is to examine and compare all existing and Asia (15). The HPAI global outbreaks from methods for disposal of dead poultry in catastrophic 1959 to 2003 resulted in the culling of less than 100 poultry disease outbreaks. It is hoped this chapter million birds (15). However since 2004, the U.N. will be a valuable resource to assist others in making Food and Agriculture Organization (FAO) estimates strategic plans for dealing effectively with future that over 200 million birds died or were culled as a outbreaks. result of HPAI (15). The disposal of bird carcasses When comparing disposal methods the major on such a global scale, in such a short period of time, concerns are preventing disease transmission and posed signifi cant challenges. pollution (air, water, and land). Once those issues Within the United States there have been a number are addressed, the next most important factors will of outbreaks of low pathogenicity avian infl uenza be cost, ease and speed of disposal, and potential (LPAI) over the past 75 to 100 years, but only four community objections. The two diseases currently outbreaks have been high pathogenicity avian infl u- posing the gravest threats for catastrophic losses to enza (HPAI); 1924–1925, 1929, 1983–1984, and the commercial poultry industries are virulent New- 2004 (see Chapter 8, Highly Pathogenic Avian castle disease and HPAI. However, other circum- Infl uenza Outbreaks in Europe, Asia, and Africa stances may require disposal of large numbers of since 1959, Excluding the Asian H5N1 Virus Out- dead or culled poultry including natural disasters breaks). Each presented with unique requirements such as fl oods, fi res, hurricanes, or tornados; chem- for disposal of poultry carcasses, both from mortal- ical contaminates such as in feed contamination; and ity in the outbreaks and preemptive culled birds. other infectious and noninfectious diseases. In these Much of the information in this chapter is based situations, the actions for disposal will be similar— upon research conducted during the last two large to dispose of large numbers of dead birds. Therefore, outbreaks of AI in the Shenandoah Valley of we will take a historical look at some of the disposal Virginia (HPAI H5N2 in 1984 and LPAI H7N2 in issues in several outbreaks of virulent Newcastle 2002). In the Shenandoah Valley in 1984, more than disease, HPAI, and LPAI in the United States. In 65 commercial poultry operations yielded 5700 tons each of these situations, no single method was of carcasses; in 2002, more than 195 depopulated exclusively used, but mixtures of methods were used operations yielded 16,920 tons (3). On both occa- to meet local needs. An example of methods used in sions, an Avian Infl uenza Task Force (AITF) was 2002 H7N2 LPAI outbreak are reported in Table organized by poultry industry, academic, and state 15.1.

Table 15.1. Different methods used in disposal of poultry carcasses during the H7N2 LPAI outbreak in Virginia during 2002. Site of Disposal Birds Tonnage %

Rockingham County Landfi ll 709,080 3,400 20.1% Page County Landfi ll 198,334 951 5.6% Frederick County Landfi ll 175,601 842 5.0% Charles City County Landfi ll 961,428 4,610 27.2% Sussex County County Landfi ll 964,557 4,625 27.3% Incineration at Rockingham Quarry 551,000 2,268 13.4% Composting 32,000 75 0.4% On-Farm Burial 15,000 128 0.8% Slaughter 590,000 21 0.1% Total 4,197,000 16,920 100%

Historical Poultry Disease Outbreaks and Contamination of the environment by pathogenic Disposal Methods agents can occur during carcass disposal by burial. The outbreak of virulent Newcastle disease in South- However, there is very limited research available ern California in 1971–1974 resulted in the loss of that has examined soils for the presence of patho- over 11.9 million birds (27). In the same region in genic microbes when unprocessed poultry carcasses 2002–2003, there was another outbreak of virulent are simply buried, but normal decomposition pro- Newcastle disease, resulting in over 3.16 million cesses destroy viruses and most pathogenic bacteria. birds being depopulated with a total eradication cost By contrast, with composting, tests for inactivation to the U.S. government of over $198 million (27). have been done in various studies. A study con- In spring of 2002, an outbreak of H7N2 LPAI virus ducted at the National Veterinary Services Labora- was eradicated in Virginia (19). This particular out- tories in Ames, Iowa, on composting carcasses break lasted 3 months, and resulted in the destruc- infected with AI virus demonstrated that the virus tion of over 4.2 million birds from 197 farms, which could not be cultured after 10 days of composting. yielded 16,920 tons of carcasses (3). Previously, Researchers calculated that the compost process within the same area, about 1.4 million birds from maintained temperatures between 100º F and 140º F 70 farms, yielding 5700 tons of carcasses, were dis- throughout the composting period (17). posed of during the 1984 H5N2 HPAI outbreak In the 1983–1984 Virginia H5N2 HPAI outbreak, (3). This earlier outbreak of H5N2 HPAI in Virginia AITF handled disposal primarily through burial on- had originated in Pennsylvania during late 1983, site. In 2002, the county landfi ll was chosen as the where it had led to the destruction of 15 million location for the bulk of disposal. This was the result birds (1). of a new law in 2002 that required on-site burials to To stop the spread of disease outbreaks, one of be recorded on the property deed of the infected the recommendations has been depopulation of sur- farm. Because no farmer wanted to risk the likely rounding fl ocks within a particular distance from devaluation of his or her land with burial sites listed infected fl ocks such as a radius of 3 miles or 5 km. on his deed, it was no longer possible to bury the Another approach is to depopulate only infected, carcasses on-site. This turn of events had not been dangerous contacts and fl ocks linked epidemiologi- expected by the AITF, who favored on-site burial cally to infected fl ocks. The successful eradication since movement of contaminated carcasses off-site of HPAI in the Netherlands in 2003 and in Canada risked spreading the disease-causing agent during in 2004 was achieved by creating a buffer zone transport to off-site disposal. through the depopulation of nonclinical poultry Unfortunately, no emergency response disposal fl ocks surrounding the HPAI-infected fl ocks (12). plans were prepared in advance of either of these AI 15 / Methods for Disposal of Poultry Carcasses 335 outbreaks. The successful containment utilizing the under cold and wet conditions, pathogen survival is respective disposal methods is a tribute to each of prolonged. The water-soluble compounds and patho- the AITFs in diligently searching for a solution gens from the waste can pose a risk to human and within the time constraints imposed by the out- environmental health if it migrates from the on-site breaks. However, the best solution is development burial and contaminates the surrounding soil and of complete disposal plans as part of a general emer- ground water. One advantage of on-site burial is the gency response plan for any catastrophic animal achievement of containing the pathogen to the indi- disease, and the development of such plans before vidual farm of origin, thus reducing the risk for the disaster occurs. spreading the virus. Generally, this method is more The principal objectives to be discussed in this economical than other methods. chapter as related to the seven disposal methods are as follows: (1) cost including transportation, labor, Landfi ll Burial materials, land-use fees, equipment usage, and This method is similar to on-site burial but done whether these costs can be offset by some saleable off-site. Biosecurity is more challenging with land- byproduct of the disposal method; (2) environmental fi ll burial, because the carcasses must be moved impact on ground water, soil, and air; (3) public from the infected premises to a landfi ll, thereby acceptance; (4) complexity of the various disposal increasing costs. However, if done in an appropriate methods, and (5) availability of the expertise and landfi ll, the negative environmental impact will be equipment. less than on-site burial. Two additional disposal methods, lactic acid fermentation and anaerobic digestion, are not discussed Composting in this chapter. These methods were described in a Ideally composting of infected birds should be done 2004 report (13). Lactic acid fermentation and on the farm of origin, preferably within the source anaerobic digestion are not practical disposal housing. Composting requires the proper mixing of methods in catastrophic poultry outbreaks, for the a nitrogen source (dead birds), a carbon source following reasons: (1) lactic acid fermentation (wood chips, straw, poultry litter, etc.) moisture, and requires the product to be rendered, and (2) the best oxygen (air). If done properly, composting tempera- raw product used commercially for optimal anaero- tures of over 140º F can be generated and maintained bic digestion is bovine manure. for weeks to months. This heat inactivates the pathogenic agent and temperature monitoring can be used Introduction to Disposal Options as a means to confi rm proper composting and inac- Seven methods of carcass disposal are examined and tivation. Biosecurity issues are also simpler to emphasis will be indicated on their applicability. control with this method since it is best done on the These methods include (1) two simple routine farm of origin. However, composting requires proper methods (on-site and landfi ll burial, both applied in attention to the details, but is not too demanding, the United States), (2) complex methods (compost- and when done properly, it will have no negative ing and incineration, both applied sparingly in the environmental impact. Also, the compost, when United States; rendering, not applied in the United cured, will serve as excellent fertilizer for land States but used in Europe), and (3) experimental application. methods (alkaline hydrolysis and “in situ” plasma vitrifi cation). Incineration The disposal options we will discuss and some of When incineration is the method used to dispose of their positive and negative characteristics are pro- carcasses of infected birds, the heat kills the patho- vided below. gens. The heat for the incineration can be produced by burning wood or fossil fuels. Because the incin- On-Site Burial eration is usually not done on the farm of origin, This has been the classic method of disposal of dead biosecurity will be more demanding than for the poultry during most disease outbreaks. Unfortu- “on-site methods.” Incineration can be quite expen- nately, knowledge is limited as to how long patho- sive and can have negative environmental impact, gens survive in burial sites, but it is known that especially on the air, and possibly on water and land 336 Avian Influenza also, depending upon the method and environmental that must be provided for and used by workers controls utilized. involved in culling, transport, or disposal of HPAI virus–infected poultry. Individual national public Rendering health organizations will determine which protective Rendering of the carcasses requires that they be equipment is needed for each situation and country. removed from the farm and taken to a rendering plant. Because the carcasses will be removed from ON-SITE BURIAL the infected premises, there will be signifi cant bios- On-site burial was the primary disposal method used ecurity risks. The rendering process mechanically during the 1984 HPAI outbreak in the Shenandoah crushes the whole carcass into 2-in3 cubes of tissue, Valley of Virginia. Of the 5700 tons of carcasses, cooks the tissue under steam, and then removes the 85% were disposed by on-site burial and the remain- moisture from the proteinaceous particles for use as ing 15% were transported to the Rockingham County a dry animal feed protein source. The heat and landfi ll. Although rendering was examined, render- drying of the process will kill the pathogen. When ing plant operators were reluctant to pursue this properly performed this process should be rather option for fear that the poultry industry would refuse environmentally friendly. In the past there has been to purchase any of their feed products due to the some reluctance by rendering companies to take processing of infected carcasses, thereby potentially known infected birds due to negative stigma for the contaminating healthy fl ocks. fi nished product, even though the process kills all Composting was not used because the technology the pathogen. at the time was not well developed. Incinerator boxes had limited availability and open burning was Alkaline Hydrolysis not acceptable to the community. A small experi- This method is done “offsite” and has all of the mental open-pit burning occurred with 1000 tons of biosecurity problems of moving the infected car- carcasses at the landfi ll site, but the unpleasant odor casses off of the premises. Alkaline hydrolysis, like and the diffi culty of fueling the fi re suffi ciently to rendering, uses steam heat in combination with a incinerate the birds caused the experiment to cease. strong base such as NaOH. However, unlike render- The partially burned carcasses were buried in the ing, there is no mechanical crushing of the tissue. landfi ll. Landfi lls were of minor use because of the The strong base dissolves the tissue using a pH acceptability of on-site burial, and because clay and exceeding 11. The water is not removed from the plastic liners were not available for leachate collec- fi nal product. tion. The Rockingham County, Virginia, landfi ll cells were not lined until 1998. In Situ Plasma Vitrifi cation Various designs and burial on-site methods were This method is also done off-site and has all of the attempted during 1984 with a standard method biosecurity problems that accompany moving emerging in May 1984. At that time, the width and infected carcasses off a premise. In in situ plasma depth of a pit were established by the AITF at 20 ft vitrifi cation (ISVP), heat is generated directly from and 10 ft, respectively. The length was calculated electricity, which then ionizes compressed air to based on the tonnage of birds because 800 lb of exceed temperatures of 7000º C and can thus vapor- carcasses required 20 ft3. Of great concern was the ize all the water from the carcass. The fi nal product potential for leachate from the carcasses to con- from ISVP is a rock like substance with 97% reduc- taminate ground water. The major factors determin- tion from the original weight. The gases released ing the suitability of a waste disposal site were the during this process can be trapped by a hood cover- soils, geology, and hydrology of the site. The crite- ing and treated by an off-gas treatment system. The ria for site selection were distances to surface water, heat generated by this process quickly kills any ground water, springs, wells, sinkholes, bedrock, pathogen. and rock outcrops (24). The burial pits were located a minimum of 500 ft from streams, springs, sink- Protection of Worker Health holes, and wells. The set-back from drainage ways Due to the concerns for protecting workers from the and rock outcrops was 100 ft. potential of contracting an HPAI infection, there are Several on-site designs were utilized in the early a number of requirements for protective equipment months of 1984 (Figs. 15.1 and 15.2). The disposal 15 / Methods for Disposal of Poultry Carcasses 337

2′ LEGEND 2.5′ COMPACTED CLAY SLOPE 3′ AS COMPACTED FILL TURKEYS 2′ REQUIRED 0.5′ LOOSE FILL 2′ UNIFORM OPEN GRADED 5′ MIN MATERIAL 20′ WATER TABLE WIDTH DEPENDENT UPON COMPACTION EQUIPMENT

Figure 15.1. Cross section of properly constructed on-site burial pit. Source: U.S. Department of Agriculture.

SLOPE AS REQUIRED

2′ 2.5′ 3′ ′ ACCESS 2 TURKEYS 0.5′ TURKEYS RAMP 2′ SLOPE AS REQUIRED WIDTH LENGTH AS REQUIRED 5′ MIN DEPENDENT UPON WATER COMPACTION TABLE EQUIPMENT

Figure 15.2. Longitudinal section of on-site burial pit. Source: U.S. Department of Agriculture. pit was required to be a minimum of fi ve ft from the tinued. As a result of decomposition and the heat water table. The carcasses were placed in pits 10 ft associated with it, and the impervious nature of the in depth, consisting of layers with a minimum of two plastic liners, gasses formed resulting in contami- ft of clay. From the top of the pit, the layers were as nated fl uids accumulating on the surface of the follows: 6 in of gravel to serve as drainage for any pit (5). leachate from the carcasses, a maximum of 2 ft of Only 23 of the 57 on-site burial pits were lined carcasses with a layer of loose fi ll of approximately with plastic. The pits were not lined with quick- 3 ft, and 2.5 ft of dirt compacted by a bulldozer fol- lime because the Virginia Department of Agricul- lowed by a layer of compacted clay. Good quality ture, in a brochure, indicated that liming should clay was often not readily available and had to be not be used to prevent interfering with the natural transported to the site. In addition, a ventilation strip acidic conditions of decomposition which would 3 ft in width of loose fi ll dirt was placed across the inactivate the virus. Quicklime had been used in center length of the pit to relieve pressure from the past to mask the odor of decomposing carcasses, decaying carcasses. to retard decomposition and to prevent insect infes- Initially, due to concern over ground and surface tation. The contaminants of most concern to the water contamination, the AITF recommendations ground water were nitrates and bacteria. However, were that the burial pits should be lined with plastic since nitrate production was an aerobic process, to prevent contamination. However, since the leach- the AITF determined that buried birds would be ate was both trapped and the volume increased more enveloped in an anaerobic environment and that than expected, it tended to percolate to the surface. minimal nitratifi cation would be produced from Plastic lining of the burial pits was therefore discon- burial. To ensure residents that their well water was 338 Avian Influenza safe, the water was tested weekly. Three tests were 1984. The carcasses had not decomposed over 18 routinely done to measure changes in well water: years but were mummifi ed. However, these mum- pH, total kieldahl nitrogen (TKN), and ammonia mifi ed carcasses tested negative for AI virus. As a

(NH3). result of this experience, the Virginia state Legisla- Prior to the digging of any burial pits, land on ture passed a law requiring landowners who wanted infected farms was surveyed for suitability of carcass to bury large numbers of dead animals on their land disposal. Approximately one-half of the infected to get a permit from the State of Virginia, and the farms had suitable burial sites. As a result, poultry permit required the deed to be amended noting the carcasses from farms that were deemed to have number and location of dead animals buried on the unsuitable land for burial pits were transported to premise. No farmer wanted to run the risk of deval- farms with pits certifi ed safe for burial. In anticipa- uing his or her land with burial sites listed on his tion that the outbreak might be greater than it sub- deed, thus eliminating use of on-site burial. This turn sequently proved to be, the AITF was issued a of events was not expected by the AITF, who were permit from the Bureau of Solid Waste Management in favor of on-site burial as the method of disposal. to use a designated 3.33-acre site in the George Washington National Forest. Clearing of land in LANDFILL the Forest for burial pits was opposed by much of Landfi lling is the oldest and most popular waste the public. Fortunately, the outbreak was contained disposal method for municipal solid wastes (MSWs), to 70 of the 600 farms in Rockingham County, having evolved from on-site burial methods. The Virginia, and additional burial sites were not ancient Greeks began landfi lling when they required necessary. citizens to take trash outside the city gates for dis- The ground water pH measurement test selected posal. Landfi lls have since slowly evolved into a by the AITF was the easiest and most reliable sur- sophisticated engineering science. Landfi lls are no rogate metric for assessment of ground-water pollu- longer big holes dug into the ground and fi lled with tion. Unlike surface water, ground water does not trash. Today, landfi lls are composed of multiple move quickly and contamination occurs only from cells, each lined with clay, special plastic, or a com- contaminated soil. If the pH was not within the 6.5 bination of both. Since 1993, Virginia has required to 7.5 range in fi eld testing, a water sample was sent that all new landfi ll cells have built-in leachate to the laboratory for further testing. Ground water management systems and gas management systems pH was measured at access points such as nearby to handle the methane gas produced as the waste wells. Decomposition of poultry generates water- decays (26). soluble organic acids that have a pH < 4 and poten- Garbage is transported to the landfi ll and crushed tially could leach into the water table. daily followed by coverage with a layer of soil to According to the 1984 DEQ AITF farm logs, a prevent access by pests and to reduce odors. These total of 15 farm wells were tested; however, only 5 facilities are regulated by state and federal laws and records were available for review. None of the must meet specifi c environmental criteria or face farms’ ground waters indicated any contamination closure. The Rockingham County, Virginia, landfi ll from the burial sites. Air contamination from on-site became a true sanitary landfi ll in 1998 (3). burials or landfi lls was negligible (3). In 1984, Virginia had three unique agencies that The costs for on-site burial disposal and landfi ll had responsibilities for addressing environmental charges in 1984 were approximately $25/ton or quality: the State Water Control Board (SWCB), the $142,000 for the entire outbreak. The total cost of Virginia Air Pollution Control Board, and the the AI outbreak in 1984 for the destruction of about Department of Waste Management (DWM). All 1.4 million birds was approximately $40 million three agencies had to come together to work on the dollars. Disposal costs account for less than 0.5% of environmental impacts of carcass disposal. These the overall cost of the AI from 1984. The major cost three agencies or boards were combined to form the of the 1984 outbreak was the loss of poultry fl ocks. Virginia Department of Environmental Quality One problem with on-site burial was recognized (DEQ) in 1993. in early 2002, as a result of construction of a new On March 29, 1984, water sampling was per- school on land where carcasses had been buried in formed on two domestic wells nearest the Rocking- 15 / Methods for Disposal of Poultry Carcasses 339 ham County Landfi ll. According to a Memorandum 15% of the 5,700 tons of poultry carcasses went to written on April 2, 1984, for the SWCB, the water the landfi ll since the risk of ground and surface sampling from these wells was the fi rst time a Vir- water contamination was serious due to poorly con- ginia agency investigated a landfi ll’s impact on the structed landfi lls. In addition, the landfi lls had no surrounding ground and surface waters. The site was capacity to contain the carcass fl uids contaminated in operation before the DWM-SWCB Memorandum with AI virus, Salmonella, and Campylobacter. In of Understanding pertaining to landfi ll approvals the outbreak of virulent Newcastle disease on the went into effect. “No engineering plans or specifi ca- West Coast affecting the states of California, Texas, tions are known to exist for this landfi ll.” According Nevada, and Arizona in 2002–2003, the U.S. Depart- to the memorandum, the site was not the most suit- ment of Agriculture’s primary method of carcass able for a landfi ll from the standpoint of impact upon disposal was by utilizing landfi lls. state waters. The site was originally used as an Landfi lling is an easy option, but the rate of unregulated dump. Because of the location of a decomposition and destruction of bird pathogens is portion of the landfi ll in two natural gullies, it was unknown. The tightly compacted earth in a landfi ll diffi cult to avoid runoff from the landfi ll draining can deprive aerobic bacteria of oxygen. In addition, into a nearby creek. Water pooled in a number of the chemicals that are in landfi lls may be toxic to ditches in newer fi lled areas. Whenever signifi cant the bacteria that are essential in carcass decomposi- rainfall or snow occurred, unrestrained runoff tion. More studies are needed to determine the effec- entered the aforementioned gullies. This runoff was tiveness of carcass decomposition in landfi lls. viewed as a hazard to surface water by the local When the AI outbreak occurred in 2002, the SWCB. Further, the landfi ll had been known to Virginia DEQ contacted all landfi lls equipped with leach during dry weather (25). The SWCB had con- leachate collection facilities. As of spring 2003, cerns regarding the landfi ll’s geologic formation about 70 landfi lls in Virginia accepted MSWs. which consisted primarily of limestone and dolomite However, not all met the defi nition of sanitary land- containing solution cavities and the SWCB deter- fi lls. Although substantial improvements had been mined the maximum depth of soil to be 30 ft. made to the Rockingham County Landfi ll since The initial use of a landfi ll for the disposal of AI- 1984, due to its small size and to keep the odor from infected poultry in the 1984 outbreak placed the becoming a problem, the landfi ll accepted only 3400 carcasses into a recently constructed trench used for tons of carcasses. The next closest landfi ll was in the disposal of domestic waste. A large outcrop of Page County, Virginia, about 40 miles distant, and limestone was exposed in the trench that extended due to its small size only 951 tons of carcasses were to within 250 ft of two domestic wells adjoining the accepted. Surrounding counties that were closer and landfi ll property. The law stated at that time that all had larger landfi lls did not accept infected carcasses on-site burials should be 300 ft from wells and 500 ft due to concerns regarding the spread of the disease from surface waters. The SWCB observed the other to uninfected poultry farms in the vicinity. Frederick thirteen carcass pits that were dug at the landfi ll but County, Virginia, accepted the least tonnage, 842 strongly advised against further landfi ll burials and tons, because of concerns that the waste treatment strongly urged the AITF to fi nd better means of plant could not handle the leachate. These concerns disposal. From that point on, detailed and accurate were proven correct. The amount of ammonia pro- records were maintained at the Rockingham County duced from the carcasses threatened to overwhelm landfi ll. the waste water treatment system, which consisted The two wells were sampled and did not indicate of water retention ponds followed by release into ground water contamination. However, heavy fecal surface waters. From April 16 to May 19, 2002, coliform contamination was found in nearby ponds. Frederick County received approximately 42 truck- There was a 240-fold increase in fecal coliform mea- loads of carcasses. The total amount of ammonia sured at surface waters near the landfi ll on April 24, tripled from normal levels of 57 mg/L to 157 mg/L 1984. and fecal coliform nearly doubled. To eliminate the In the 2002 AI outbreak in Virginia, 85% of possibility of contaminating the local surface water 16,900 tons of poultry carcasses went to landfi lls supply with leachate rich in ammonia and coliform (Fig. 15.3). In the 1984 AI outbreak in Virginia, only bacteria, regulations were enacted to contain the 340 Avian Influenza

Figure 15.3. Map of 1984 and 2002 AI outbreak farms in Virginia. Source: U.S. Department of Agriculture.

pond water. The pond water’s ammonia levels mea- million in tipping fees were paid to the two landfi lls, sured in March 2003 were too high for release into and had the outbreak lasted 60 more days, it would the local surface waters. Frederick County landfi ll have cost another $1 million. However, the major was the only landfi ll that was required to assess disposal cost was for transportation and not landfi ll leachate because the leachate did not go directly into fees ($45/ton). The transportation costs were quite a waste water treatment facility. Apparently, the high due to the distance traveled (4-hour round trip) waste water treatment plants at the other landfi lls to these two facilities with each truck averaging only were large enough that the leachate did not need to two loads of carcasses per day. These trucks had to be measured at those facilities. meet special requirements to haul carcasses, spe- Due to the lack of local landfi ll space, the remain- cifi cally, each truck was required to have a special, ing 64% of total carcass tonnage was shipped over leak proof rubber seal on the rear latches and a cover 160 miles to two large landfi lls, in Charles City and that could be tightly sealed to prevent feathers and Sussex County, Virginia. Charles City received debris from blowing out during transport. 4610 tons and Sussex County received slightly over As extra precautions to reduce the risk of trans- 4625 tons; both landfi lls were prepared to receive up mission during transport, carcasses were fi rst placed to 10,000 tons each. The AITF had 60 days reserve in large plastic bags and sealed with duct tape. The capacity, if the outbreak had continued at a disposal truck beds were lined with sawdust to absorb any rate of 188 tons of carcasses per day. Almost $1 carcass fl uids that leaked from tears in the plastic. 15 / Methods for Disposal of Poultry Carcasses 341

At the landfi ll, the bag was placed into small box- 1 like cells of various dimensions dug in old existing Air Burners, LLC trash sites and not in areas of active garbage disposal. These cells were created by excavating Air trenches, 6 ft deep by 4 ft wide and fi lling with car- 4 casses to a depth of 4 ft with the existing garbage 2 used to refi ll the trench. A cover of compacted soil was placed on top. Trucks were disinfected before 5 leaving the landfi ll area. 3 INCINERATION Incineration of MSWs is an old concept with appli- Figure 15.4. Diagram of box incinerator. cation varying from a simple open-pit burning to air Source: Norbert Fuhrmann, Air Burners, LLC, curtain incinerators to complex industrial incinera- www.airburners.com. tion plants. In its simplest form, the cost for incineration includes the costs of transporting the carcasses and excavating a hole. However, fuel to burn carcasses is expensive and not always avail- addition of accelerants such as diesel fuel, which has able. The cheapest fuel is often used, such as a BTU value of roughly 21,500 BTU/lb, in order to unwanted pieces of forest wood or commercial heat fresh-cut or wet wood enough so that it can wood that is no longer of resale value, or has a low evaporate moisture and burn effi ciently. British thermal unit (BTU) value due to high mois- Landfi ll disposal was being negotiated and not ture content. available when the 2002 outbreak fi rst occurred. There are fi ve combustible classifi cations of Desperately seeking an alternative to on-site burial, wastes. On a scale of 0 to 4, 0 (<10% moisture such a company that specializes in the clearing of woods as very dry wood or paper) is the best and 4 (>70% for land development was called upon to dispose of moisture such as carcasses) is the worst (8). Poultry the carcasses with air curtain incinerators (T-350 carcasses have a 70% moisture content, and thereby trench burner). a low combustibility of 1000 BTU/lb. However, the An air curtain incinerator is essentially a pit, or a greater the percentage of animal fat in the carcass, large metal box (lined with a refractory material), the more effi cient a carcass will burn, for example that has combustion air forced across the top and ruminants and swine have more body fat than poultry into the hole or box containing a burning mixture of and also burn more effi ciently. Animal fat has a fuel and carcasses (Fig. 15.4). This “air-curtain” of BTU value of 17,000 BTU/lb and is considered an high velocity air blowing across the top and into the excellent fuel source. For example, rendering (to be box (or hole) promotes a high temperature fi re and discussed later) can extract animal fats and resell more complete combustion of the carcasses. them as excellent grade 2 fuel. Therefore, some These boxes were effectively used to dispose of animals will burn more effi ciently than others based thousands of dead hogs that drowned when Hurri- on their fat content. However, poultry, especially of cane Floyd completely submerged hog farms in the age and body condition of most of the birds in North Carolina. Early in the 2002 Virginia outbreak, commercial broiler operations, have relatively low four air curtain incinerator boxes where promptly fat content. delivered to the selected incineration site, an active Incineration was attempted at the Rockingham rock quarry that was determined to be distant enough County, Virginia, landfi ll in 1984 as a pilot project from residential areas so that the odor of burning using open pit burning. However, the heat generated carcasses would have minimal impact on humans. by the fresh fi rewood at the site did not incinerate The incinerator boxes used wood as a primary carcasses at an effi cient, controllable rate. Freshly fuel source, but a petroleum fuel was used at the cut wood or wet wood has a 20% moisture content, outset to get the wood to burn due to poor-grade which lowered the wood’s BTU value from 8500 to wood transported from the landfi ll. After a few days, 6500 BTU/lb. Thus, open-pit burning requires the there was a suffi cient amount of higher-quality wood 342 Avian Influenza to begin burning the carcasses. Several tons of car- In order for incineration of carcasses to be practi- casses had accumulated on-site to be burned and cal and effi cient, a consistently high BTU fuel source liquid from the carcasses had leached and contami- needs to be available. Propane can incinerate car- nated a small pond located at the quarry. The pond casses at a faster rate as well as not contribute to the was quickly sterilized with 1000 lb of chlorine to weight of the ash. One ton of propane can incinerate ensure total destruction of the AI virus. 3 tons of poultry carcasses (28). Even at that, using After burning several tons of carcasses at an the above fi gures and fi guring 4.3 lb/gallon of extremely slow rate, it was determined that wood propane, it would require 756 tons of propane to from the landfi ll was not a good fuel source due to incinerate 2268 tons of birds. At a cost of $1.50/ its high moisture content. The incinerator boxes are gallon of propane, it would cost $529,000 for the specially designed with electric fans to blow air onto propane. wood to make the wood burn faster and smokeless Another experience at incineration of poultry car- (Fig. 15.4). However, due to the high moisture casses was at the Devil’s Lake, North Dakota, New- content of both the birds (which had become wet castle disease outbreak in August 1992. A task force from rain) and the wood, the birds created a terrible of U.S. Department of Agriculture–Animal and odor that drifted several miles. People living nearby Plant Health Inspection Service (USDA/APHIS) had to be moved temporarily into hotels. and North Dakota Department of Agriculture per- It was determined by trial and error that the best sonnel attempted to burn 27,000 mature turkey car- method of burning the carcasses was by layering casses. This was done in a large trench dug with a them upon wood pallets, thus allowing suffi cient air track hoe. The trench was approximately 6 ft deep, circulation to burn effi ciently. A combination of 20 ft wide, and 150 ft long. Wooden pallets and bales forest wood and pallets were used. The only draw- of hay/straw were placed under the carcasses and back in using pallets was the nails that remained in gelatinized gasoline and diesel fuel was applied to the ash. The nails were required to be removed by the pile and then set on fi re. The U.S. Forest Service using a power screener when the ash was to be reap- assisted the APHIS task force by applying the gela- plied to land as a rich source of nutrients. In addi- tinized fuel mixture which is used by the Forest tion, care had to be taken to prevent water collection Service for forest controlled burns. The fi re burned in the boards from rain or other sources. quite well for a number of hours. Unfortunately, An average of 78 tons of birds were burned daily after a few hours, it began to rain and continued for for 29 days. A total of 2268 tons were incinerated 36 hours. The fi nal result was charred carcasses only using 3023 tons of wood fuel at a cost of $317,616 partially consumed. Also, this burning produced a ($105/ton), including transportation costs. The total great deal of smoke, which would be unacceptable cost of transport of the carcasses from the farms to in most outbreaks areas. the quarry was estimated to be $267,908. A total of Although not used in the United States for carcass 113 truckloads transported 2,268 tons of poultry car- disposal, large industrial incineration plants have casses a distance averaging 15 miles ($2.5/mile). been used in Europe to dispose of poultry carcasses. Trucks were rented at $45/hour and worked an These are very effi cient plants that provide a consis- average of 1.5 hours. The rental costs of four incin- tent burning of the carcasses but typically use expen- erators, along with the cost of labor to maintain the sive fossil fuels. Incineration can be combined with fi res, to place carcasses into the fi re boxes, and to rendering which will inactivate the virus and produce remove the ashes was $810,389. The rental of the a dry meat meal byproduct of low moisture content power screener and its operation for several weeks and burns effi ciently. of screening ash cost an additional $75,283. A total of 4080 tons of strained ash was removed and used COMPOSTING as fertilizer on local farms at a cost of $173,466. One Composting is an aerobic process requiring a con- ton of carcasses cost approximately $477 to incin- stant supply of oxygen to maintain a proper tem- erate and required 1.35 tons of wood. Due to the perature to breakdown the carcasses. Composting high costs of incineration, and community com- is used as a year-round on-site carcass manage- plaints from the odor, air curtain incineration was ment option on many poultry farms to handle dis- terminated. posal of daily mortality. Composting can be managed 15 / Methods for Disposal of Poultry Carcasses 343 successfully on nearly any scale as long as the several proven designs for in-house composting of basic needs of the microbes—moisture, food, entire depopulated fl ocks. One mortality study oxygen, and temperature—are maintained. In the undertaken by the Maryland Agriculture Extension United States, composting of entire deceased Service demonstrated that in-house composting fl ocks has been undertaken for limited fl ock-disease would successfully dispose of birds infected with AI outbreaks. (22). However, there was reluctance from some In 1984, during the fi rst AI outbreak in the farmers to tie up poultry houses with compost, which Shenandoah Valley of Virginia, composting was demands several weeks of curing, and further mainly used to handle daily mortality on individual entailed applying the compost to the land in order to farms. However, the technology has evolved, reap its nutrient benefi ts. Having suffered sudden and has been used as a means of disposal in massive losses due to the outbreak and depopula- the United States of poultry on individual farms tion, commercial poultry operations favored decon- from H7N2 LPAI outbreaks in 2004. In Canada taminating the houses as soon as possible and a during the 2004 HPAI outbreak in the Fraser Valley return to production. In the initial phases of the of British Columbia, many of the infected fl ocks outbreak, one site was selected and used to compost were successfully composted on the farms of 50 tons of diseased turkeys in-house. Due to incor- origin. rect composting, the compost row dried out within Recent research indicates that composting has a month and was not successful. The AITF investi- widespread agricultural benefi ts, above and beyond gated other forms of composting, particularly one waste disposal. Compost-enriched soil can suppress that could be used outdoors with minimal impact plant diseases that attack root systems and ward off upon ongoing operations. A commercial windrow pests such as specifi c types of nematodes (11). In plastic bag composting system was investigated controlled studies, compost administered on fi elds (Ag-Bag, Warrenton, Oregon; www.ag-bag.com), has shown to increase crop yields. Thus, the benefi - which could compost 50 tons of carcasses inside a cial uses of compost can help farmers save money, plastic bag that was sealed except for a few vent reduce the use of some types of pesticides, and con- holes (Fig. 15.5). The plastic bag system was fi tted serve natural resources. with plastic pipes that run inside the bag and air was In 2002 in response to the AI outbreak in the forced through every few minutes by a timed electric Shenandoah Valley of Virginia, the AITF had blower. The system was tested on two premises: a

Figure 15.5. Canada H7N3 HPAI outbreak. (A) Loading machinery with accompanying plastic windrow composting bags. (B) Vents in plastic windrow composting bags allow exit of CO2 gas; 4-ft probe thermometer indicating proper composting temperature of 160° F. Source: J. Hahn. 344 Avian Influenza turkey farm and an egg-layer farm with 25 tons of composts should have been better monitored for infected chickens. moisture content, yet only the temperature was Moisture is an essential ingredient in composting. tracked and monitored. In order for composting to The presence of moisture is necessary for microor- work effectively, temperatures should be maintained ganisms to utilize organic material effectively. The between 57º and 63º C (135º to 145º F) (22). moisture content of a compost pile will be defi nitive Another important factor neglected in the turkey as to whether it is an aerobic or anaerobic process. and the egg-layer composts was the proper mix of Aerobic composting is best for the disposal of animal carbon and nitrogen, for the bacteria need a balanced carcasses, due to the fact that it is quicker and emits mixture of both. To be an acceptable and cost- fewer odors. A level of 40% to 50% moisture content effi cient means of carcass disposal, composting reis best for aerobic composting. If the moisture quires a minimum ratio of carbon to nitrogen (C : N). content exceeds 65%, the composting starts to Without such ratios, the compost is not viable as a become anaerobic (10). Moisture content below fertilizer and may produce intolerable odors. With a 35% starves composting bacteria and prevents ade- ratio below 15 : 1, bacteria will not use all of the quate composting. nitrogen and ammonia. If the ratios are greater than Subsequent tests of both the turkey and egg-layer 30 : 1, it takes longer for the composting process to composts revealed excellent nutrient value for fertil- occur (10). Improper ratios are most often attribut- izers (Table 15.2). Early tests for any evidence of able to uneven mixing of carcasses and litter, and in viable AI virus were negative. Samples were taken the cases of the turkey and egg-layer composts, there 1 year later to measure the nutrient levels in the are reasons to suspect that the compost components respective composts. After harvest of the material were loaded into the composting bags in a nonuni- from the windrow plastic bag system, further tests form manner. Using a mixer would produce a more were run to track any changes to the nutrients and uniform product going into the plastic windrow bag. compost. The moisture content of both composts At the turkey farm, the dried, transplanted in-house exceeded 65%, which is higher than recommended, compost constituted an unknown mix. At the egg- and could have been prevented by a supplemental layer facility, the only equipment available on-site carbon source such as wood shavings or straw to the was a front-end loader. The euthanized birds were mix. Once the plastic bags were opened, additional piled on the ground on top of a layer of litter, with effort was required to fi nish the curing process. Both a subsequent layer of litter covering the poultry pile.

Table 15.2. Results of Compost testing collected on March 2003. Chicken Compost lb/ton Turkey Compost lbs/ton

Ammonium nitrogen 1.06% 21.20 1.06% 21.20 Total nitrogen 1.99% 3,968 1.99% 3,968 Available nitrogen 30.29 30.29 Available surface nitrogen 21.81 21.81

Phosphorus as P2OS 1.99% 39.82 1.01% 36.14

Potassium as x2o 1.01% 20.29 0.51% 10.13 Calcium 1.45% 29.12 146% 29.15 Magnesium 0.29% 6 0.20% 4.00 Sulfur 0.24% 4.79 0.12% 2.48 Zinc 283.86 ppm 0.57 200.16 ppm 0.40 Copper 259.89 ppm 0.52 116.80 ppm 0.23 Manganese 349.29 ppm 0.70 220.17 ppm 0.44 Sodium 1542.16 ppm 3.08 987.49 ppm 1.97 Aluminum 471.56 ppm 3.94 1882.97 ppm 3.77 Moisture 69.28% 65.54% 15 / Methods for Disposal of Poultry Carcasses 345

The front-end loader scooped all three layers and time and temperature that were considered suffi cient dumped them into the plastic windrow bagging were determined from a table published by Lu et al. machine, which slowly fi lled the 200-ft bag. in 2003 (14). As a practical matter, in one study, at The cost of composting was calculated to be least 30º C (86º F) for a minimum of 3 consecutive approximately $60/ton. The way to successfully days has been shown to inactivate LPAI viruses in compost is to utilize an experienced commercial artifi cially added manures. However, inactivation is company that specializes in the compost process and not only temperature dependent but infl uenced by to monitor the moisture, temperature, and carbon concentration of virus and moisture content. With and nitrogen contents on a continual basis. As the high virus concentrations and moist conditions, 30º C compost matures, it will yield a valuable byproduct for 3 days may not be adequate to inactivate all AI to offset the cost. Other important advantages to virus in manure. In addition, proper composting proper composting include improved biosecurity greatly exceeds minimum temperature requirements because infected birds do not leave the farm. to inactivate AI virus. However, it is important that before composting Stage 2 was accomplished on the same farm and carcasses, local offi cials be contacted to fi nd out if outside the houses. It was usually started around day there are specifi c rules and/or permits needed for 7 after euthanasia. A compost pile was constructed composting. between two rows of highway dividers 15 ft apart In the Virginia outbreak during 2002, the poultry and as long as necessary to hold the composting industry elected for quick removal of the infected material. Passive aeration was utilized by laying per- carcasses from their premises; however, composting forated plastic pipe through the compost material. also offers a middle-ground disposal option. While The temperature in the pile was monitored for 28 in-house composting yields its valuable byproduct days. The temperature was expected to be a minimum only after a considerable period of time, the AITF of 30º C for at least 10 consecutive days to have a has shown that it does destroy the AI virus in a high degree of confi dence that the virus had been relatively short time, perhaps within 2 weeks. There- inactivated (21). However, proper composting after, the compost can be safely transported to should achieve between 57º and 63º C (135º to another site to complete the composting or fi nal dis- 145º F), which is more than adequate to inactive AI posal by burial or land application. viruses. In February through May 2004, there was an out- It seems that some compost piles did not “mature” break of HPAI in the Fraser Valley, around Abbots- as well as planned (to complete composting), due to ford, British Columbia, Canada. The 1.25 million inadequate aeration. Therefore, on June 1, 2004, the birds from the infected farms were disposed of by policy was instituted to turning many of the compost composting (46%), incinerating off the farm (40%), piles to improve aeration. and landfi ll usage (14%). As part of their effort to stop the spread of the For the composting, there were two different uses virus, all commercial poultry in the entire control of the composting process. One was used for infected area were to be depopulated (18,900,000 birds). This fl ocks and the other was used for disposing of nonin- was accomplished by the industry via controlled fected fl ocks that they were unable to send to slaugh- slaughter of birds from noninfected farms. Most of ter. For infected premises, a two-stage composting the birds were salvaged for their meat. However, process was used. Stage 1 was usually started a day approximately 1 million birds were not able to be or two after euthanasia and was done in the poultry salvaged and their carcasses were disposed of via house in windrows approximately 5 ft high and 10 ft composting in a central composting facility. This wide at the base. These piles were then covered with central composting facility was run by a contractor wood shavings. The piles were monitored for tem- that used a windrow plastic bag—type composting perature and had the objective of decreasing the system. This worked extremely well, in part due to amount of live virus through the initial heating of the the experience of the contractor. contaminated birds and litter. When the compost had reached a suffi cient temperature over a suffi cient RENDERING period of time to inactivate the AI virus, it was Rendering is a type of recycling (Fig. 15.6). Render- moved outside the barn to a stage 2 compost pile. The ing plants are facilities that process deceased animals, 346 Avian Influenza

NON-CONDENSABLE GASES

Dead Stock Carcasses Shop Fat and Bone CONDENSER ODOR CONTROL ENTRAINMENT SEPARATOR TO SEWER RAW MATERIAL RECEIVING SYSTEM Perc Vent Exhaust Vapor

Screw Press Vent CRUSHER

Unpressed Tankage SCREW PRESS Steam – 25 – 75 PSI COOKER BLOW TANK PERCOLATOR DRAIN P AN Pressed Jacket Condensate Screen Cracklings

Free Run Fat Screw Press Fat Oversize PRECOAT LEAF FILTER

CENTRIFUGE PROTEIN GRINDER CRUDE MEAL ANIMAL STORAGE ANIMAL FAT FAT Solids to Screw Press HOPPER SCREEN STORAGE TANK TANK

Figure 15.6. A ﬂ ow chart for the rendering process from raw to ﬁ nal product. Source: D.A. Franco, The Original Recyclers, 1996.

abattoir remains, and excess meat from grocers and In 1984, several rendering plants in the Shenan- eating establishments into a reusable byproduct. doah Valley, Virginia, declined to render infected These products are used in animal feed for numerous carcasses. In 2002, the same rendering plants species, mostly in the form of bone meal and animal approached the AITF and offered to render carcasses fat added to animal feed. In 1983, rendering plants to assist in the disposal crisis. The AITF elected not produced 7.9 billion pounds of product utilized in to render and chose landfi ll disposal for the majority pet food (34%), poultry feed (34%), hog feed (20%), of carcasses. At the time, the AITF was not familiar and cattle feed (12%) (18). Rendering plants have with rendering methods and was concerned with the in-place systems to recycle air and water wastes and cost and maintaining acceptable biosecurity at the the emissions of odors are minimal. rendering plant. Rendering plants are busy facilities The plants are equipped with devices to crush with a constant fl ow of large trucks moving in and whole carcasses into 2-in3 pieces, which allows for out of the plants coming from various types of farms. effi cient cooking. The pieces are steam cooked at Although, the fi nal product is sterile, there is a risk 250º to 275º F (121º to 132º C) for 90 to 120 minutes. of the feed becoming contaminated with infectious This temperature range has been proven to kill most agents during the cooling down phase after the feed infectious microorganisms including AI and ND is removed from the cookers, but this can be managed viruses. Avian infl uenza virus is destroyed at 140º F through separation of equipment and people involved in under 30 minutes (14). in the fi nal versus raw product. 15 / Methods for Disposal of Poultry Carcasses 347

Rendering of infected carcasses has been an effec- the infected zone, carcasses were stored in freezers tive tool in disposing of animal carcasses in Europe. until there was suffi cient rendering capacity. The On March 16, 2001, a rendering plant was opened carcasses could remain in the freezers for up to 1 in Widnes, Cheshire, England, to assist with the year. Some of the carcasses from noninfected farms disposal of livestock euthanized during the foot and in the infected zone were incinerated at an incinera- mouth disease outbreak. The plant’s sole purpose tion plant, while some carcasses were transported to was to render carcasses 24 hours a day, 7 days a a landfi ll enclosed in bags with a capacity of approx- week (4). imately 1000 kg. This is a compelling example of the capabilities The Netherlands used one rendering plant, located rendering plants can provide during a catastrophic in Son (Noord-Brabant). The total capacity of Son outbreak necessitating the disposal of large numbers was 8500 tons of high-risk or specifi c-risk carcasses of animal carcasses. Such plants can dispose of disper week. To facilitate rendering infected carcasses, eased carcasses in a sterile, effi cient manner. Ren- the Dutch increased the operating hours of the plant dering plants may be used to reduce diseased from 108 hours to 143 hours per week and sent high- carcasses to a low-moisture form, which is easier to risk material from slaughterhouses within the non- incinerate rather than the necessity of burning intact restricted areas to rendering plants in foreign carcasses. countries. When classic swine fever (CSF) was diagnosed in Germany on October 17, 2001, the German author- ALKALINE HYDROLYSIS ities euthanized 2054 animals of 651 confi rmed The term “alkaline hydrolysis” refers to the process cases in order to control and eradicate the outbreak. of thermally sterilizing under pressure and using an The majority of the hogs were disposed of at render- alkali solution (e.g., KOH or NaOH) to dissolve ing plants and the rendered product was then animal carcasses into an amino acid/peptide solu- destroyed. On May 23, 1997, the Netherlands used tion. The only solid structure remaining after the rendering as an option to dispose of hog carcasses processing of the carcass is bone “shadows,” com- infected with CSF (9). posed of calcium phosphate that no longer contain In spring 2003, the Netherlands, Belgium, and collagen and can be easily crushed by hand to form Germany were confronted with an outbreak of powder. Many veterinary diagnostic laboratories HPAI. The outbreak began in the Netherlands in across the United States use these machines to February and in less than a month had spread to dispose of low-volume infectious and noninfectious Belgium and Germany (20). By the end of the out- carcasses. break, 30 million birds had been euthanized in the Although the concept of alkaline hydrolysis is not Netherlands. This resulted in more than 54,000 tons new, recently designed machines can provide safe of poultry and 6,000 tons of eggs for disposal. Of removal of highly infectious material ranging from this, 46,000 tons were processed at rendering plants. birds infected with AI virus to cows having bovine However, the end-products of the rendering process spongiform encephalopathy (BSE) (23). Several were incinerated and not used for feed products. One types of machines are commercially available (WR2; thousand tons were sent to an incineration plant Waste Reduction, Inc., Indianapolis, Indiana) to directly, and 5500 tons were stored in cold storage handle differing amounts of waste materials. for eventual rendering. In addition, 1500 tons were In 2003, the U.S. Department of Agriculture transported to a landfi ll site. (USDA) purchased two mobile tissue digestors In the Netherlands 2003 outbreak, poultry car- (MTDs) to be used in various animal disease out- casses were rendered at a temperature of 133º C breaks (Fig. 15.7). Currently, an MTD is operating (271º F) for 20 minutes. All high-risk or specifi c-risk at the Veterinary Diagnostic Laboratory at the Uni- material was processed into meat-and-bone meal versity of Wisconsin. The unit has processed 575 (MBM) and tallow, which were incinerated at special tons of deer carcasses infected with or suspected of facilities. However, rendering plant capacity was not being infected with chronic wasting disease agent. suffi cient to meet the immediate demand. Therefore, Although proposed as a mobile system, the system the Dutch had to resort to alternative disposal works best if it is encased in a building and has a methods. For poultry from noninfected farms within temporary storage tank to handle the effl uent. There 348 Avian Influenza

Figure 15.7. Mobile alkaline hydrolyzer unit for carcass disposal. Diagram courtesy of WR2, Inc., www.wr2.net. are three different types of products in the effl uent: It appears the MTD is best suited for use in smaller (1) a slurry of small peptide chains, monosaccha- disease outbreaks. This appears to be because of the rides, fatty acids, and triglycerides, (2) the mineral relatively small amount of carcasses processed and portion of bone, and (3) the slow digesting cellulose. the cost of operating the machine. This can be illus- The slurry is disposed of by the city sewage district, trated by using the fi gures from the 2002 LPAI out- and the mineral and cellulose portions are sent to a break in Virginia. landfi ll. In the Virginia outbreak they had to dispose of Each MTD has the capacity to process 4000 lb of 16,920 tons of carcasses over a 90-day period. This carcasses in a cycle, and it is possible to process two is an average rate of 188 tons/day. One MTD unit cycles in 24 hours. The supplies needed to process can process 4 tons of carcasses/day (2 tons/cycle and 4000 lb of carcasses in a single cycle include: 2 cycles/day). Therefore, it would have required 47 MTD units to have processed the 188 tons/day 1. 4000 lb (480 gal) of water (16,920 tons over 90 days). 2. 560 lb of KOH (45% solution) or 400 lb of NaOH The cost to process the carcasses with the MTD (50% solution) units, using the cost of $0.20/lb ($400/ton), would 3. Steam for the heat and pressure cycle is produced have been $6,768,000. And, at a cost of $975,000/ by a steam generator, which can be fueled by unit, it would have cost another $45,825,000 to pur- multiple fuels, i.e., propane or fuel oil when chase the 47 MTD units needed. mobile, or natural gas if the unit is stationary (also if the unit is stationary, the steam may be IN SITU PLASMA VITRIFICATION supplied by a central steam supply) In situ plasma vitrifi cation uses a plasma torch that 4. Electricity produces a fl ame that exceeds 7000º C and is gener- 5. Sewage disposal ated from direct current electricity. This process 6. Solids disposal (landfi ll) ionizes the air to produce an extremely hot fl ame in an effi cient and cost-effective manner. In the 1980s, The cost of these supplies varies greatly depend- the technology was adapted with the realization that ing upon local availability, types of suppliers, etc. the torch not only could be used to test heat shields The Wisconsin Veterinary Diagnostic Lab estimates manufactured for NASA space shuttles, but also that their total cost of operating their MTD is about used to dispose of MSWs through a pyrolysis $0.20/lb ($400/ton) (16). process, “ex situ” plasma process, which does not 15 / Methods for Disposal of Poultry Carcasses 349 release the environmentally unfriendly gases as The in situ plasma vitrifi cation technology has been occurs when burning fossil-based fuels. It has been specifi cally modifi ed to theoretically accommodate determined that by treating MSWs by ex situ plasma carcass disposal (6). The modifi ed plasma process processing, energy is developed in the form of requires burial of the carcasses followed by the use of hydrogen and carbon monoxide gas. Carbon mon- the plasma torch to pyrolize the carcasses within the oxide is a combustible gas, and is used as a fuel soil to generate gaseous effl uent (Fig. 15.8). In situ when it is an effl uent from the plasma gasifi cation plasma vitrifi cation has the potential to safely dispose process. Theoretically, both gases, hydrogen and of carcasses from diseased animals in a timely, effi - carbon monoxide, can be used to generate the elec- cient, and safe manner that could meet environmental trical power required by the torch, and in addition, emissions and solid residue regulations (6). generate enough electricity for sale to the electrical As an example of the effi ciency of plasma torch grid. This increase in electrical energy results from technology, consider the disposal of 188 tons/day of the large volume of gases created from the gasifi ca- infected poultry carcasses. Using four 2.4 MW Mark tion of the MSWs. The resulting pyrolyzed MSWs Eleven torches manufactured by Westinghouse cools into a rock-like or sand-like residue, which can Plasma Corporation ($2.5 million per torch), oper- be sold as road gravel or processed into brick. The ated by fi ve persons, and available electricity, MSW plant is offset by three income streams: the response time to an outbreak could be minimal. Col- MSW tipping fees, the sale of the gas or electricity, lection hoods have been developed by the U.S. and the sale of the rock or sand residue. A commer- Department of Energy to neutralize harmful gases. cial MSW plasma gasifi cation plant is currently Purchase of the gas treatment system is $1.5 million under construction in Ottawa, Canada. The facility plus $0.5 million for one hood. Thus, the total start- will process about 94 tons/day of MSWs and produce up costs for plasma torch technology is $12 million enough electricity to power 3800 households. The (7). There are several benefi ts of in situ plasma vit- electricity produced will be sold to the electrical rifi cation disposal, including (1) no odor is emitted, grid (2). (2) low risk of groundwater contamination, and (3) Because there is no leachate from the residue, the minimal air pollution when using a collection hood. plasma technology has tremendous potential in Because the vitrifi cation can be done on-site, bios- reducing a variety of organic waste products includ- ecurity risk is low and eliminates concerns about ing poultry carcasses to a nonharmful construction how fast and thorough the carcasses will decompose product. using on-site burial alone.

Plasam Torch

Plasma Torch

Hood Off-Gas Treatment Subsidence

Backfill

Subsidence Pipe Pile Vitrified Zone Contamination Melting Zone

Figure 15.8. Diagram of in situ plasma vitriﬁ cation system. Source: L. Cicero, Georgia Technical Research Institute. 350 Avian Influenza

COMPARATIVE ANALYSIS OF destruction of carcasses and the AI virus uses a DISPOSAL METHODS natural aerobic decomposing process, and (3) there Determining the most appropriate method or is a favorable public perception when done properly. methods to use in disposal is dependent upon analy- The least complex method of disposal is on-site sis of fi ve critical factors: (1) fi nancial costs, (2) burial. A simple pit is excavated, carcasses are environmental suitability, (3) public and industry buried, and no more work is required. The condi- perceptions, (4) complexity, and (5) availability. tions of the soil greatly infl uence the rate of decom- First, the overall cost of the disposal method must position. If the soil is dry and rich in clay, it may be examined and should include costs of raw mate- take many years for the carcasses to completely rials needed for processing, equipment, labor, trans- decompose. However, local, state and/or national portation of carcasses to a disposal site; energy regulations may prohibit or discourage on-site burial required to dispose of carcasses; and actual process- for environmental reasons. ing costs. Second, the environmental impact factors Incineration has many advantages but requires must be assessed and should include assessment of special attention to be paid to the raw materials. potential ground water and surface water contamina- Such as, when using special air curtain incinerators, tion, and air pollution generated under practical only dry solid fuel or readily available liquid fuel biosecurity controls. Third, the perceptions of public should be used, and the animal carcasses should not and industry must be evaluated and will include odor be wet. If incineration is done incorrectly, air quality and visual issues and public criticism of the industry and air pollution will be unpleasant byproducts and approach. Fourth, the complexity of the disposal will result in prohibited use. process must be assessed and determined it is fea- If carcasses must be disposed of off-site, care sible with particular attention to any potential dis- must be taken to bio-contain the contaminated car- posal problems of byproducts, re-salability of casses during removal and transportation. Landfi ll products generated by the process, and any unwanted disposal is the simplest solution, but requires spe- wastes of initial disposal requiring further process- cially constructed and designed landfi lls to prevent ing. Finally, the availability of each method needs leachate from contaminating surface or ground to be assessed for location. For example, some local- water. Contingency contracts between local and ities may have legal regulations that prohibit on farm state governments and landfi lls should be entered burial thus making on farm burial unavailable. Also, into prior to outbreaks. alkaline hydrolysis and in situ plasma vitrifi cation Rendering is a viable off-site disposal option. technologies are considered experimental possibili- However, rendering plants will need to have con- ties and will need further development before they tracts established and emergency operational bios- can be available for large-scale use. ecurity procedures to avoid cross-contamination of Comparative analysis of each of the fi ve factors the fi nal sterile produce with incoming raw material. is a subjective decision-making tool based on actual This fi nal product can be used as a sterile protein experience, oral histories, and the research, whereby meal in animal feed, an alternative source of fuel, or a case-by-case method may be assessed differently. perhaps incorporated into fertilizer. Plasma vitrifi cation and alkaline hydrolysis have CONCLUSION great potential for future use if large-scale equip- Each method of disposal has advantages and disad- ment can be constructed and maintained. However, vantages based on location circumstances. Gener- at this time, they are not feasible for large-scale ally, the greatest cost in disposal of animal carcasses animal carcass disposal from catastrophic events. is transportation to a disposal site. Disposing on the The best strategy for carcass disposal is to plan farm greatly reduces the need for transportation and ahead. Each local, state, and national government associated costs and decreases the risk of a biosecu- should develop disposal plans, which must include rity breach. backup plans, as part of emergency and disaster On-site composting is the most environmentally preparedness. The lines of communication and safe disposal method and it provides the highest re- cooperation must be kept open between the various use value of the residue. With this method there is (1) parties involved, especially the animal industries, virtually no anthropogenic energy requirement, (2) and the state and federal government entities. 15 / Methods for Disposal of Poultry Carcasses 351

ACKNOWLEDGEMENTS acdisposal.html. EchoChem: Delia, Alberta, The use of trade, fi rm, or corporation names in this Canada. Accessed March 3, 2007. page and linked publications is for the information 11. EPA Website. 1997. Innovative uses of compost: disease control for plants and animals. EPA530-F- and convenience of the reader. Such use does not 97-044. Available at www.epa.gov/epaoswer/non- constitute an offi cial endorsement or approval by the hw/compost/disease.pdf. U.S. Environmental University of North Carolina, Chapel Hill, or the Protection Agency: Washington, DC. Accessed U.S. Army of any product or service to the exclusion March 3, 2007. of others that may be suitable. 12. Harder, T.C., and O. Werner. 2006. Avian infl u- enza. Infl uenza report. Available at http://www. REFERENCES infl uenzareport.com/ir/ai.htm. Flying Publisher: 1. Bean W.J., Y. Kawaoka, J.M, Wood, J.E. Pearson, Tygerber, South Africa. Accessed March 3, 2007. and R.G. Webster. 1985. Characterization of 13. Kansas State University, Purdue University, and virulent and avirulent A/chicken/Pennsylvania/83 Texas A&M University. 2004. Carcass disposal: a infl uenza A viruses: potential role of defective comprehensive review. Available at http://fss. interfering RNAs in nature. Journal of Virology k-state.edu/research/books/carcassdispfi les/ 54:151–160. Carcass%20Disposal.html. National Agricultural 2. Bryden, R. 2007. PlascoEnergy Trail Road Dem- Biosecurity Center, Kansas State University: onstration Facility. Available at http://www. Manhattan, KS. Accessed April 21, 2007. zerowasteottawa.com/docs/PlascoEnergy 14. Lu, H., A.E. Castro, K. Pennick, J. Liu, Q. Yang, Group12apr07.pdf. Plasco Energy Group: Ottawa, P. Dunn, D. Weinstock, and D. Henzler. 2003. Ontario, Canada. Accessed 20 April 2007. Survival of avian infl uenza virus H5N7 in SPF 3. Brglez, B. 2003. Disposal of poultry carcasses in chickens and their environments. Avian Diseases catastrophic avian infl uenza outbreaks: a compari- 47:1015–1021. son of methods (technical report for Master of 15. Monke, J. 2006. CRS Report for Congress, Public Health). University of North Carolina: Avian Infl uenza: Agricultural Issues, Order Chapel Hill, NC, p. 105. Code RS21747. Available at http://fpc.state.gov/ 4. Brown, P. 2001. Slaughter policy brings fresh documents/organization/65002.pdf. Congressional problems, Special report: Foot and mouth dis- Research Service, The Library of Congress: ease. Available at http://www.guardian.co.uk/ Washington, DC. Accessed March 5, 2007. footandmouth/story/0,7369,457597,00.html. Guard- 16. Rutta, S. 2007. Veterinary Diagnostic Laboratory, ian Unlimited: London. Accessed March 3, 2007. University of Wisconsin, personal communication, 5. Buish, W.W. 1984. Letter from AI Task Force April 13, 2007. Director to the Executive Director of the Virginia 17. Senne D.A., B. Panigrahy, and R.L. Morgan. 1994. State Water Control Board. U.S. Department of Effect of composting poultry carcasses on survival Agriculture: Riverdale, Maryland. of exotic avian viruses: HPAI virus and adenovirus 6. Circeo, L.J. 2004. Plasma disposal and decontam- of egg drop syndrome-76. Avian Diseases 38:733– ination of diseased animal carcasses. Georgia Tech 737. Research Institute: Atlanta, GA. 18. Shah, P.K. 2005. Recycling of slaughterhouses 7. Circeo, L.J. 2007. Georgia Tech Research Insti- waste (rendering plants). Available at http:// tute. Personal communication, April 4, 2007. jainsamaj.org/literature/recycling-301002.htm. 8. CMTS Incinerators. 2007. Frequently asked ques- Federation of Jain Associations in North America: tions. Available at http://www.cmtsproduct.com/ Raleigh, NC. Accessed March 5, 2007. enquiry/faq.asp. CMTS: Selangor, Malaysia. 19. Smeltzer, M.A. 2003. Control of low pathogenic Accessed March 3, 2007. avian infl uenza in Virginia. In: Proceedings of 9. de Klerk, F. 2007. National Inspection Service 52nd Western Poultry Diseases Conference. Uni- for Livestock and Meat/Food and Consumer versity of California: Davis, CA, pp. 3–4. Product Safety Authority, Ministry of Agricul- 20. Stegeman, A., A. Bouma, A.R.W. Elbers, M.C.M. ture, Nature and Food Quality. The Hague, de Jong, G. Nodelijk, F. de Klerk, G. Koch, Netherlands: personal communication, March 14, and M. van Boven 2004. Avian infl uenza virus 2007. (H7N7) epidemic in the Netherlands in 2003: 10. EcoChem. 2007. Composting: an environmentally Course of the epidemic and effectiveness of control safe option for animal and poultry carcass disposal. measures. Journal of Infectious Diseases 190:2088– Available at http://www.ecochem.com/t_ 2095. 352 Avian Influenza

21. Stepushyn, K. 2007. Canadian Food Inspection 26. Virginia State. 1993. Virginia Waste Management Agency, personal communication, March 23, 2007. Act, Chapter 10.1-1413.2. Available at http:// 22. Tablante, N.L., G.W. Malone, F.N. Hegngi, L.E. www.deq.state.va.us/waste/wastereg2000st.html, Carr, P.H. Patterson, G. Felton, and N. Zimmer- Virginia Department of Environmental Quality: mann. 2002. Guidelines for in-house composting Richmond, VI. Accessed March 6, 2007. of catastrophic poultry mortality, Fact Sheet 801. 27. Whiteford, A.M., and J.A. Shere. 2004. California Available at http://www.agnr.umd.edu/MCE/ experience with exotic Newcastle disease: a state Publications/PDFs/FS801.pdf. Maryland Coopera- and federal regulatory perspective. Proceedings of tive Extension: College Park, MD. Accessed April the Western Poultry Disease Conference 53:81– 15, 2007. 84. 23. Taylor, D.M. 2000. Inactivation of transmissible 28. Wineland, M.J., T.A. Carter, and K.E. Anderson. degenerative encephalopathy agents: a review. The 2006. A cost comparison of composting and incin- Veterinary Journal 159:10–17. eration as methods for mortality disposal. PS Facts 24. Virginia State Water Control Board. 1984. On-site #25. Available at http://www.ces.ncsu.edu/depts/ disposal for poultry waste. Memorandum, January poulsci/tech_manuals/composting_incineration. 26, 1984. html. North Carolina Cooperative Extension 25. Virginia State Water Control Board. 1984. On-Site Service: Raleigh, NC. Accessed March 5, Disposal for Poultry Waste. Memorandum, April 2007. 23, 1984. 16 Farm and Regional Biosecurity Practices

Carol J. Cardona

WHAT IS BIOSECURITY? have protected the fl ocks or herds. The costs depend Biosecurity practices are series of human behaviors, on the disease, the value of the animals, and the type procedures, and attitudes that create barriers for of production system involved. disease agents. In public health parlance, actions The alternatives to protecting birds or animals with the same goals are called social distancing. In with biosecurity include vaccinating where possible, writings for lay audiences, biosecurity practices are treating diseases when they occur, frequent depopu- often divided into categories under three goals: isolations, or living with disease. Vaccination, when lation, sanitation, and traffi c control (10). Isolation used in the absence of biosecurity, is really a way to refers to the confi nement of animals within a con- sustain economical production while living with trolled environment. Traffi c control includes both infection. When vaccination is used in conjunction the traffi c onto the farm, off the farm, and the traffi c with biosecurity, the strategies may well be additive patterns within the farm. Sanitation addresses the and can jointly be an effective barrier to infection cleaning and disinfection of materials, people and (29). Treatment, although widely used as an alterna- equipment entering the farm, and the cleanliness of tive to biosecurity, has many limitations. For many the personnel on the farm. These divisions are useful diseases, especially those caused by viruses, there in that they allow poultry producers to evaluate their are no treatments available. For bacterial diseases, own biosecurity programs based on an assessment the use of antibiotics can limit the marketing of eggs of progress toward the goal of the cumulative or meat from treated animals. The result is that treat- actions. For example, under sanitation, a biosecurity ments, if available, are expensive when one consid- plan should include all of the procedures for the ers not only the cost of medications but also the loss cleaning and disinfection of the various types of of productivity. Maintaining a rapid turnover rate is equipment, buildings, and vehicles used on the farm. another option to biosecurity. Although this tactic is To assess the implementation of sanitation goal of not possible in most production farms, it is indeed farm’s plan, a producer has only to inspect cages or the strategy for disease control employed by many vehicles for cleanliness. bird wholesalers and retailers. By having a rapid turnover rate, live poultry marketers who bring birds ALTERNATIVES TO BIOSECURITY together from many sources, avoid having birds that Not all animal populations are protected by biosecu- appear unhealthy, although many of them may be rity. In fact, worldwide, the vast majority of domes- infected or become infected as a result of mixing. ticated animals are not protected by enough The fi nal alternative to biosecurity is simply what biosecurity to prevent most occurrences of disease. most of the world does, and that is to live with For commercially raised animals, the alternatives to disease. If this is the accepted approach, then pro- biosecurity are often considerably more expensive duction systems will always be small and use than the cost of the biosecurity actions that could resources ineffi ciently.

THE ELEMENTS OF BIOSECURITY for the farm. In many situations, farm employees Biosecurity for a farm or an operation is achieved may be called upon to make decisions that can affect by addressing all three of its goals: isolation, sanita- biosecurity. For example, when someone arrives at tion, and traffi c control. This is done through the the gate of the farm asking to purchase eggs, the combined use and consideration of the farm envi- farm hand will ultimately make the decision of rons, the physical structure of the farm, supplies, and whether to allow the visitor to come in. The chances farm and nonfarm personnel. These elements of that the employee will make the right decision for biosecurity can be categorized as conceptual, struc- biosecurity can be greatly improved by educating tural, and operational components (31). A complete the worker about the biosecurity plan and its goals. discussion of these biosecurity designations is In the classifi cation of biosecurity proposed by included in Chapter 23 (Control of Low Pathogenic- Shane (31), this falls under the category of concep- ity Avian Infl uenza). tual biosecurity. The geographic location or environs of a farm can The elements of biosecurity are used together to infl uence biosecurity. Farms in remote areas may achieve the goals of isolation, sanitation, and traffi c not need fences or signs to achieve isolation. In control for any given premises. The actions, people, contrast, for farms in densely populated poultry supplies, and structures that create biosecure barriers areas, fences, locked gates and signs may still not for a farm should then be compiled to create a bios- be suffi cient to achieve isolation. An excellent dis- ecurity plan. cussion of considerations in the selection of a site and design of a poultry farm are reviewed in Zander DEVELOPING A BIOSECURITY PLAN (41). Biosecurity can be applied to a broad variety of Supplies and physical changes made to the farm poultry and other avian species in varied environ- to enhance biosecurity are often the only elements ments with diverse production systems or environ- enumerated in biosecurity surveys and checklists. ments. But, because each is different, and thus how Included in the list of supplies should be disinfec- disease agents might enter differs, every facility or tants appropriate to their proposed function, high operation must have its own specifi c biosecurity pressure sprayers, dedicated clothing for employees, plan. Every plan should consider the management pest control products, protective clothing for visi- style of the farm, the geographic location of the tors, etc. Although people sometimes think they are farm, the types, proximity and density of neighbors, the central need for an effective program, the sup- the value and replaceability of the fl ocks, and the plies needed to create biosecure barriers are limited resources of the farm. and, for many, there are effective substitutes. A A biosecurity plan should be both general, for common question that arises on the topic of biosecu- emerging disease threats, and specifi c, for current rity supplies is about disinfectants. Many people disease threats. For general threats, addressing the focus on making sure they are using the proper dis- goals of biosecurity is probably all anyone can do. infectant when their real concern should be about However, for specifi c disease threats, one should the correct use of the disinfectant. Once the focus is consider how the agent is transmitted. As an example, one of when and where a disinfectant can be effec- understanding that rodents are major transmitters of tive, then it is clear that downtime or changes in Salmonella enterica serovar enteritidis (SE) is traffi c patterns can substitute in situations when a important (19), and therefore, an effective SE bios- disinfectant is not available or cannot be used. ecurity plan will include rodent control. For low The fi nal and most critical element of biosecurity pathogenicity avian infl uenza (LPAI) viruses, is people. Among the people who contribute to a knowing the lessons learned in Minnesota in the farm’s biosecurity program are employees of the 1980s and 1990s, where waterfowl interacting with farm and the company, neighbors, business partners, free-range turkeys resulted in frequent infections, is vendors, and regulatory personnel. The most impor- important (Chapter 23). Thus, an LPAI virus bios- tant of these in determining the success or failure of ecurity plan should include ways to prevent interac- a farm’s biosecurity program are the employees tions between free-fl ying waterfowl and domestic working on the farm. These employees must be poultry populations. This is especially important for knowledgeable about the specifi c biosecurity plan species that are closely related to free-fl ying birds 16 / Farm and Regional Biosecurity Practices 355 that carry avian infl uenza (AI) viruses, like domestic determine. Deciding when it is safe to allow for ducks, and for those species known to be susceptible interactions and when it is not should be based on to infection with waterfowl origin AI viruses, like considerations of disease agent stability in the envi- turkeys (see Chapter 23, Control of Low Pathoge- ronment and on mechanical vectors, prevalence of nicity Avian Infl uenza, for more information on risk infection and shedding of disease agents by infected assessments and biosecurity audits). hosts, and complete knowledge of where and how To determine which factors should be addressed human traffi c moves, or a best guess. to prevent exposure to a specifi c disease agent, risk Once interactions that will be allowed is deter- assessments conducted during or after previous out- mined, either because they pose little risk to the breaks can be very helpful. Determination that ren- birds or because they are necessary, then each type dering was a risk factor for H7N2 LPAI during the of visitor should be specifi cally addressed in the Virginia outbreak of 2002 was helpful in pointing biosecurity plan. Some visitors may be allowed out that bird disposal could be associated with when the risk of disease is low but excluded if the disease transmission (23). However, these types of farm is placed on alert. The critical needs for the studies are fairly specifi c to the outbreak time and farm are strategies to lower the disease risks posed place and may not be more than just generally by needed visitors and those should be a part of the helpful in creating a disease-specifi c biosecurity biosecurity plan. So, to use the earlier example, the plan. What is truly needed is an expert risk assess- plan should list how the risk associated with feed ment for each premises in its current state. A much- delivery will be mitigated, i.e., that protective cloth- needed general risk assessment tool has been created ing and foot coverings will be provided for the (15) and is fully described in Chapter 23. In addi- drivers, and the trucks will be cleaned and disin- tion, a commercial farm biosecurity audit tool is fected at the farm entrance. described in Chapter 17 and Appendix 17.1. These Finally, the biosecurity plan for a farm should be risk assessment tools can be adapted for use with practical to create, modify, and ultimately to use for most avian pathogens and to all poultry facilities, the people who need it. Many operators have com- but like all tools, it should be used by people with puters and are adept at using modern technical some level of expertise. So, one of the best advancements and so they can generate biosecurity approaches to developing a disease-specifi c, prem- plans that have a very professional appearance. ises-specifi c, species-specifi c biosecurity plan, is to However, there are many bird production sectors enlist the help of a qualifi ed poultry veterinarian to where managers do not have computers and need apply these tools to a specifi c premises. simpler ways to make, and modify biosecurity plans. For most producers and veterinarians, preventing One such method is used by producers participating the interactions that may result in disease transmis- in the Live Bird Market LPAI control plan in sion is conceptually easy. For example, if a disease California. The individual plans consist of note- is being transmitted through a region by the move- books kept by the producers outlining their biosecu- ments of people, then conceptually, cutting off any rity policy according to a set of standardized issues visitors to a farm should protect the fl ocks. However, (Table 16.1). At the top of a page or a section, the there may be essential visitors who need to come to issue to be addressed is listed. The practices used on the farm, like off-farm employees or the feed truck the farm are then handwritten onto the pages of the driver. Determining which visitors or interactions notebook by the farmer or employee. The notebooks are dangerous is tricky. In the end, one can never provide a fl exible format that is easy for small pro- know for sure. Additionally, it is often the essential ducers to use. If the plan is a part of a quality assur- visitors who are the biggest disease risk to the farm. ance program, then additional issues to be addressed Unfortunately, the decisions about what exposures can be added to the notebooks. to prevent are often based on convenience or fi nan- A biosecurity plan should be developed and cial considerations and probably less frequently on written for every premises. A written biosecurity science. Particularly in commercial poultry opera- plan helps farm operators and employees maintain tions, there are always practical and fi nancial con- consistency in their policies and, once recorded, straints on the implementation of biosecurity, making others can review and contribute to, and possibly where lines are drawn complicated and diffi cult to improve, the plan. A written biosecurity plan is a 356 Avian Influenza

Table 16.1. Small producer biosecurity plan template. Category Issues addressed in the plan

General Who is responsible for the plan? Description of the farm and operation, location Describe how you monitor the health of your fl ocks. Isolation Describe how farm and delivery personnel are separated. What is the source of birds, and how are they introduced to the farm/fl ock? How often do you check the testing records of the source fl ock to ensure that they are free of avian infl uenza? (You need to keep copies of tests provided by your supplier in your records.) Describe your fl y and rodent control programs. How are they monitored? Traffi c control Describe signs to keep visitors out (how many and where are they placed), logbooks used, protective clothing used (type and source), and visitor policy, i.e., a visitor is not allowed if they have been around birds in 24 hours. Sanitation Describe cleaning and disinfection programs for buildings, equipment, and vehicles (frequency and site of cleaning and disinfection activities for all equipment and vehicles) Disinfectants used, concentrations

tremendous aid to regulatory agencies charged with in organic substrates may survive for 2 weeks or controlling foreign animal diseases. A written plan longer. These fi ndings are consistent with fi eld tests allows outsiders to see how biosecurity is main- conducted during the 2002–2003 exotic Newcastle tained using the farm’s available resources and in disease (END) eradication campaign in which the the farm’s unique manner. END virus was found to survive in poultry manure for 17 days (20). To create a science-based biosecu- Developing a Biosecurity Plan Based on Science rity recommendation from these data, one might determine that complete cleaning and disinfection is Pathogen Survival in the Environment required for any equipment that comes onto the One of the key considerations in developing a farm. It might also be determined that poultry farms science-based biosecurity plan is pathogen survival should not allow feces-contaminated cages, egg outside of its host. Generally speaking, as enveloped racks and trays, vehicles, and equipment to come RNA viruses, AI viruses will not persist in the envi- into contact with their fl ocks unless they have been ronment for long periods of time. The exception to rested for at least 2 weeks. A failure to remove fecal this general rule is survival in cold climates, where material from cages used to haul spent fowl or birds the virus may remain viable over months in a cold headed for the live bird markets (LPMs) is not and wet or frozen state. Previous studies have docu- uncommon and, thus, they are a likely source of mented survival under refrigerated laboratory condi- viruses that infect fl ocks on the farm to which they tions of up to 4 weeks (35). Additionally, there is are returned. anecdotal evidence that AI viruses may be transmit- In contrast to the studies conducted with high ted from the environment to wild waterfowl in their titers of virus in larger volumes, AI viruses on sur- natural setting after overwintering (18). With these faces do not survive as long. Studies in human hos- exceptions fi rmly in mind, it can be said that the pitals have demonstrated survival times of 24 to 48 virus is inactivated within days or weeks under most hours on porous and less than 8 to 12 hours on non- environmental conditions. Figure 16.1A shows the porous surfaces (3). Similar studies have also been gradual decline in viral titer in allantoic fl uid kept at conducted with LPAI viruses under laboratory con- room temperature under laboratory conditions (37). ditions using materials commonly found on poultry These data would suggest that viruses in high titer farms (37). Times are grouped for infectious virus 16 / Farm and Regional Biosecurity Practices 357

A 1.00E+08

1.00E+07

1.00E+06

1.00E+05

1.00E+04 virus titer virus 1.00E+03

1.00E+02

1.00E+01

1.00E+00 0 1 2 3 9 12 15 18 time (hrs incubation)

B 1.00E+04

1.00E+03 Absorbent non-absorbent 1.00E+02 virus titer virus

1.00E+01

1.00E+00 0 24 48 72 144 216 time (hrs incubation)

Figure 16.1. Infl uenza A virus survival times under laboratory conditions at room temperature. (A) Recovery of infectious virus from allantoic fl uid over time. (B) Infl uenza A virus survival times on absorbent and nonabsorbent surfaces. (Adapted from Ref. 37.)

survival on absorbent (cotton and polyester fabric tible birds can occur when commercial egg produc- and wood) and nonabsorbent surfaces (steel, plastic, ers swap pullet hauling equipment or when egg tiles, gumboot, etc.) in Figure 16.1B. These data collection equipment is not cleaned and disinfected indicate that viruses in small volumes survive longer before its return from the egg processing plant. on nonporous than on porous surfaces but not beyond a week. Designing a biosecurity plan with these Pathogen Transmission studies in mind, a farm should have a policy not to The means by which a disease agent is transmitted use equipment from another without cleaning and between susceptible populations is one of the key disinfecting it or resting it for a few days or more, pieces of information that should be considered even if it looks clean. The transmission of small when designing a prevention strategy. Avian infl u- amounts of infectious virus on surfaces to suscep- enza viruses are primarily transmitted to naïve hosts 358 Avian Influenza through indirect contacts with infected birds of the usually linked through their environment. So, con- same species. In the case of commercial poultry tamination of bodies of water with feces is among fl ocks, these indirect contacts often result in the the most likely means of AI virus spread through movement of infectious fecal material. Fecal mate- a fl ock of free-fl ying birds. A compilation of the rial can and does move on improperly cleaned ways through which AI viruses are transmitted equipment, on feet not covered with clean boots, or within and between avian populations is shown in on vehicles coming onto a farm without being dis- Figure 16.2. infected, among other means. The movements of infectious material between farms of domesticated Populations Associated with Disease Risk birds, usually follow the movements of people and An important part of a biosecurity plan should equipment because that is how the farms are linked include how a fl ock or farm is isolated from those to each other. In contrast, wild birds in a colony are birds that might be infected. Determining which

3 2 1 6 4 5 2 3 7 8 f a b 1 2 4 9 6 b a f g b b a c d e

POULTRY WILD BIRDS c e e g c e f g h g d h a f b 3 5 7 9 10 f 1 2 4 10 Barriers to transmission 8 9 1 Hand-washing / good hygiene Sources of infection 2 Personal protective equip. / dedicated clothing a Feces 3 Vehicle & equipment disinfection b Contaminated environment / body of water 4 Enclose birds / avoid attractants c Contaminated equipment / vehicles 5 Restricting movements on and off premises d Eggs & meat 6 Depopulation e Secretions 7 Vaccination f Carcasses / live infected birds 8 Proper disposal of carcasses g Pests (insects, rodents, cats, etc.) 9 Pest control h Contaminated clothing / footwear 10 Avoiding contaminated environment / carcasses

Figure 16.2. The modes of transmission of AI viruses and the barriers to spread. The likelihood of transmission is indicated as either small, medium, or large, depending on the size of the circle. The impact of a specifi c barrier on the likelihood of transmission is indicated by the size of the triangle: small, medium, or large. 16 / Farm and Regional Biosecurity Practices 359 populations present a risk, is a critical step in the Birds that link populations process. Among the populations that should gener- Birds that provide a link between one or more oth- ally be avoided are those that have been involved in erwise separate populations should always be con- previous outbreaks, populations that interact with sidered at high risk of infection. Of particular more than one other group, birds that travel long interest, are birds that link domestic and interna- distances, and birds in which infection may go tional populations (Fig. 16.3). Gamefowl, such as undetected. fi ghting cocks, link domestic and international populations, and historically, they have been associated Historical risk with the importation of foreign animal diseases (25). One of the best measures of the potential for a pop- Similarly, pet birds are both theoretically and his- ulation to be diseased or infected, is to examine the torically high risk (16, 26, 39). Birds that move, pose historical record of outbreaks (examined in Chapter a risk to the populations they link; therefore, they 23). However, industry practices, personnel, and should be surveyed frequently (22). In addition, markets are not uniform across all regions, so the special care should be taken to prevent the direct and information is usually not equally applicable. Even indirect contacts between birds from these popula- specifi c farms that have been involved in previous tions and more stable groups. outbreaks change and, thus, the disease risk can Most poultry do not travel far from their places change. For example, although LBMs are a known of hatch but there are some notable exceptions. reservoir of LPAI viruses in the eastern United Gamefowl may travel extensively both domestically States (6) and in other parts of the world (32, 40), and internationally for contest events or for breeding they have not been important AI virus reservoirs in purposes. Among commercial poultry sectors, the all places where they exist. Additionally, as lessons elimination of spent fowl plants throughout the about disease transmission are learned, industry best United States has forced the egg production and practices are modifi ed to incorporate practices that broiler breeder industries to move birds over great would better protect poultry fl ocks. distances (Table 16.2). Poultry raised for LPMs may

Figure 16.3. Interactions between avian populations in densely populated poultry regions that have resulted in the transmission of AI or exotic Newcastle disease (solid arrows with references cited) or are connections that could result in disease transmission (dashed arrows). The populations within the dashed circle are those that are commonly present in densely populated poultry regions. 360 Avian Influenza

Table 16.2. Travel by spent fowl to reach episode, to our knowledge, but it certainly created a slaughter facilities. link between populations that were and should be Origin Destination Length of trip maintained as distinct. As mentioned, most poultry move only short dis- Southern Central California 5 to 7.5 hours tances over their lives, and thus their disease trans- Californiaa mission capabilities are limited to the local Iowab Minnesota, 2 to 8 hours populations with which they interact. In contrast, Nebraska, free-fl ying migratory birds may travel long distances Indiana and interact with both international and domestic Minnesotac Minnesota 1 to 8 hours avian populations that only move long distances Pennsylvaniad Virginia, New 2.5 to 8 hours with the assistance of humans. Usually, birds that Jersey, Ontario are moved have an economic or other value that cannot be or is not transferred through methods a Source: C. Cardona. besides the movements of live animals. Psittacine b Source: D. Trampel. and other caged pet bird species come to the United c Source: D. Halvorson. States or into other countries because there is a large d Source: E. Gingerich. market for them as pets. They may arrive in the United States through both legal methods, which also travel relatively long distances to LPMs. involve quarantine, and illegal means (16). Zoos Markets are usually located in urban areas while the import birds and mammals from long distances for birds are grown in more rural areas. The typical breeding and conservation purposes following legal transport in California is 2 to 4 hours, transport from channels. The methods of quarantine used to legally farms in Pennsylvania to markets in New York or import exotic birds into the United States have been New Jersey is 3 to 4 hours, and from Iowa farms to very effective in preventing AI viruses from entering Minnesota LBMs is also between 3 and 4 hours of the United States. Birds imported from outside of transit time. Movement alone does not present a the United States legally are required to enter a risk to biosecurity. What does create a risk when U.S. Department of Agriculture (USDA)-licensed birds travel over routes that are in close proximity quarantine facility where the birds are tested and to the farms of another region, or when the same then held for 30 days (38). transport trucks and cages are also used by produc- Interestingly, samples from birds entering quaran- ers in another region without proper cleaning and tine stations demonstrate that AI virus–infected disinfection. birds do enter quarantine (8, 33). The fact that legally Poultry also move great distances when there has imported birds have not been associated with disease been a disruption in supply and demand. This is not outbreaks is a good indication that this system is uncommon during the recovery period after a disease effective. In contrast, there have been outbreaks outbreak when a large portion of a commercial associated with the unrestricted importation of pet industry has been depopulated. After H7N3 high birds (39) and detection of AI virus (16) and END pathogenicity avian infl uenza (HPAI) was eradi- virus (5) in illegally imported birds. cated in Italy in 1999, poultry producers imported large numbers of hatching eggs to rebuild their Trojan horses industry from international sources. Eggs were In public health circles, the asymptomatic preclini- imported from multiple sources, including one cal period of a disease is when pathogens are thought through which END was also imported (9). Simi- to be most effi ciently transmitted to new hosts (12, larly, California egg producers after the 2003 END 21). This is probably due to a failure to (1) detect during which 22 commercial egg-laying fl ocks were infection and (2) apply personal protective proce- depopulated (5), imported pullets from Iowa, 29 dures, like hand washing, social avoidance, and hours away, because the demand for pullets was respiratory etiquette at all times. With AI in birds, a greater than the supply available from local sources. similar situation occurs, both with preclinical infec- No disease agents were transmitted in this last tions and with subclinical infections. Subclinical and 16 / Farm and Regional Biosecurity Practices 361 preclinical infections with AI viruses occur in avian combine is considered to be a single biosecurity species because (1) some species do not show clini- unit. In other words, birds within a club or combine cal signs of disease when infected, (2) the popula- are treated as if they belong to the same loft. When tion is managed to avoid clinical disease, (3) the a bird comes in from a race, it can be introduced infecting virus is nonpathogenic, or (4) the popula- immediately back into the loft because it has just tion is at least partially immune. As with asymptom- been out fl ying with its loft-mates. This method is atic humans in early disease, bird owners in contact easy to apply for most loft owners because it requires with subclinically ill birds may underestimate their no additional quarantine for birds returning from a contagious capacity and fail to use adequate biose- race. However, any member of the club can intro- curity measures to prevent transmission. duce disease into the club and all lofts practicing this Domestic ducks and birds in LPMs have been method of isolation will be at risk. The other strategy associated with the spread of H5N1 HPAI viruses is to consider all lofts other than your own as sources (34), probably for the same reasons that preclinically of disease. All birds returning from a race are quar- ill humans are effi cient spreaders of disease. These antined for 14 days before they are reintroduced to specifi c populations are very likely to appear healthy the loft. This method puts the owner into control of because they are resistant to clinical disease, as is the fate of his loft and, overall, it is a safer method. the case with ducks, or they are managed to avoid There is a certain amount of extra labor and planning the appearance of disease, as happens in LPMs. If that is required to achieve this type of biosecurity. active surveillance is not in place for these popula- Pigeon racing enthusiasts have recognized the need tions, infections may not be detected until they are for disease prevention in their sport and require loft transmitted to fl ocks that do show clinical disease. registration, which includes the implementation of Domestic ducks and LPMs may share feed sources, biosecurity for some activities (2). rendering companies, meter readers, etc., with more This same approach to biosecurity can be used for stable and less resistant domestic poultry popula- other birds that are intermingled and then returned tions. When barriers are not biosecure, disease to their source fl ocks, or to other fl ocks. That said, agents can fl ow between poultry populations, result- avian populations that intermix should be consid- ing in outbreaks of clinical disease. Biosecurity ered at high risk for infection (22). In addition to should be applied to all vehicles and people entering racing pigeons, gamecocks and exhibition poultry a poultry farm, but the tendency is to relax the rules also fall into this category of high-risk poultry pop- when there is no perceived risk of exposure much ulations for the reasons outlined in the preceding like the failure to use social distancing around section. healthy-appearing people. BALANCING COST AND PROTECTION Biosecurity Plans for Unusual Populations Finances are always a concern when it comes to Every farm needs its own individually tailored bios- adding biosecurity, because the return on dollars ecurity plan because every ranch is different, but spent to prevent disease is neither certain nor con- some populations seem to defy biosecurity because sistent (36). Unless there is a real and proximal of the very purpose of the birds. Pigeon lofts that are threat of a catastrophic disease, then investing in involved in racing are one such group. The indi- biosecurity may not be cost effective, especially in vidual birds involved in racing are commingled with the short term. The bottom line dictates many aspects other competitors in the transport truck; they are of biosecurity because most biosecurity practices then released at some distant point and return directly have an associated cost. Most of the reported costs to their home lofts. For disease transmission, it is of disease prevention are associated with physical not hard to imagine that a pathogen could be trans- changes to a farm or facility such as the building of ferred from a single fl ock to many lofts in this fences to control traffi c or installing showers or process. change rooms for employees. However, in some Despite these diffi culties, many racing lofts have cases, it may be diffi cult to quantify the costs of been able to achieve reasonable biosecurity in one biosecurity. As an example, a remote location can of two ways. In the fi rst strategy, the racing club or certainly isolate a farm far better than a fence, but 362 Avian Influenza the cost of that isolation (such as the cost of the where expenditures on disease prevention can result additional fuel needed to reach the site) is usually in improved profi t margins. not listed as a cost of biosecurity. In fact, the only recorded costs of biosecurity associated with an AI REGIONAL BIOSECURITY virus outbreak in Pennsylvania were those for cleaning and disinfection (11). Movement Patterns Other effective biosecurity practices that do not There are predictable patterns that essentially result appear on most spreadsheets include a reduction in in limitations on the movements of birds. For the number of visitors to a poultry farm, keeping example, although migratory birds move long dis- visitor log books, managing feed delivery routes, tances, they generally stay within fl yways. There are and maintaining proper on farm and off farm traffi c notable exceptions when birds are blown off course patterns. These practices do have costs in employee but the very existence of distinct clades of AI viruses time, and require the help of the farm’s workforce, i.e., North American and Eurasian, would support neighboring farms, and of allied industries to accom- that these trends are generally true. Human-assisted plish. The consequences of poor employer–employee bird movements are also predictable, although they relations such as high turnover rates and employees may not seem logical to all who look at them. For who are neither knowledgeable nor committed to the example, to outsiders, it may seem that gamefowl farm’s policies, including biosecurity, can be disas- are moved haphazardly; however, if one understands trous. However, employee benefits and rewards are gamefowl breeding practices and the associations rarely considered as a part of a biosecurity plan or through which birds are tested, their movements are as a biosecurity cost, although they should be. predictable. The predictability of avian movements The return of profi t on an investment in biosecu- makes it possible to make assumptions about the rity measures can be very good or very bad in any risks of disease transmission posed by specifi c given fiscal period because exposure to disease groups based on historical data (see discussion on agents is not certain for any fl ock, especially when Populations Associated With Disease Risk). one considers a sporadically occurring disease like AI. Sometimes the return on profit is determined by Voluntary Movement Control Programs performance ranking systems, but it is certainly not Many poultry farms are located close to other poultry as simple to determine as a treatment that alters feed farms to take advantage of shared resources. These conversion. Generally speaking, biosecurity costs resources include feed mills, rendering plants, are most justifiable in circumstances where the like- slaughter facilities, and, of course, markets. In turn, lihood of exposure to a disease agent is high, espe- concentrations of commercial poultry support the cially if a likely route of transmission can be growth of human population sectors, which support determined (36), if the costs of production are high, animal agriculture. The large numbers of low-pay, or if the value of the birds is especially high or they low-skill positions in animal agriculture often attract are irreplaceable, as in the case of breeder stock new immigrants as workers. These new immigrants (24). may also engage in activities, which involve live Although the costs of biosecurity are frequently birds such as cockfi ghting and the purchase of birds diffi cult to justify, especially in the short term, pre- for food at LPMs. The former was implicated in the ventative measures do pay off when and where there spread of END in California (27), and the latter have is a disease challenge. In the wake of a foreign been involved in the spread and maintenance of animal disease outbreak, farms that do not have H7N2 LPAI virus in the northeastern United States effective biosecurity lose their income for the short (7) and in other parts of the world (32, 40). term and, frequently, over the long term, their In areas where poultry farms are on adjoining or markets. So, although biosecurity is not always nearby properties or in areas tied together by fi nancially justified, in a given fiscal period, it is the common waterways, roads, or fl yways, the risk of only option to make agriculture sustainable. Addi- disease spread from one farm to another is greatly tionally, biosecurity practices have been incorpo- increased no matter what type of biosecurity is prac- rated into several quality assurance programs that ticed by individual farms. In fact, in regions with are linked to brands. This strategy results in a system dense poultry populations, AI viruses can spread so 16 / Farm and Regional Biosecurity Practices 363 easily and rapidly that it can seem to poultry owners becomes important. In some instances, routes may that it is spreading in the air. In reality, spread likely be designated as clean or dirty only during specifi c occurs through the numerous connections that exist times, but only if dirty loads are properly contained between fl ocks in the same community, including in order to prevent contamination of the route. In everything from insects to mailmen. The direct and addition to separating clean and dirty routes in a indirect interactions of avian species in a densely region, it is critically important to consider all roads populated poultry region that have resulted in or near a poultry farm to be off limits to traffi c from may result in the spread of AI or END are shown in other poultry farms. Figure 16.3. All poultry owners have the same goals of protecting their fl ocks or birds from infection and Traffi c Flow disease. Most poultry producers and owners know To prevent the movement of disease agents between that their fl ocks may well share disease agents with avian populations, the movements of poultry, neighboring fl ocks (4, 32, 40). However, they do not manure, and contaminated people and/or equipment always recognize that preventing disease in their must be controlled. During a foreign animal disease neighbor’s fl ocks should also be their priority. In outbreak, these movements off the farm are regu- fact, there is often substantial mistrust between the lated through the use of quarantine authority exer- groups of people who own birds in densely popu- cised by federal or state authorities. At times when lated poultry regions. Regional biosecurity efforts there are no foreign animal disease outbreaks, coor- rely on the cooperation of competitors and partners, dinating movements is important both to prevent friends and enemies, regardless of their ability to infections with non–exotic disease agents and to work together for any other reason. Because the prevent the spread of a foreign animal disease in the poultry owners in a region may not have the same early phases of an outbreak, i.e., before it is detected ideas about best management practices, a third party, and/or reported to authorities. The major difference like a cooperative extension agent or advisor can be is that during nonquarantine times, all poultry pro- an excellent convener and mediator for the group. ducers must voluntarily honor established routes in Education, motivation and reminders are critical order to protect poultry fl ocks from exposure to parts of keeping a voluntary regional biosecurity disease agents. In addition, they must all agree to be program in place. Again, cooperative extension transparent about their disease status, even with personnel can provide materials to achieve these competitors. During federal or state quarantines, goals. which movements are permitted is determined by Unfortunately, most efforts to develop regional regulatory authorities and they ascertain how and biosecurity plans fail to include all of the parties when they will occur. who own poultry or other domestic species suscep- Regulatory or voluntary regional biosecurity is tible to the disease agents of poultry. Regional bios- largely achieved through the separation of clean and ecurity programs usually only include members dirty poultry traffi c in a specifi ed geographic area. from a single commodity group, such as the Examples of clean traffi c include pullets, feed, Minnesota turkey plan (13). These plans include chicks, poults, or clean supplies. In contrast, dirty those owners most likely to be affected by the traffi c includes manure trucks, rendering trucks, actions of other members of the group, and have a spent fowl, and live haul trucks. The dirty and clean good chance at achieving longevity because these traffi c in a region should be separated, because dirty owners also have other interactions. The problem is traffi c has a high likelihood of being contaminated that they exclude a number of people involved with with disease agents that could spread to clean traffi c. other susceptible bird populations. Exclusion has Clean traffi c, if contaminated, can introduce infec- resulted in instances of disease spread to nonmem- tious agents into poultry fl ocks. Where possible, bers (C. J. Cardona, personal observation), and it specifi c roads and/or routes should be designated as certainly can add to animosity between owners in a clean or dirty. However, sometimes it is not possible region. The California North Valley taskforce is an to consistently designate a route as off limits without interesting group that meets in the densely populated hardship for other producers. Hence, coordination Central valley of California to discuss regional bio- and communication between poultry producers security. They include any commercial producers or 364 Avian Influenza other poultry owners willing to attend and discuss Viruses, for more information). However, as LPAI biosecurity. It is sometimes diffi cult to maintain the viruses, their control remains, at least partially, in interest and energy in a group of people that share the hands of state agencies. little besides a common disease prevention interest, States vary in their approaches to the control of but these types of groups are easiest to maintain LPAI viruses and they may apply different types or when the perceived risk of infection is high. levels of controls to diverse types of premises. For example, an infected farm may be quarantined and Communication then allowed to move birds to slaughter after recov- Communication among regional neighbors can ery using the process of controlled marketing (see occur in many ways. Some use a telephone or a Fax Chapter 23 for more information about this strat- tree to communicate about disease outbreaks. An egy). In contrast, an LPM may have to completely example of this is the poultry industry–operated depopulate its inventory and pass an inspection in disease hotline in California. This hotline is designed response to a positive LPAI virus test. It is diffi cult, to rapidly inform all participating members of the but nonetheless important, not to treat one type of occurrence of designated contagious diseases in poultry owner in a state more or less favorably than poultry so that spread of the disease may be halted another type. Measuring and comparing the costs of as quickly as possible. When a laboratory diagnoses lost production or sales as a percentage of annual a case of one of the diseases that the poultry indus- revenue for farms or markets is one way to deter- try has agreed to report on a farm or in a market, mine if a response plan is evenhanded to all types they will call the state department of agriculture, of commercial entities. The intangible costs associ- which will in turn initiate the hotline. Every market ated with losses of pets or rescued animals are dif- owner or producer will receive a Fax or telephone fi cult to determine and, really, cannot be compared call with all of the information that they need to to expenses incurred by businesses. However, that know about the case. Because the information on the does not minimize the need to treat all affected hotline can be sensitive, participants have agreed owners fairly. Future regional disease control efforts that hotline information is not to be repeated to depend on the perception by all parties that they sources outside the state or to the media. were regarded with respect. In addition to the receipt of disease reports by hotline, some poultry industry companies communi- Controls During a Foreign Animal cate their movements of clean materials and ask Disease Outbreak others to respect those routes. Some groups use a HPAI is a foreign animal disease, and there are coordinator to help with communication. Other several factors used to determine the movement con- groups meet regularly to discuss movements and trols that have to be put in place during a foreign disease status in a region. Whatever method of com- animal disease outbreak. Some of these controls are munication is used, it is essential that it is inclusive set out in the Code of Federal Regulations for the of all neighbors and that it facilitates open and United States, and similarly regulations in other honest interactions. countries, or are part of an international trade organization policy such as the OIE. However, to some Regulatory Control of Poultry Movements degree much of what happens (i.e., how large quarantined areas are, which species are monitored and Controls During an Outbreak of Low restricted, and if regionalization can be used) is Pathogenicity Avian Infl uenza determined by a nation’s trading partners and what Regulatory controls may or may not be instituted for they require. cases or even outbreaks of LPAI depending on the The purpose of a regional quarantine in the control viral subtype and regulations of the state. LPAI of an outbreak is to place general restrictions on viruses of the H5 or H7 subtypes are treated differ- movements prior to full knowledge about where ently than other LPAI viruses by international and cases are or how a pathogen is spreading. The place- domestic regulatory agencies, because they can ment of a regional quarantine is followed by inten- become HPAI viruses (see Chapter 2, Molecular sive surveillance efforts to determine the extent of Determinants of Pathogenicity for Avian Infl uenza infection in an area. Once the extent of infection in 16 / Farm and Regional Biosecurity Practices 365 a region has been established, then quarantine ous contacts. In addition, diseases that are transmit- authority is generally used to limit movements on ted to nonagricultural animals, like cats, dogs, and and off of infected or suspect premises. horses, may well either be unregulated or may fall Regional quarantines will remain in place until under the regulatory control of health services if surveillance can establish freedom from infection. they pose a threat to human health. During the time that a regional quarantine is in place, the movements of known fomites and poten- CONCLUSION tially infectious products within the region will be Effective biosecurity is an essential part of the sus- conducted only under permit and the movement of tainable production of poultry today. The specifi c these materials outside of the zone is strictly con- biosecurity plans of poultry farms and facilities may trolled by federal authorities. achieve the goals of biosecurity (isolation, sanita- Although movements of people are often directly tion, and traffi c control) in very diverse ways. correlated with the movement of the pathogens of However, even the most successful of on-farm bios- importance in animal agriculture, state and federal ecurity programs can fail if regional biosecurity is agencies do not stop human movements as a part of not in place. This is especially important in densely regional quarantines. This is because pathogens, populated poultry regions, where disease agents can including AI viruses, are moved by fomites that are spread very rapidly. Regional biosecurity programs moved by humans. Quarantines generally stop or can take the form of voluntary agreements between regulate the movements of known fomites, such as producers or movements that are fully regulated by equipment, vehicles, supplies and products. In the use of quarantine authority, depending on the response to disease outbreaks where humans them- infection status of the area. Properly used, biosecu- selves may be fomites, like foot-and-mouth disease, rity is a very powerful and cost-effective tool for a regulatory agency may well institute a mandatory disease prevention and control. decontamination site for all human traffi c leaving a region. Otherwise human traffi c is unregulated REFERENCES except on and off of specifi c quarantined premises. 1. Akey, B.L. 2003. Low-pathogenicity H7N2 avian The movements of species and their potentially infl uenza outbreak in Virgnia during 2002. Avian infectious products known to be involved in the Diseases 47(3 Suppl.):1099–1103. spread of a disease agent are regulated. Additionally, 2. American Racing Pigeon Union, Inc. 2003. AU390 the movements of species that have not been associ- Loft certifi cation program. In: Board Policies, Pro- ated with disease spread in the current outbreak but cedures And Rules of the American Racing Pigeon that are known to be susceptible to infection may Union, Inc. Available at http://www.pigeon.org/ loftregistration.htm. American Racing Pigeon also be regulated and moved only under permit Union: Oklahoma City, OK. during a quarantine period. These two statements are 3. Bean, B., B.M. Moore, B. Sterner, L.R. Peterson, rather open ended and can result in questions that D.N. Gerding, and H.H. Balfour, Jr. 1982. Survival come up during an outbreak. For example, when of infl uenza viruses on environmental surfaces. does a product, like feathers, go from the infectious The Journal of Infectious Diseases 146(1):47–51. to noninfectious category? Those types of questions 4. Bermudez, A.J., and B. Stewart-Brown. 2003. are answered by a scientifi c advisory board. The Disease prevention and diagnosis. In: Y.M. Saif, scientifi c advisory board may be composed of aca- H.J. Barnes, J.R. Glisson, A.M. Fadly, L.R. demic and/or government experts in the fi eld. Their McDougald, and D.E. Swayne, eds. Diseases of recommendations on the multitude of gray areas that Poultry, 11th ed. Iowa State Press: Ames, IA, arise during an outbreak may be advisory to the pp. 17–60. 5. Breitmeyer, R.E., A.M. Whiteford, and J.A. Shere. leadership of an eradication effort or the regulatory 2003. California experience with exotic Newcastle agency in charge. disease: a state and federal regulatory perspective. An outbreak of a zoonotic pathogen in agricul- In: Proceedings of 107th United States Animal tural animals would likely involve multiple regula- Health Association, pp. 65–70. tory agencies, and quarantines are likely to be 6. Bulaga, L.L., L. Garber, D. Senne, T.J. Myers, R. imposed by local, state, or federal human health Good, S. Wainwright, and D.L. Suarez. 2003. agencies on suspected human cases or their danger- Descriptive and surveillance studies of suppliers to 366 Avian Influenza

New York and New Jersey retail live-bird markets. H7N2 in Pennsylvania (1996–98). Avian Diseases Avian Diseases 47(3 Suppl.):1169–1176. 47(3 Suppl.):1022–1036. 7. Bulaga, L.L., L. Garber, D. Senne, T.J. Myers, R. 18. Ito, T., K. Okazaki, Y. Kawaoka, A. Takada, R.G. Good, S. Wainwright, S. Trock, and D.L. Suarez. Webster, and H. Kida. 1995. Perpetuation of infl u- 2003. Epidemiologic and surveillance studies on enza A viruses in Alaskan waterfowl reservoirs. avian infl uenza in live-bird markets in New York Archives of Virology 140(7):1163–1172. and New Jersey, 2001. Avian Diseases 47(3 19. Kinde, H., D.M. Castellan, D. Kerr, J. Campbell, Suppl.):996–1001. R. Breitmeyer, and A. Ardans. 2005. Longitudinal 8. Butterfi eld, W.K., R.J. Yedloutschnig, and A.H. monitoring of two commercial layer fl ocks and Dardiri. 1973. Isolation and identifi cation of myxo- their environments for Salmonella enterica serovar viruses from domestic and imported avian species. enteritidis and other Salmonellae. Avian Diseases Avian Diseases 17(1):155–159. 49(2):189–194. 9. Capua, I., P.M. Dalla, F. Mutinelli, S. Marangon, 20. Kinde, H., W. Utterback, K. Takeshita, and M. and C. Terregino. 2002. Newcastle disease out- McFarland. 2004. Survival of exotic Newcastle breaks in Italy during 2000. Veterinary Record disease virus in commercial poultry environment 150(18):565–568. following removal of infected chickens. Avian 10. Cardona, C.J., and D.R. Kuney. 2002. Biosecurity Diseases 48(3):669–674. on chicken farms. In: D.D. Bell and W.D. Weaver 21. Klimov, A.I., E. Rocha, F.G. Hayden, P.A. Shult, (eds.). Commercial Chicken Meat and Egg Pro- L.F. Roumillat, and N.J. Cox. 1995. Prolonged duction, 5th ed. Kluwer Academic Publishers: shedding of amantadine-resistant infl uenza A Norwell, MA, pp. 543–556. viruses by immunodefi cient patients: detection by 11. Davison, S., D. Galligan, T.E. Eckert, A.F. Ziegler, polymerase chain reaction-restriction analysis. The and R.J. Eckroade. 1999. Economic analysis of an Journal of Infectious Diseases 172(5):1352–1355. outbreak of avian infl uenza, 1997–1998. Journal of 22. Marangon, S., L. Bortolotti, I. Capua, M. Bettio, the American Veterinary Medical Association and M. Dalla Pozza. 2003. Low-pathogenicity 214(8):1164–1167. avian infl uenza (LPAI) in Italy (2000–2001): 12. Hall, C.B., and R.G. Douglas, Jr. 1975. Nosoco- epidemiology and control. Avian Diseases mial infl uenza infection as a cause of intercurrent 47(3 Suppl.):1006–1009. fevers in infants. Pediatrics 55(5):673–677. 23. McQuiston, J.H., L.P. Garber, B.A. Porter-Spald- 13. Halvorson, D. 1986. Avian infl uenza: a Minnesota ing, J.W. Hahn, F.W. Pierson, S.H. Wainwright, cooperative control program. In: Proceedings of D.A. Senne, T.J. Brignole, B.L. Akey, and T.J. Second International Symposium on Avian Infl u- Holt. 2005. Evaluation of risk factors for the spread enza, pp. 327–336. of low pathogenicity H7N2 avian infl uenza virus 14. Halvorson, D., D. Karunakaran, D. Senne, C. among commercial poultry farms. Journal of the Kelleher, C. Bailey, A. Abraham, V. Hinshaw, and American Veterinary Medical Association 226(5): J. Newman. 1983. Epizootiology of avian infl u- 767–772. enza—simultaneous monitoring of sentinel ducks 24. Morris, M.P. 1995. Economic considerations in and turkeys in Minnesota. Avian Diseases 27(1): prevention and control of poultry disease. In: S.M. 77–85. Shane, D.A. Halvorson, D. Hill, P. Villegas, and 15. Halvorson, D.A., and W.D. Hueston. 2006. The D. Wages, eds. Biosecurity in the Poultry Industry. development of an exposure risk index as a rational American Association of Avian Pathologists: guide for biosecurity programs. Avian Diseases Kennett Square, PA, pp. 4–16. 50(4):516–519. 25. Nolen, R.S. 2002. Exotic Newcastle disease strikes 16. Hawkins, M.G., B.M. Crossley, A. Osofsky, R.J. game birds in California. Journal of the American Webby, C.W. Lee, D.L. Suarez, and S.K. Hietala. Veterinary Medical Association 221(10):1369– 2006. Avian infl uenza A virus subtype H5N2 in a 1370. red-colored Amazon parrot. Journal of the American 26. Pearson, G.L., and M.K. McCann. 1975. The role Veterinary Medical Association 228(2):236–241. of indigenous wild, semidomestic, and exotic birds 17. Henzler, D.J., D.C. Kradel, S. Davison, A.F. in the epizootiology of velogenic viscerotropic Ziegler, D. Singletary, P. DeBok, A.E. Castro, H. Newcastle disease in southern California, 1972– Lu, R. Eckroade, D. Swayne, W. Lagoda, B. 1973. Journal of the American Veterinary Medical Schmucker, and A. Nesselrodt. 2003. Epidemiol- Association 167(7):610–614. ogy, production losses, and control measures asso- 27. Pedersen, J.C., D.A. Senne, P.R. Woolcock, H. ciated with an outbreak of avian infl uenza subtype Kinde, D.J. King, M.G. Wise, B. Panigrahy, and 16 / Farm and Regional Biosecurity Practices 367

B.S. Seal. 2004. Phylogenetic relationships among 35. Swayne, D.E., and D.A. Halvorson. 2003. Infl u- virulent Newcastle disease virus isolates from the enza. In: Y.M. Saif, H.J. Barnes, J.R. Glisson, A. 2002–2003 outbreak in California and other recent M. Fadly, L.R. McDougald, and D.E. Swayne outbreaks in North America. Journal of Clinical (eds.). Diseases of Poultry, 11th ed. Iowa State Microbiology 42(5):2329–2334. University Press: Ames, IA, pp. 135–160. 28. Quinn, J., and D. Marshall. 2003. Summary of 36. te Winkel, G.P. 1997. Biosecurity in poultry pro- avian infl uenza in North Carolina: March–April duction: where are we and where do we go? Acta 2002. In: Proceedings of 107th United States Veterinaria Hungarica 45(3):361–372. Animal Health Association, pp. 516–519. 37. Tiwari, A., D.P. Patnayak, Y. Chander, M. Parsad, 29. Sen, S., S.M. Shane, D.T. Scholl, M.E. Hugh- and S.M. Goyal. 2006. Survival of two avian respi- Jones, and J.M. Gillespie. 1998. Evaluation of ratory viruses on porous and nonporous surfaces. alternative strategies to prevent Newcastle disease Avian Diseases 50(2):284–287. in Cambodia. Preventative Veterinary Medicine 38. U.S. Department of Agriculture–Animal and Plant 35(4):283–295. Health Inspection Service (USDA/APHIS). 2006. 30. Senne, D.A., D.L. Suarez, D.E. Stallnecht, J.C. Avian infl uenza (bird fl u), Release No. 0511.05. Pedersen, and B. Panigrahy. 2006. Ecology and Availabe at http://www.usda.gov/wps/portal/!ut/p/_ epidemiology of avian infl uenza in North and s.7_0_A/7_0_1OB/.cmd/ad/.ar/sa.retrievecontent/. South America. Developments in Biologicals c/6_2_1UH/.ce/7_2_5JM/.p/5_2_4TQ/_th/J_2_ (Basel) 124:37–44. 9D/_s.7_0_A/7_0_1OB?PC_7_2_5JM_contentid= 31. Shane, S.M. 1997. Prevention of diseases. In: 2005%2F11%2F0511.xml&PC_7_2_5JM_ Handbook on Poultry Diseases. American Soybean parentnav=AI_FACTSHEETS&PC_7_ Association: St. Louis, MO, pp. 20–23. 2_5JM_navid=AI_FACTSHT. USDA/APHIS: 32. Shortridge, K.F., N.N. Zhou, Y. Guan, P. Gao, T. Washington, DC. Ito, Y. Kawaoka, S. Kodihalli, S. Krauss, D. 39. Utterback, W.W., and J.H. Schwartz. 1973. Epizo- Markwell, K.G. Murti, M. Norwood, D. Senne, L. otiology of velogenic viscerotropic Newcastle Sims, A. Takada, and R.G. Webster. 1998. Char- disease in southern California, 1971–1973. Journal acterization of avian H5N1 infl uenza viruses from of the American Veterinary Medical Association poultry in Hong Kong. Virology 252(2):331–342. 163(9):1080–1088. 33. Slemons, R.D., R.S. Cooper, and J.S. Orsborn. 40. Webster, R.G. 2004. Wet markets—a continuing 1973. Isolation of type-A infl uenza viruses from source of severe acute respiratory syndrome and imported exotic birds. Avian Diseases 17(4):746– infl uenza? The Lancet 363(9404):234–236. 751. 41. Zander, D.V. 1995. Location and design of farms 34. Songserm, T., R. Jam-on, N. Sae-Heng, N. to promote biosecurity. In: S.M. Shane, D.A. Meemak, D. J. Hulse-Post, K.M. Sturm-Ramirez, Halvorson, D. Hill, P. Villegas, and D. Wages and R.G. Webster. 2006. Domestic ducks and (eds.). Biosecurity in the Poultry Industry. Ameri- H5N1 infl uenza epidemic, Thailand. Emerging can Association of Avian Pathologists: Kennett Infectious Diseases 12(4):575–581. Square, PA, pp. 25–30. 17 Farm Biosecurity Risk Assessment and Audits

David Shapiro and Bruce Stewart-Brown

INTRODUCTION tion in general. Programs instituted in response to Modern commercial poultry producers make daily emergencies or high-profi le poultry diseases usually determinations as to the relative importance of provide collateral benefi ts by concomitantly reduc- various husbandry, housing, nutritional, and bird ing the risk posed by other, less-prominent diseases. health inputs. These decisions are based on past experiences, experimental evidence, and the recom- DEFINITIONS mendations of experts. The inputs are applied and The terms related to risk analysis in this chapter monitored in order to correlate the results with those have been distilled from the offi cial defi nitions used decisions. Adjustments to poultry production pro- by the United States Department of Agriculture and grams are then made, justifi ed by successes and fail- the World Organization for Animal Health, more ures, but also tempered by economic considerations. commonly known as the Offi ce International des Biosecurity, like any other essential poultry pro- Epizooties (OIE) (15, 21). duction input, must be evaluated for both quality and quantity. The objective of biosecurity is to reduce Risk analysis is defi ned as the combined processes the risk of disease. To apply it effectively, one must of “hazard identifi cation, risk assessment, risk rationally assess the existing risks and determine the management, and risk communication.” In this ability of any specifi c biosecurity variable to increase case, the hazard identifi cation task is quite or decrease that risk. The level of risk must then be straightforward. The primary hazard is the balanced against the cost of any particular biosecu- introduction of the AI virus into a poultry fl ock. rity measure. Assessing biosecurity risks logically Risk assessment involves the scientifi c evaluation and auditing procedures designed to reduce risk of the various risks and their potential to increase allow poultry producers to apply biosecurity proce- or decrease the probability of the primary hazard dures in a cost-effective manner. Reliable risk occurring. Risk assessment of biosecurity-related assessment information also provides poultry pro- risks associated with AI at a commercial poultry ducers with a tool for long-range planning, construc- site is the primary focus of this chapter. tion, and improvements. Risk management is the formulation and im ple- Although this book focuses on avian infl uenza mentation of measures aimed at reducing the risk (AI), the concepts and tactics presented in this chapter in question. Risk communication is the sharing of can be applied to most transmissible poultry dis- the risk analysis information with all relevant eases. The implementation, improvement, or review parties. of biosecurity programs is often driven by the threat A biosecurity audit, like any type of audit, is simply of serious diseases such as AI, velogenic Newcastle a methodical examination and review of the disease, or zoonoses. However, a valid biosecurity biosecurity procedures or lack thereof existing at risk assessment will apply to poultry disease preven- a specifi c poultry site.

HISTORY dures are largely eliminated. An overall level of risk Although the term biosecurity did not become part for a specifi c site is determined based on the many of normal poultry production parlance until the individual biosecurity-related parameters. The risk 1980s (20), some of its basic concepts and prefera- assessment determines the overall risk for a particu- ble practices (age separation, isolated farm location, lar site. The utility of an individual biosecurity quarantine for sick birds, proper disposal of poultry parameter is judged after its incorporation into the carcasses and waste, and effective disinfection) were total risk assessment score, not by debating the cost/ clearly described in early poultry disease texts (8, benefi t of a specifi c procedure to prevent a specifi c 16). Informal monitoring of these basic biosecurity disease. The site manager makes biosecurity deci- practices undoubtedly occurred at around the same sions on specifi c procedures based on cost or feasi- time, but examples of more formal biosecurity audits bility in relation to how the changes could impact did not occur until later (22). These early attempts the overall site risk. The company receives the same to assess the level of risk frequently took the form benefi t and is also given information to assist with of checklists. Points were often assigned for specifi c long-term planning. answers, and the biosecurity status was determined Risk assessments, when applied routinely and by the resulting score. Although such checklists pro- consistently, are powerful training and educational moted sound practices, they were not comprehen- tools. A company may fi nd consistently high-risk sive and often arbitrarily assigned relative values to practices within an area and focus training, educa- different biosecurity parameters. tion, and fi scal resources on mitigating the weak- Formal risk assessments become more com- nesses (risks) identifi ed. Rational assessment can monplace in the late 1980s and 1990s (5, 18). By transform annual biosecurity training from a general, this time, more care was taken to assign risk based redundant, nonspecifi c, and unproductive seminar to on experimental, epidemiological, or experiential a focused, interactive discussion. When used in this evidence. way, all involved parties better understand the issues Currently, most commercial operations have and priorities, with the common goal being a reduc- detailed biosecurity procedures for any sites involved tion of overall risk rather than rote memorization of with live production or processing. Internal verifi ca- biosecurity tasks. tion procedures range from minimal to extensive. The most direct benefi t of the biosecurity audit Formal comparative risk assessment of individual process is to check compliance. An audit may also farm sites, however, remains the exception rather be done before formal biosecurity procedures are than the rule. instituted in order to provide a baseline. Both situations provide immediate feedback to the site manager JUSTIFICATION FOR BIOSECURITY RISK and the poultry company. The site manager is able ASSESSMENTS AND AUDITS to make immediate decisions to change procedures The primary reason for assessing disease risk and its within his or her control to improve compliance. relation to biosecurity is prioritization. There are an For operations that use different levels of biosecu- almost infi nite number of possible biosecurity pro- rity (e.g., low or high; level 1, level 2, or level 3) in cedures, policies, and structures that can be applied response to disease outbreaks, risk assessments can to a poultry operation. Each of these biosecurity be used to determine if the varying degrees of bio- options can be implemented at varying levels of security stringency are justifi ed. It may be deter- stringency. Just as with housing, husbandry, and mined that the change in procedures from low nutrition, biosecurity-related decisions must be biosecurity to high does not actually impact overall made, not to guarantee maximum performance but risk appreciably. Conversely, it could become appar- to achieve an optimal economic outcome while ent that more stringent biosecurity measures reduce maintaining accepted norms of bird health and risk to such a degree that it is better to implement welfare. Risk assessments are used to prioritize bios- them on a permanent basis. ecurity procedures by their contribution to risk Biosecurity risk assessments and biosecurity reduction and to rank farms by their overall risk for audits contribute to risk reduction through rational occurrence of disease outbreaks. methods based on scientifi c information as opposed Another advantage of assessing risk is that con- to haphazard application of individual biosecurity troversies regarding individual biosecurity proce- procedures. 17 / Farm Biosecurity Risk Assessment and Audits 371

GENERAL CONSIDERATIONS obtained by different assessors evaluating the same Both risk assessments and biosecurity audits must site. be fair and objective. The assessor should be blinded Ideally, a formal disease and risk analysis should insofar as knowledge of previous disease outbreaks be done prior to establishing a site’s standard operat- on that site. Preferably the assessor would be an ing procedures (SOPs) for biosecurity. Normally, independent third party, not affi liated directly with however, every site already has at least some sem- the poultry production company or the farm site. If blance of a biosecurity policy. Cataloging all the a reputation for fairness precedes the site visit, the routinely used biosecurity procedures and variables level of cooperation and compliance increase. present in an area should be made prior to a thorough Advance warning of the visit, along with some risk analysis or biosecurity audit. Copies of any explanation to the site manager beforehand regard- existing biosecurity SOPs must be made available to ing the procedures and objectives, is desirable. the assessor. Any forms used must be written in concise, unam- A postassessment or postaudit debriefi ng should biguous language. Use of colloquial poultry termi- be held with the farm site manager. Although more nology is acceptable as long as it is well understood detailed discussions can be held later, an informal in the region where it is to be applied. debriefi ng should be held at the end of the farm visit. Neither risk assessments nor biosecurity audits In the case of a risk assessment, the variables and can be properly conducted by telephone, mail, Inter- how they contribute to overall risk should be net (email, online), or other indirect techniques. The explained. Differentiation should be made between farm site must be visited in person by a trained the variables that the farmer can control to reduce assessor. Assessors should be well versed in live risk and those that cannot easily be changed. After production of poultry, especially for the production a biosecurity audit, the site manager should be made class being evaluated. They should have the ability clearly aware of any defi ciencies and lauded for to communicate comfortably with persons accus- compliance with good biosecurity practices. tomed to life on a poultry farm. Every attempt should be made to arrive punctually for the farm visit and RISK ASSESSMENTS suffi cient time must be allowed to adequately evalu- There is a paucity of detailed information or estab- ate each site. The assessor is responsible for bring- lished procedures with regard to risk assessment of ing any equipment necessary for a risk assessment biosecurity at poultry operations. However, the stan- or biosecurity audit. Besides documentation, the dardization of such risk assessment is addressed in most commonly used piece of equipment is a hand- the 1994 “SPS Agreement” as part of the “Final Act held global positioning satellite (GPS) device. A of the Uruguay Round of Multilateral Trade Nego- GPS device is invaluable for accurately confi rming tiations” signed in Marrakesh on April 15th, 1994. the location of sites and for determining distances It states, “Members shall ensure that their sanitary between critical points (e.g., distance from poultry or phytosanitary measures are based on an assess- houses to roads, distance between poultry farms, ment, as appropriate to the circumstances, of the etc.) In some cases, binoculars are useful in deter- risks to human, animal, or plant life or health, taking mining the livestock content of surrounding farms. into account risk assessment techniques developed The assessor must develop a consistent method and by the relevant international organizations.” Article language for asking questions and should avoid 5 of this document summarizes the factors that leading the respondent or suggesting answers. should be considered when performing risk assess- Risk assessments must be tested prior to formal ments relating to animal health (25). These are out- use. Pretesting of biosecurity audits, as straightfor- lined in Table 17.1. ward compilations of biosecurity procedures, is not While these precepts are intended as guidelines as crucial. Testing uncovers obvious defi ciencies, for international trade, most of the considerations reveals extraneous components, and provides the are very appropriate for assessing the risk of AI on assessor with valuable experience. Certain aspects poultry farms. Regardless of a particular poultry of an assessment or audit, which seemed quite operation’s size or marketing niche, diseases with logical when formulated in an offi ce, may seem epizootic or zoonotic potential have graphically impossible or impractical once one is standing on a emphasized the international interrelation between farm. Testing should include comparison of scores all poultry-producing regions. 372 Avian Influenza

Table 17.1. Basis for risk assessment of sanitary protection measures with regard to animal health. Methodology and information Animal disease factors Economic considerations Key trade issues

Make use of available Disease prevalence Potential for loss in case Human health risks scientiﬁ c information Presence of disease- of outbreak should be given Take current production free areas Probability of spread or exceptional methods Environmental establishment of the consideration Consider existing conditions disease Established measures inspection, sampling, Quarantines Cost of control or must not be arbitrary and testing procedures Treatments eradication or unjustiﬁ able Cost-effectiveness of Overall objective alternative approaches should be to to limit risk minimize trade impact in case of outbreak Adapted from Article 5 of the 1994 SPS Agreement.

The information necessary to quantify risk associ- ers was also made (2). Results such as these allow ated with each biosecurity parameter or procedure risk assessors to assign varying risk levels to differ- can be obtained using three methods: experimen- ent types of footwear precautions. tal evidence, retrospective analysis, and expert While virus inactivation studies have been per- recommendation. formed in the laboratory for most poultry viral Existing experimental data alone are insuffi cient pathogens, studies examining virus persistence to support a complete risk assessment of the many under fi eld conditions are more valuable when possible biosecurity parameters or procedures in all developing risk assessments. The AI virus has been their diversity. Differing local conditions make it shown to survive and remain infective for up to 32 diffi cult to extrapolate from a defi ned experiment to days at 15º to –32º C in chorioallantoic fl uids but varying local farm conditions. Nonetheless, concrete loses infectivity after 6 days when mixed with fi eld scientifi c facts contribute signifi cantly to all biose- chicken manure (11). Such data can be used to esti- curity programs and their risk assessments. The mate risk assessment values for poultry house down- general body of microbiological knowledge and the times and clean-out procedures. known virology of the AI virus play a role in many In developed countries, live poultry markets of our decisions relating to disinfectant choice, bio- (LPMs) are well accepted as reservoirs of infection logical vector traffi c, farm clean-out procedures, and for many poultry pathogens, including the AI virus. other basic biosecurity procedures. Whenever appli- Studies comparing isolation rates with market cable, experimental data should be applied, espe- bio security measures, clean-out procedures, and cially if it provides a quantifi able comparison or “monthly rest days,” where the market is entirely ranking of risk factors. Many examples of practical emptied of poultry and cleaned 1 day each month, experimental data exist. A study using post treat- clearly show a reduced risk as measured by AI virus ment bacterial counts on boots that had been con- isolation frequency (4, 10). Epidemiological differ- taminated with pig manure showed that boot ences are seen in LPMs for AI virus and the New- scrubbing was superior to standing in disinfectant. castle disease virus (NDV). It appears that the AI Even scrubbing boots in untreated water was supe- virus is introduced to LPMs at a relatively low fre- rior to standing in disinfectant and as effi cacious as quency but is maintained well, especially in LPMs scrubbing in disinfectant under some conditions. A without rest days or good biosecurity measures. The comparison of different disinfectants as boot sanitiz- NDV, in contrast, is reintroduced regularly from 17 / Farm Biosecurity Risk Assessment and Audits 373 supplier fl ocks, making biosecurity measures less of actual outbreaks is small and all the farms report effective at reducing NDV incidence at LBMs (10). the same boot wash policy. Finally, even if a vari- Such distinctions support making different risk able is shown not to be statistically signifi cant, it assessments depending on the relative threats from could still be epidemiologically signifi cant. In the different pathogens. In this case, the change in a case of AI, the farm or company may be willing to particular biosecurity variable might affect the institute an inexpensive biosecurity procedure even overall risk for AI virus infection differently than if it only offers a slight reduction in risk. the risk of NDV infection. When neither experimental nor retrospective data The degree to which poultry pathogens can be are available, it is advisable to review the literature spread through the air has long been a subject for for veterinary disease outbreaks that may be par- debate. Negative instances provide seemingly little tially analogous. There is substantial information peace of mind, while instances of apparent airborne available regarding risk factors for diseases such transmission are often met with skepticism due to velogenic ND or foot and mouth disease (FMD) the diffi culty of ruling out nonairborne vectors. from which AI applicable information can be However, because distance between poultry trans- extrapolated. port routes and other livestock-related sites are key Expert surveys or individual recommendations to a risk assessment, an attempt to quantify AI virus from experts are not mutually exclusive from the airborne transmission risk must be made. In one experimental data or retrospective studies. In addi- well-controlled study, in a room designed and main- tion to their clinical experiences, experts’ views are tained to prevent disease transmission other than tempered by their familiarity with scientifi c data and airborne, an high pathogenicity avian infl uenza published reports. Seventy-two poultry health spe- (HPAI) virus was shown to be transmitted 100% to cialists in North America were surveyed in order to naïve direct contact chickens from inoculated chick- prioritize biosecurity risks (23, 24). The results are ens; however, no birds in a pen 3 m distant became summarized in Table 17.3. infected despite the fact that room airfl ow was When composing a risk assessment form and directed from the inoculated bird pen to the pen 3 m assigning risk levels to different parameters, it may away (6). Other studies had similar results and be necessary to customize forms for the different suggest overall that airborne transmission is not a poultry production classes. While many variables major risk factor with regard to AI virus (3, 9, 14). will be the same, the traffi c patterns, bird fl ow, and Retrospective review of previous AI outbreaks is housing can be different enough to warrant different perhaps the most reliable predictors of risk when forms. Even in an integrated broiler operation, it analyzed rigorously and objectively. Their utility is is advisable to use different assessments for broiler limited only by the fi nite number of examples and and breeder sites. The primary diseases for which lack of appropriate recordkeeping. Published articles hazards are being identifi ed must be decided in that describe AI outbreaks in a purely narrative advance. A risk assessment for AI in a broiler oper- fashion, while generally informative, do not provide ation would differ signifi cantly from a risk assess- substantial risk assessment information. Those ment for Salmonella enteritidis in a multiple age papers that attempt to objectively identify factors layer complex. Depending on local regulations and that help control an outbreak, reduce the rate of indemnifi cation policies, even risk assessments for spread, or increase the chances of further infections HPAI and low pathogenicity avian infl uenza (LPAI) are more valuable. Some examples are given in might look appreciably different. Table 17.2 (1, 7, 12, 13, 19). All biosecurity variables can be categorized as One must view such conclusions cautiously. Any either tasks or circumstances. They are either pro- survey administered to large numbers of people can cedures that humans must do or refrain from doing be misinterpreted no matter how carefully written. (tasks), or they are existing physical structures or Respondents may give inaccurate answers that they situations affecting risk (circumstances). Changing feel are more acceptable despite promises of ano- into biosecurity garb would be a task. A perimeter nymity. Sample size and lack of variation can also fence or proximity to other poultry farms would be confound such studies. It is diffi cult to statistically circumstances. Generally, poultry producers have analyze the utility of using a boot wash if the number more control over tasks than circumstances. 374 Avian Influenza

Table 17.2. Examples of observations relevant to risk assessment from published accounts of selected AI outbreaks. Key factors believed to Key factors believed to Factors not believed Location, date, type of have increased have decreased to have inﬂ uenced outbreak, and reference transmission risk transmission risk transmission risk

Hong Kong, 2002, Live poultry markets Quarantine — HPAI H5N1 (19) Increased biosecurity Depopulation of infected and contact farms Limited use of killed AI vaccines Virginia, West Virginia, Live poultry markets Testing and depopulation Wild or backyard North Carolina, USA Human, fomite, and birds (initial observations), equipment trafﬁ c by Airborne transmission 2002, LPAI H7N2 vehicle (1, 17) Delays in depopulation Rendering related transport Virginia, West Virginia, Disposal of birds by — Number of birds on North Carolina, USA rendering farm (farm survey and Age of birds greater Perimeter fencing statistical analysis), than 10 week Wild fowl observed 2002, LPAI H7N2 Nonfamily caretakers near poultry houses (13) on farm Farm owners or family members with other off-site jobs Mammalian wildlife observed near poultry houses Italy, 2000–2001, LPAI — Limited use of killed AI — H7N1 (12) vaccines Testing and depopulation Increased biosecurity and trafﬁ c control Pennsylvania, USA, Depopulation of nearby Quarantine — 1996–1998, LPAI farms Controlled marketing H7N2 (7) Live poultry markets Increased biosecurity Lapses in on-farm biosecurity

The biosecurity risks for existing sites that are The second active risk is performing a procedure affected by these biosecurity variables can be placed that does not reduce risk. The error in this situation into one of four classes, two being passive failures, is the opportunity cost of lost resources that could and two being active failures. have been applied to a risk-reducing procedure. The ﬁ rst active risk is any detrimental action, such The ﬁ rst passive risk is failing to perform a risk as traveling to an LBM and returning to the farm. reducing procedure such as boot washing. The 17 / Farm Biosecurity Risk Assessment and Audits 375

Table 17.3. Highest priority biosecurity risks as determined by a survey of 72 poultry health specialists in North America. People Related Issues Location and Trafﬁ c Related Issues Animal Contact Related Issues

Farm employees also own High farm density Poultry of multiple ages (e.g. poultry Presence of backyard fl ock within brooding and growout) in Farm employees attend 400 meters same building cock fi ghts Central bird disposal location used Multiple ages of poultry on Relatives of farm by multiple growers same farm employees have contact Local rendering traffi c going farm Presence of rats or mice during with other poultry to farm daylight Grower or farm employee Wild birds in house when visits other poultry farms poultry are present Farm employee owns pet More than one poultry species birds on farm Leaving live birds on site after shipout Pets with access to poultry houses Based on Vaillancourt (23, 24).

Table 17.4. Examples of the four basic biosecurity risk types. Active Passive

Increases risk Unnecessary travel to neighboring poultry Failure to change footwear farms before entering farm premises Fails to reduce risk Disinfection of car windshield with formalin Farm location next to a live prior to reentering farm premises poultry market second passive risk is circumstantial and occurs the farm would have caused or experienced an because of an inherently high-risk situation, such as increase in all four types of biosecurity-related a farm being located next to an LBM. Active failures risk. or risks are usually corrected by passive actions (e.g., cease visiting LBMs) while passive failures Risk Assessment Form can often be corrected by active correction (e.g., Appendix 17.1 is an example of a risk assessment institute a footwear policy). Some passive risks, form developed for use on North American broiler especially those related to location, cannot be easily farms to assess the risk of AI infection by quantify- remedied through by opposing actions. It is not ing the relative risk reduction associated with a practical to move a farm. This type of risk must wide variety of biosecurity procedures and situa- either be accepted, the farm closed, or overall risk tions. It was developed by poultry veterinarians, live lowered through manipulation of other biosecurity production personnel, and other poultry industry variables. experts. Simple actions or omissions can impact biosecu- The initial part of the form is for farm identifi ca- rity risk in all four categories (Table 17.4). For tion. Such sections can be tailored to an individual example, a grower who travels to an LBM located operation. However, accurate contact information, next to his farm returns without a change of clothes precise location, and housing inventory are essential but washes his car’s windshield prior to reentering minimum requirements. In addition to a farm 376 Avian Influenza address, a more meticulous measure, preferably lon- density have repeatedly been shown to strongly cor- gitude and latitude as measured by a handheld GPS relate with the risk of disease outbreak. This is sup- device is recommended. ported by axiomatic concept that the best predictor On the left side of the form, each variable is listed of the future is the past. Although corrective mea- as an implied question or concise descriptive state- sures often cannot be applied in areas where the risk ment. Up to four scenarios which answer these is very high, quantifying the area biosecurity risk implied questions are listed to the right. These allows one to better assess the relative importance answers are in order of decreasing risk with a score of variables in the FARM and HOUSE sections. The of 64, 16, 4, or 0 assigned, respectively. The expo- strong emphasis on AREA may initially appear exag- nential scale is used to clearly differentiate the dis- gerated. However, both clinical experience and logic crete levels of risk. A higher score denotes a higher support this weighting. There is no amount of prac- relative risk. tical on-farm or poultry house biosecurity that can It is crucial that the individual variables and sce- reduce the overall risk to acceptable levels if the narios are as unambiguous and discrete as possible. farm is located in a poultry-dense region riddled This largely eliminates subjective judgment which with all types of infectious poultry diseases. Con- often occurs when survey responders are asked to versely, a farm located in an area devoid of any other rate the quality or character of biosecurity variables poultry farms or poultry traffi c may not signifi cantly on large, almost continuous scales (e.g. on a scale reduce its overall risk by instituting draconian bio- of 1 to 10 where 1 is most biosecure and 10 is least security measures. biosecure). The authors of this form, in fact, seri- The fi rst part of the AREA section is an inventory ously considered using a maximum of only three of the recent AI or velogenic, viscerotropic New- levels. For many variables, there are only two or castle disease (VVND) outbreaks within 2 and 6 three scenarios. The criteria for the four levels of miles of the farm site. The rest of the AREA section risk were as follow: takes inventory of other poultry related sites within a 2-mile radius of the farm being assessed. Although 0: Zero risk or as close to zero risk as could these distances are somewhat arbitrary, they are practically be expected under commercial based on distances circumstantially linked to epizo- poultry conditions otic poultry disease transmission and typical human, 4: Acceptable risk within a framework of good poultry, wildlife, and vehicle traffi c patterns in farm practices poultry-producing regions. For the most part, they 16: High risk assume that purely airborne transmission of AI is not 64: Unacceptable risk signifi cant. The criteria for each site type should be defi ned on a separate instruction sheet to ensure The assessment portions of the form are divided consistent evaluation by assessors. into three major areas: AREA, FARM, and HOUSE. The farm site manager may not have suffi cient Each individual biosecurity variable is placed into information for the assessor to complete the disease one of these three categories based on whether it history portion of the AREA section. Often, the primarily impacts the risk of disease introduction company veterinarian, local poultry diagnostic labo- into the surrounding area, the farm site, or the ratories, or state authorities must be consulted. Both poultry buildings. The AREA variables make up direct observation, while traveling in the surround- approximately 50% of the risk assessment, with the ing area, and discussions with a locally knowledge- FARM and HOUSE variables contributing roughly able person are usually necessary to reliably equally to the remaining 50%. determine which poultry sites and poultry traffi c While the AREA variables are often not under exist within the 2-mile radius. Either a handheld direct control of the site manager, they are nonethe- GPS device or an accurate map is essential for less important variables in establishing the overall making such distance determinations. risk to the site. Such area-specifi c variables, outside The FARM section of the assessment focuses on of the farmer’s control, have often been omitted those biosecurity variables that impact the risk of a from traditional biosecurity evaluations. The epide- pathogen gaining access to the farm proper. Dis- miological history and surrounding poultry site tances to risks in the immediate vicinity of the farm 17 / Farm Biosecurity Risk Assessment and Audits 377 site are measured, and the existence of other farm Proximity of other livestock, location of signs, pres- entry barriers is quantifi ed. The risk level posed by ence of biosecurity garb, etc., can all be determined mechanical vectors, such as machinery and vehicles, to some degree by observation. While a tour of most are assessed. Human traffi c, including both the farm of the farm site and entry to poultry house anterooms workers and persons with whom they associate or are necessary to properly complete an assessment, it live, is evaluated. The presence of human residences is usually not essential to enter the areas where live that might indirectly increase risk-associated traffi c poultry are held. to the farm site is noted. On the form in Appendix 17.1, the answers to the The HOUSE section assesses biosecurity risk questions are arranged in four columns. After the variables related to direct exposure of poultry inside assessment is completed, the scores for each section the houses. General management variables such as are totaled, yielding a risk assessment score for downtime, water sanitation, clean-out procedures, AREA, FARM, and HOUSE. Although it is tempting dead bird disposal, presence of multiple ages of to add a variety of explanations or narratives to an birds, and warning signs are checked. Personal bio- assessment, the score remains the single objective security measures necessary for entry into the houses measure by which sites can be compared. Narratives are scored. The total number of birds on a site are summarizing the key parameters or sections that inventoried, as well as any other animal species that have contributed most strongly to a particular score could gain access to the house. are more appropriate than excessive notes describ- This document is an example of a form intended ing exceptions or nonessential details. After the fi rst for a typical U.S. broiler integrator to assess the risk use of an assessment form on a large number of on contract broiler farms. Such forms might need farms, a correlation should be made with historical modifi cation for use by other companies with differ- information to establish how predictive the risk ing policies. For example, certain biosecurity risks assessment might be. There should be a strong cor- might not only be considered universally bad prac- relation between the assessment scores and any his- tices (e.g., presence of pet birds on farm) but could torical data on disease incidence by farm. Once also be offenses for which the grower’s contract disease outbreaks occur in an area where risk assess- could be terminated. In this case, the score received ment scores are available, then again the assessment for such a risk would be secondary to the practical should be evaluated as to its predictive value. Each considerations. The scores and possible scenarios outbreak should be used as an opportunity to add should also be adjusted appropriately for different knowledge to the risk analysis and improve any risk poultry production classes. Few contract broiler assessment forms. farms in the United States would be completely fenced, allowing entry of humans or vehicles only Practical Applications of Risk through a locked gate. This type of barrier would be Assessment Results more typical for a hatchery or breeder site. Addi- A valid risk assessment procedure can be a valuable tional levels of fencing scenarios and scores could business tool for a poultry production company. The be added for such sites. Some variables and sce- business advantages can be divided into two catego- narios would be universal but others would need to ries: (1) ability to manage the existing risk more be customized for different poultry producing effectively and (2) opportunity to reduce future risk. regions. Table 17.5 describes examples of both. It is essential that the farm site manager be present Risk assessments allow for rational policy changes during the assessment. It is not necessary to go with regard to high-risk sites. If AI has been identi- through each question in a regimented fashion as fi ed in an area, chick placement schedules can be long as all questions are answered. The assessment effectively managed using the biosecurity risk can be expedited and the site manager put at ease by assessment score with chick placements only on adopting a conversational and observational meth- farms scoring lower than a specifi ed level until the odology. For example, if the site manager’s pet dog AREA threat level is reduced. Company personnel follows the manager into the breeder house during can be directed to increase site visit frequency (e.g., the farm visit, that particular risk question has been for increased monitoring) or decrease visits (e.g., to answered without the need for verbal interrogation. reduce traffi c-related risk). Overall, surveillance on 378 Avian Influenza

Table 17.5. Examples of practical use of biosecurity risk assessment. Use of biosecurity risk Tool allows company assessment and site owner to . . . Method of application

Determine priority for chick Manage current risk Farms with lower scores will continue to placement during high-risk receive chick placements in high-risk time events periods, whereas high-risk farms may not. Determine servicing Manage current risk High-risk farms are serviced by company prioritization and personnel at higher or lower levels. Farm sequencing visits may be restricted to traffi c by dedicated service personnel operating at a higher level of biosecurity. Determine surveillance Manage current risk High-risk farms may be tested on a higher frequency frequency or higher sample size. Determine priority for Manage current risk If a high-risk situation presents itself, high- vaccination (if applicable) risk farms may receive vaccination fi rst. Allows a criteria for zoning Manage current risk High-risk farms are compartmentalized together and have a separate and specifi c biosecurity program. Criteria to consider before Reduce future risk Each farm considering expansion must adding additional houses have a risk assessment score (after (capacity) to existing farms expansion) below the average for the complex. Criteria to consider before Reduce future risk New farms must have a biosecurity risk building houses on a new assessment score lower than the average for site the complex (although if not built yet, the answers to most questions can be determined through analysis of plans and current standard operating procedures). Utilize scores to determine Reduce future risk Those farms with lower scores may be given premium incentives for premium ranking with regard to fi nancial or low-risk farms farm classifi cation purposes. Utilize scores to demonstrate Reduce future risk When done on an annual basis, scores can be continuous improvement compared from year to year. Utilize overall performance Reduce future risk After assessment of the farms, determine the on each question to select issues that represent the most accumulation educational topics of points.

high-risk farms can be increased to provide a higher a single group for the purposes of international trade level of confi dence of the disease status. If vaccina- decisions. In the past, such zoning policies were tion is an available tool, risk assessment scores can based on geographic considerations (regionaliza- be utilized to prioritize farms where immunization tion). Because of the wide variation in types of would have the greatest chance of reducing overall poultry farms, number of livestock sites, diverse risk. levels of biosecurity, and the prevalence of wildlife Risk assessment can be used to support zoning in any geographic region, regionalization policies programs based on compartmentalization, where were not always effective. The preference for com- one or more livestock subpopulations under common partmentalization stems partially from the ability to management or biosecurity programs are treated as document the management, biosecurity, and risk 17 / Farm Biosecurity Risk Assessment and Audits 379 level similarities within a group of livestock sub- the risk of disease introduction into a poultry facil- populations. Good risk assessments are part of this ity. It gives immediate feedback and acts as a check justifi cation. In situations where zoning is not rec- on compliance. Although straightforward in execu- ognized or where public opinion threatens to accel- tion, a biosecurity audit is by no means foolproof. erate depopulation schemes beyond what is A written procedure and available biosecurity sup- necessary, good risk assessment data can help in plies do not guarantee that any particular key task is making a case for more prudent control and eradica- being performed reliably. Instead of performing tion programs. comprehensive audits at regular intervals, spot Examples of using risk assessment scores to checks of key procedures may be a more effective improve the risk profi le of a group of farms going means of auditing and ensuring biosecurity proce- forward are also listed in Table 17.5. Scores can dures and structures. Any existing biosecurity SOPs support decisions regarding expansion and new farm must be available to the auditor well in advance of site locations as well as those relating to site layout the audit and the auditor must have permission to and poultry house design. A decision to expand or view all parts of the premises relevant to the audit. build a farm with a poor existing or predicted risk The output of a biosecurity audit is a list or nar- assessment score presents an opportunity to correct rative of biosecurity defi ciencies and assets in com- some issues associated with the FARM and HOUSE parison to the written SOPs. Short-term and questions that resulted in the elevated score. long-term recommendations should also be part of a Contract growers can be rewarded for low scores biosecurity audit. Although some biosecurity audit just as they are rewarded for farm improvements and forms and checklists may include a score, this is no updated equipment. This reward might be in the more important to a biosecurity audit than a fi nal form of a premium contract or simply more depend- balance correction would be to the audit of a fi nan- able and frequent placement of fl ocks on the site. cial account. Including scores may give the false Contract growing sites are commonly classifi ed as impression that a risk assessment is being performed “class A” or “class B” based on the quality of the when actually the degree of compliance with a rela- feeding, watering, ventilation, or lighting equipment tively infl exible set of SOPs is being ascertained. or on other house construction criteria. A risk assess- A good biosecurity audit reveals not only the ment score could be used in the same way. Once this degree of compliance with existing SOPs but also premium is established, high-risk sites are motivated indicates the practical feasibility of those same to move toward a lower level of risk. Good data SOPs. Procedures that are unreasonably stringent or from risk assessments combined with reliable disease impossible to execute are exposed. Some site man- outbreak information also allow the discontinuation agers will undoubtedly fi nd more effi cient ways to of costly or inconvenient biosecurity measures that achieve the same biosecurity tasks. These improved are not effective in lowering risk. methods can be transferred to other sites. Companies working to better manage biosecurity have a need for demonstrating progress (or lack FIELD USE OF RISK ASSESSMENTS thereof) and identifying areas that need attention, In the United States, a localized poultry operation which then are translated into educational initiatives which works with a common management team, for the upcoming year. The biosecurity risk assess- common hatchery, common feed mill, and some- ment process provides both. If done annually, trends times even a common processing plant is routinely can be established. After assessing a group of sites, referred to as a complex. In the following example common weaknesses might become apparent from the fi eld, risk assessments were performed on through a consistently high score for a specifi c ques- four different complexes and the averages for all tion or group of questions on the risk assessment. sites within a complex are shown in Figure 17.1 (B. Appropriate focus on the practice identifi ed results Stewart-Brown, unpublished data, 2006). in accelerated improvements and lower overall risk. Complexes 1 and 3 show lower average levels of risk among the participating sites than complexes 2 BIOSECURITY AUDITS and 4. Although specifi c biosecurity variables should A biosecurity audit is a complete accounting of all always be examined, it may be appropriate to biosecurity tasks and structures designed to reduce increase the overall education and oversight to the 380 Avian Influenza

300

250 244 237

200 191 189

150

100

Average Site Score for Complex 0 Complex 1 Complex 2 Complex 3 Complex 4

Figure 17.1. Average biosecurity risk assessment scores determined for four different broiler production complexes.

Sample Complex, 209 Farms Mean = 190

100 90 81 80 70 60 48 50 40 36 30 20 Number of Sites 20 12 5 4 10 0 2 1 0 0

0 0 0 0 0 0 0 0 0 50 5 0 5 0 5 0 5 0 5 < 1-100 - 4 - 5 5 01 - 1 51 - 2 01 - 2 51 - 3 01 - 3 51 - 4 01 51 01 - 5 1 1 2 2 3 3 4 4 5 Risk Assessment Scores

Figure 17.2. Distribution of biosecurity risk assessment scores within one company. two complexes with the higher averages. If the same Twelve sites had scores greater than 300 and there- company operates all four complexes and expansion fore warrant further analysis. If the scores are high is needed or being considered, complexes 2 and 4 due to FARM and/or HOUSE associated variables, might be better suited to handle additional housing signifi cant improvement may be possible with edu- from a biosecurity risk perspective. cation, changes in management practices, or minor In another fi eld example from a large commercial structural changes. These farms would also be can- broiler operation, a single complex of 209 sites was didates for enhanced surveillance if and when an assessed. The risk assessment data are shown in infectious disease risk was recognized in the geo- Figure 17.2 (B. Stewart-Brown, unpublished data, graphic vicinity of this complex. Although the 300- 2006). point threshold is arbitrary, scores over 300 mean The average risk assessment score for all the sites that a signifi cant number of inquires were answered in this complex was 190. Twelve sites appeared to as “high risk” or “unacceptable risk” (16 or 64 point be very low risk with scores between 51 and 100. answers). 17 / Farm Biosecurity Risk Assessment and Audits 381

10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 Total Points Contributed by Each Question 0 1 4 7 10131619222528313437404346495255586164 Question #

Figure 17.3. Relative contribution of individual questions to biosecurity risk assessment scores.

The frequency at which individual questions (repre- Biosecurity risk assessment scores ranged from a senting specifi c biosecurity variables) contribute to low of 228 to a high of 644. The strain of H7N2 scores can also be evaluated. A compilation of total LPAI present in this area, resulted in 5 of the 33 sites points attributed to each question were compiled by in this complex becoming positive for AI. The bio- individual question and shown in Figure 17.3. This security risk assessment scores of the fi ve sites biosecurity risk assessment represented 637 total infected were 396, 512, 580, 608, and 636. Four of sites (B. Stewart-Brown, unpublished data, 2006). the fi ve sites were clearly in the higher range of risk, It is apparent that questions 14, 39, and 59 were at least partially substantiating the validity and pre- the three inquires generating the most points of the dictive value of this particular risk assessment (B. total 66 questions. These questions associated with Stewart-Brown, unpublished data, 2006). these numbers were: CONCLUSION 14. A high concentration of sites that raised birds A thorough biosecurity risk analysis, a comprehen- for other integrated companies sive disease risk assessment, and the meticulous 39. Sites where manure spreading was in close auditing of biosecurity procedures are necessary proximity to the poultry houses parts of any disease prevention program. These 59. Sites where poor visitor dress policy practices analyses must be based as much on scientifi c fact as were in place possible, supported by retrospective data, and augmented by well-considered recommendations from This utilization of the data allows management experienced poultry veterinarians and other poultry to focus on specifi c practices that can reduce risk health professionals. Direct observation through with improved education and minor adjustments in farm visits by an experienced assessor is crucial. practices. Ongoing review and correlation of existing assess- Following an AI outbreak, a complex utilizing a ments and procedures with actual disease incidence biosecurity risk assessment document similar to the allow for constant improvement of biosecurity pro- one described earlier in this chapter had the oppor- grams and continuous refi nement of the associated tunity to evaluate the predictive value of the biose- risk assessments. curity risk assessment. In Figure 17.4, a histogram Although biosecurity programs have become a of the 33 site scores from this complex is shown. routine part of most modern poultry operations, they 382 Avian Influenza

Risk Assessment Scores

Not Infected with AI Infected with AI 8

7 1 6

3 6 5 5 11 Number of Sites 2 2 3 3 1 22 1 1 0 0 0

00 50 1-200 1-250 1-300 1-350 1-4 1-450 1-500 1-550 1-600 1-6 1-700 15 20 25 30 35 40 45 50 55 60 65 Range of Risk Assessment Scores

Figure 17.4. Histogram of the 33 site biosecurity risk assessment scores from a poultry complex assessed following an avian inﬂ uenza outbreak.

will not be entirely effective until poultry producers 6. Forman, A.J., I.M. Parsonson, and W.J. Doughty. have the tools to assess factors that contribute to risk 1986. The pathogenicity of an avian infl uenza and audit biosecurity procedures with the same dili- virus isolated in Victoria. Australian Veterinary gence and attention to detail applied currently to the Journal 63(9):294–296. measurement of other poultry inputs such as nutri- 7. Henzler, D.J., D.C. Kradel, S. Davison, A.F. Ziegler, D. Singletary, P. DeBok, A.E. Castro, H. tion, equipment, housing, poultry health products, Lu, R. Eckroade, D. Swayne, W. Lagoda, B. and labor. Schmucker, and A. Nesselrodt. 2003. Epidemiol- ogy, production losses, and control measures asso- REFERENCES ciated with an outbreak of avian infl uenza subtype 1. Akey, B.L. 2003. Low-pathogenicity H7N2 avian H7N2 in Pennsylvania (1996–98). Avian Diseases infl uenza outbreak in Virginia during 2002. Avian 47:1022–1036. Diseases 47:1099–1103. 8. Holmes, J.H. 1944. Hygiene and sanitation with 2. Amass, S.F., B.D. Vyverberg, D.R. Ragland, C.A. the poultry fl ock. In: H.E. Biester and L. Devries Dowell, C.D. Anderson, J.H. Stover, and D.J. (eds.). Diseases of Poultry. The Collegiate Press: Beaudry. 2000. Evaluating the effi cacy of boot Ames, IA, pp. 85–93. baths in biosecurity protocols. Swine Health Prod- 9. Homme, P.J., B.C. Easterday, and D.P. Anderson. ucts 8(4):169–173. 1970. Avian infl uenza virus infections II. Experi- 3. Beaudette, F.R. 1925. Observations upon fowl mental epizootiology of infl uenza A/turkey/ plague in New Jersey. Journal of the American Wisconsin/1966 virus in turkeys. Avian Diseases Veterinary Medical Association 67:186–194. 14:240–247. 4. Bulaga, L.L., L. Garber, D. Senne, T.J. Myers, R. 10. Kung, N.Y., Y. Guan, N.R. Perkins, L. Bissett, T. Good, S. Wainwright, and D.L. Suarez. 2003. Ellis, L. Sims, R.S. Morris, K.F. Shortridge, and Descriptive and surveillance studies of suppliers to J.S.M. Peiris. 2003. The impact of a monthly rest New York and New Jersey retail live-bird markets. day on avian infl uenza virus isolation rates in retail Avian Diseases 47:1169–1176. live poultry markets in Hong Kong. Avian Dis- 5. Dekich, M.A. 1995. Principles of disease preven- eases 47:1037–1041. tion in commercial integrated broiler operations. 11. Lu, H., A.E. Castro, K. Pennick, J. Liu, Q. Yang, In: S.M. Shane, D. Halvorson, D. Hill, P. Villegas, P. Dunn, D. Weinstock, and D. Henzler. 2003. and D. Wages (eds.). Biosecurity in the Poultry Survival of avian infl uenza virus H7N2 in SPF Industry. American Association of Avian Patholo- chickens and their environments. Avian Diseases gists: Kennett Square, PA, pp. 90–94. 47:1015–1021. 17 / Farm Biosecurity Risk Assessment and Audits 383

12. Marangon, S., L. Bortolotti, I. Capua, M. Bettio, (eds.). Biosecurity in the Poultry Industry. Ameri- and M. Dalla Pozza. 2003. Low pathogenicity can Association of Avian Pathologists: Kennett avian infl uenza (LPAI) in Italy (2000–2001): Epi- Square, PA, p. 2. demiology and control. Avian Diseases 47:1006– 19. Sims, L.D., Y. Guan, T.M. Ellis, K.K. Liu, K. 1009. Dyrting, H. Wong, N.Y.H. Kung, K.F. Shortridge, 13. McQuiston, J.H., L.P. Garber, B.A. Porter- and M. Peiris. 2003. An update on avian infl uenza Spalding, J.W. Hahn, F.W. Pierson, S.H. Wain- in Hong Kong 2002. Avian Diseases 47:1083– wright, D.A. Senne, T.J. Brignole, B.L. Akey, and 1086. T.J. Holt. 2005. Evaluation of risk factors for the 20. Stewart, R.G. 1987. ABUS: systems approach to spread of low pathogenicity H7N2 avian infl uenza preventive medicine. 1987. Poultry International virus among commercial poultry farms. Journal of 26(3):46–48, 50. American Veterinary Medical Association 226(5): 21. U.S. Department of Agriculture. Risk analysis. In: 767–772. Food Safety and Inspection Service background- 14. Narayan, O., G. Lang, and B.T. Rouse. 1969. A ers/key facts. U.S. Department of Agriculture: new infl uenza A virus infection in turkeys. Archive Washington, DC. Virusforschung 26:149–165. 22. University of California, Agricultural Extension. 15. Offi ce International des Epizooties. 2006. General 1973. Disease Prevention Checklist. University defi nitions. In: OIE Terrestial Animal Health Code. of California, Agricultural Extension: Davis, Chapter 1.1.1. pp. 9. CA. 16. Salsbury, J.E. 1937. Sanitation. In: Poultry Dis- 23. Vaillancourt, J.-P., and A. Martinez. 2001. Rela- eases and Related Subjects. Book III. Chapter VIII. tive importance of biosecurity measures. A Delphi Dr. Salsbury’s Laboratories: Charles City, IA, study. Annual Meeting of the American Associa- pp. 150–154. tion of Avian Pathologists Poster Session, July 17. Senne, D.A., T.J. Holt, and B.L. Akey. 2005. An 14–18, Boston, MA. overview of the 2002 outbreak of low-pathogenic 24. Vaillancourt, J.-P. 2002. How do you determine H7N2 avian infl uenza in Virginia, West Virginia the cost-benefi t of a biosecurity system? VIII and North Carolina. In: R.S. Schrijver and G. Koch Seminario International de Patologia y Producciòn (eds.). Avian Infl uenza Prevention and Control. Avicola, October 9–11, Santiago, Chile. Springer: Dordrecht, pp. 41–47. 25. World Trade Organization. 1994. Agreement on 18. Shane, S.M. Introduction. 1995. In: S.M. Shane, the application of sanitary and phytosanitary mea- D. Halvorson, D. Hill, P. Villegas, and D. Wages sures. GATT 1994: Uruguay. 384 Avian Influenza

Appendix 17.1

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—AREA

Farm Name: Farm Address:

Complex:

Contact Person:

Phone:

GPS Coordinates and Grid Number: Date:

Number of Houses in Use: Total Sq Ft in Use:

1 Number of Standing Houses not in use: A unit is 40,000 sq ft, # of units (to nearest /2):

Assessor: Signature:

DISEASE HISTORY 64 16 4 0 Circle the correct response with regard to the most recent disease outbreaks (including this farm) within 6 miles of the farm: ILT — last 12 months last 3 years >3 years Avian Infl uenza in commercial poultry last 12 months last 3 years last 10 years >10 years (chickens, turkey, ducks, etc)—low path Avian Infl uenza in commercial poultry last 12 months last 3 years last 10 years >10 years (chickens, turkey, ducks, etc)—other than low path Avian Infl uenza in avian species other — last 12 months last 3 years >3 years than commercial poultry Exotic ND or VVND in commercial last 12 months last 3 years last 10 years >10 years poultry (chickens, turkey, ducks, etc) Exotic ND or VVND in avian species — last 12 months last 3 years >3 years other than commercial poultry Circle the correct response with regard to the most recent disease outbreaks (including this farm) on or within 2 miles of the farm: ILT — last 12 months last 3 years >3 years Avian Infl uenza in commercial poultry last 12 months last 3 years last 10 years >10 years (chickens, turkey, ducks, etc)—low path Avian Infl uenza in commercial poultry last 12 months last 3 years last 10 years >10 years (chickens, turkey, ducks, etc)—other than low path 17 / Farm Biosecurity Risk Assessment and Audits 385

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—AREA Circle the correct response with regard to the most recent disease outbreaks (including this farm) within 2 miles of the farm: Avian Inﬂ uenza in avian species other — last 12 months last 3 years >3 years than commercial poultry Exotic ND or VVND in commercial last 12 months last 3 years last 10 years >10 years poultry (chickens, turkey, ducks, etc) Exotic ND or VVND in avian species — last 12 months last 3 years >3 years other than commercial poultry 386 Avian Influenza

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—AREA Circle the number of each of the types of sites within a two mile radius of this farm: Broiler farms that contract with Company — — 3 or more 2 or less Broiler farms that contract with other companies — 3 or more 1 or 2 none Broiler breeder or pullet farm contracting with — — 1 or more none Company Broiler breeder or pullet farm contracting with — 3 or more 1 or 2 none other companies Table egg pullet farm 2 or more 1 — none Table egg layers 1 or more — — none Turkey farm 1 or more — — none Backyard poultry/hobby poultry/fi ghting chickens 1 or more — — none Poultry live market/poultry fl ea market 1 or more — — none Livestock farm of any type (besides chicken, turkey, — — 1 or more none goats, or hog) Goats or hog farm — 1 or more — none Litter processing site 1 or more — — none Live haul, rendering, or (used) litter route used ≥ 5 1 or more — none times weekly Wildlife preserve or other natural site with 1 or more — none waterfowl or wild birds Diagnostic lab — 1 or more — none Poultry company offi ce, allied industry offi ce, or — — 1 or more none poultry equipment company offi ce Processing/rendering/live haul site #1 1 none Processing/rendering/live haul site #2 1 none Processing/rendering/live haul site #3 1 none Feed mill — — 1 or more none Hatchery — — 1 or more none Livestock fed poultry litter from other farms 2 or more 1 — none 17 / Farm Biosecurity Risk Assessment and Audits 387

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—FARM With regard to the topics in the left column, circle the one description on the right which best describes the parts of this farm under the direct control of the caretaker: PHYSICAL Shortest distance to <500 feet 500–1000 feet >1000 feet next poulty house under different management Shortest distance from — ≤100 feet 100–500 feet >500 feet any of the poultry houses to the nearest road If less than 500 ft from None road, vegetative/ physical buffers (berms or trees) Water On The Site natural creek, pond, natural creek, no open water of any lake, or permanent pond, lake, or kind or it is greater than standing water on permanent 500 ft from house site ≤100 feet from standing water on houses site 100–500 ft from houses Farms / Manure — houses within 500 — houses further than 500 feet of farmland on feet of any farmland on which litter or which litter or manure is manure is spread, spread, hauled, or fed hauled, or fed Machinery outside tools, outside tools, outside tools, farm is self-sufﬁ cient in equipment or equipment or equipment or all tools, equipment, and machinery are machinery are machinery are machinery; none are brought on to farm brought on to farm brought on to ever brought on to farm or shared with or shared with other farm or shared or shared with other other farms once a farms more often with other farms farms week or more than once a month only when out of often birds Fencing — — no fencing on some fencing around property houses or at property boundary Warning Sign At Farm — none sign in English sign in English and Entrance only Spanish and/or other appropriate languages Farm Entrance — multiple entrances one entrance, not one or more entrances to to farm, none ever gated farm, but always gated gated Trash Removal — dumpsters are within dumpsters are no dumpsters 100 ft of house present Vehicles — — vehicles can park vehicles must park within 100 feet of further than 100 feet chicken houses from chicken houses or without being must be washed before washed — parking within 100 feet of houses 388 Avian Influenza

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—FARM PEOPLE Anyone associated with Has multiple Has multiple farms Has multiple Has only one farm farm (owns, lives, or poultry farms of placed within 7 days farms within 7 works) different ages and of each other and days of each visits them when visits them when other but only birds are present birds are present visits in-between ﬂ ocks Owner or Family involved in any involved with any involved with any with no other business Members other poultry- other (nonpoultry) other business involvement related business agriculture-related that comes in business direct contact with birds (live or dead and includes processed birds) or poultry manure Anyone else associated involved in any involved with any involved in any this farm is the only with farm (lives or other poultry- other (nonpoultry) other business business of associated works) related business agriculture-related people that comes in business direct contact with birds (live or dead and includes processed birds) or poultry manure Family / Employees more than one —one non-family no outside or non-family non-family outside employee employees employee anytime On-Farm Residence — caretaker lives off- caretaker is not caretaker is farm owner site the farm owner and lives on-site but lives on-site Multiple Residences — More than one — Only residence on farm residence on the is that of the caretaker farm within 500 feet or primary farm of houses manager 17 / Farm Biosecurity Risk Assessment and Audits 389

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—HOUSE With regard to the topics in the left column, circle the one description on the right which best describes this farm:

PHYSICAL

Warning Sign At — none sign in English at least, House Door other appropriate languages are beneﬁ cial and suggested

Downtime In the last 5 In the last 5 fl ocks, 2 Everything else In the last 5 fl ocks, at fl ocks, 2 or or more fl ocks had 7 least 4 fl ocks had 14 more fl ocks days or less days or more had 3 days or less

Source of Water Surface water City or Well water

Water Sanitation — — drinking water not drinking water regularly sanitized sanitized

Cleanout Cake not removed or Cake removed from house Cake removed from destroyed in approved and stored in open within house and immediately manner 500 feet of house removed from farm, stored in manure shed, or covered until removed from farm

Visitor Log Nothing or not current Exists and kept up to date

PEOPLE

Caretaker / — caretaker/employees farm dedicated footwear stepover changing area Employee Dress may enter live bird (or shoe coverings) and is the only way to Policy area without any clothing is donned by proceed onto farm or biosecurity caretaker/employees within 100 feet of precautions before entering live bird houses area

Visitor Dress visitors may farm dedicated farm dedicated footwear stepover changing area Policy enter live bird footwear (or shoe (or shoe coverings) and is the only way to area without coverings) worn by all clothing is donned by proceed onto farm or any biosecurity visitors visitors before entering within 100 feet of precautions live bird area houses

ANIMALS

Number Of Birds Greater than 100,000 to 200,000 50,000 to 100,000 birds 50,000 birds or less On Site 200,000 birds

Other Livestock — any other type of any other livestock no other livestock on (non poultry) with livestock with contact allowed but not within farm Contact To to chicken houses or 100 feet of chicken houses Chicken House(s) within 100 feet

Pets any avian pet any non-avian pets any non-avian pets on no pets of any kind on anywhere on allowed in chicken farm property the site (subject houses to contract termination in some areas) 390 Avian Influenza

SAMPLE FARM BIOSECURITY RISK ASSESSMENT FORM—HOUSE Rodent Control no formal rodent bait stations placed but no signs of vermin, control plan, clear not routinely maintained comprehensive bait evidence of rodents station setup

Wild Birds — wild birds seen in wild bird presence or clear no wild birds ever in or houses evidence (e.g. nests) right next to houses thereof seen on or right next to houses

GENERAL MANAGEMENT

All-in / All-Out Live poultry of live haul picks up a live haul picks up a All-in / All-out policy; any kind not portion of the house, portion of the house, live farm is devoid of from current live haul and haul and associated chickens during ﬂ ock associated people/ people/equipment are downtime; single age equipment not sanitized prior to catch facility, single catch of sanitized prior to catch all birds

Dead Bird carried off site all mortality — all mortality composted, Disposal in any way or composted, incinerated, or buried picked up by incinerated, or buried (regularly and properly) renderer on site but there is on site clear evidence (e.g. piles of birds, etc.) that it is not done regularly

Additional Notes and Description of Dead Bird Disposal Process (method, location, routine used): 18 Methods for Inactivation of Avian Influenza Virus in the Environment

Nathan G. Birnbaum and Bethany O’Brien

INTRODUCTION process that prepares the items for safe handling This chapter covers cleaning and disinfection (C&D) and/or further decontamination (2). Cleaning is a of poultry production facilities and equipment under major part of sanitation procedures and should be routine conditions and under special conditions as employed frequently to maintain biosecure animal part of the response to an avian infl uenza (AI) event facilities. on the premises. Proper C&D practices and good Decontamination refers to the disinfection or ster- hygiene help to prevent disease occurrence and to ilization of infected articles by removing potentially control disease spread. This chapter is directed harmful materials and pathogens to make these suit- toward regulatory personnel, poultry growers and able for use (2). Decontamination involves physical their employees, and contractors involved in disease and chemical means to eliminate or remove patho- prevention, control, and eradication activities. This genic microorganisms allowing the location or the chapter is not intended to serve as an offi cial AI item to be returned to its normal use. C&D policy but is instead intended to supplement Disinfection is the destruction or inactivation of the facility, local, regional, national, and industry pathogenic and other kinds of microorganisms by C&D policies. thermal or chemical means on inanimate surfaces. Sterilization is a process in which all microorgan- Terminology isms are destroyed. Disinfectants are chemical or There is a variety of terms related to C&D. Biosecu- physical agents that destroy or inactivate most forms rity refers to the procedures and practices followed of harmful microorganisms. to prevent the spread of infectious diseases. It In response to an AI event, the poultry industry, includes activities that reduce the chances of an agricultural community, and emergency response infectious disease carried onto or off of premises by community will work cooperatively sharing man- people, animals, equipment, or vehicles (28). Bio- power, equipment, materials, and knowledge regard- security involves not only C&D, but also all other ing biosecurity and decontamination procedures and practices that prevent the contamination by infec- processes. All poultry facilities should have their tious microbial pathogens of facilities, equipment, own emergency response plan for an AI event. There supplies, or personnel. Poultry workers should know is no one best C&D plan that will fi t all situations. about and follow biosecurity practices to prevent the Responsible offi cials may designate specifi c AI introduction of AI into a poultry fl ock (29). C&D response procedures that differ from those Cleaning refers to the removal, usually with employed in other jurisdictions. Some locales allow detergent and water, of adherent visible soil, organic the use of specifi c chemical liquid and gaseous dis- material, and other debris from the surfaces, crev- infectants while the use of these same chemicals is ices, serrations, joints, and lumens of instruments, prohibited by other locales. Variations also may devices, and equipment by a manual or mechanical exist in the recommended post-C&D destocking

Avian Influenza Edited by David E. Swayne 391 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 392 Avian Influenza period (i.e., down time) in which the premises must directions, will be effective against the H5N1 strain. be left without susceptible species following an AI Users should look for an EPA registration number event, but a minimum of 21 days has been recom- on the label (e.g., EPA Reg. No. 123.45). Each mended (17). manufacturer’s name and telephone number are provided so that potential product users may contact the Regulatory Framework manufacturer. Users should carefully follow the dis- In the United States, disinfectants are regulated by infection directions on the label on how to handle the Environmental Protection Agency (EPA) under and safely use the pesticide product to avoid harm the authority of the Federal Insecticide, Fungicide, to human health and the environment. The approved and Rodenticide Act (FIFRA), as amended. Under label of a product can be found in the Pesticide FIFRA, any substance or mixture of substances Product Label System (PPLS) database label search intended to prevent, destroy, repel, or mitigate any site (8). To obtain a product label, enter the EPA pest (including microorganisms but excluding those Registration Number of the primary product in the in or on living humans or animals) must be regis- search query boxes (i.e., the company identifi cation tered prior to sale or distribution. To obtain a disin- number and the product number) of the PPLS data- fectant registration, a manufacturer must submit base. Information about the Pesticide Product Label specifi c physical/chemical, toxicological, environ- System (PPLS) database is posted on the PPLS mental, and effi cacy data to the EPA concerning the homepage. safety and the effectiveness of each disinfectant. If EPA concludes the product may be used without Cleaning and Disinfection Considerations causing “unreasonable adverse effects,” then the Following an Avian Infl uenza Event product and its labeling are registered. The manu- Following the depopulation of birds on premises due facturer may then sell and distribute the product in to an AI event, carefully performed C&D proce- the United States. The EPA also has the authority dures play a vital role in eliminating the pathogen under Section 18 of FIFRA to issue emergency from the environment and controlling the disease exemptions to permit the sale or distribution of spread. It is of the utmost importance to thoroughly unregistered disinfectants or unregistered uses of C&D contaminated objects with detergent and rec- registered disinfectants, provided certain conditions ommended disinfectants (24). The C&D processes are met. Finally, FIFRA requires users of products incorporated following an AI event may be similar to exactly follow the use directions and safety pre- to those incorporated during routine terminal C&D, cautions on each product label. Using a product “in with a few signifi cant differences. These differences a manner inconsistent with its labeling” would be a are primarily increased emphasis on compliance, violation of FIFRA. Not following the specifi ed use increased government oversight, and increased dilution, contact time, method of application, or any public scrutiny. Due to the signifi cant animal and other label-specifi ed condition of use would be con- human health concerns as well as the economic considered misuse and potentially subject to enforce- cerns related to an AI event, C&D must be per- ment action under FIFRA. formed in strict accordance with offi cial government The EPA has registered approximately 100 disin- policies. fectant products intended for use against AI viruses Some of the differences include the following: on hard, nonporous inanimate surfaces (7). These Because of the zoonotic potential of AI, protection products are typically used by the poultry industry of worker health and safety requires additional pre- to disinfect their facilities. The label indicates that cautions. In contrast to routine C&D where the the product is effective against “avian infl uenza A” primary concern is exposure to chemical disinfec- and specifi es the sites (e.g., poultry houses and farm tants, an increased level of personal protective premises) for application of the product to kill or equipment (PPE) to prevent exposure to the infec- inactivate the AI virus. Although there are no anti- tious agent must be used. On biosecure facilities, microbial products registered specifi cally against the only authorized personnel routinely have access; H5N1 subtype of AI viruses, EPA cites scientifi c however, during an AI outbreak, even stricter access information that the currently registered AI prod- regulations will be enforced. Routine C&D includes ucts, when applied in strict accordance with the label removal of live animals and litter before the process 18 / Methods for Inactivation of Avian Influenza Virus in the Environment 393 begins. AI C&D requires removal of dead birds and Cleaning and Disinfection litter following in-house composting. Before C&D Planning Considerations begins, one or more negative virus isolation tests of It is important to develop a facility-specifi c C&D the composted material must be completed. As part plan in conjunction with the various stakeholders in of the precleaning phase, the in-house temperature advance of a disease outbreak. Planning consider- should be raised and maintained per regulatory ations should include availability and cost of disin- direction. A light spraying of the poultry facility fectants, C&D application equipment, poultry with a detergent solution or with a disinfectant solu- facilities structure, environmental persistence of the tion just prior to the start of the dry cleaning phase target microorganisms, the potential for disinfec- may reduce the presence of dust in the air. During tants to adversely affect ground or surface water, the dry cleaning phase, the use of high pressure air containment and disposal requirements, and the currents may cause aerosolization of particulate health and safety of personnel performing the C&D. matter. Disinfectants should only be used that have The natural processes of time, dehydration, warm a label claim for AI effi cacy or are approved by the temperature, and sunlight should be considered in proper regulatory authorities. As always, disinfec- planning as these can enhance the decontamination tants must be used as indicated on the label or as operation (9). instructed by offi cial regulatory authorities. Caution should be taken when removing equipment outside PHYSICAL METHODS FOR INACTIVATING of the facility for C&D due to environmental con- AVIAN INFLUENZA VIRUS tamination concerns. Various natural or artifi cial physical forces that reduce the pathogen load in the environment are Routine Cleaning and important and can often be used to inactivate patho- Disinfection Considerations gens (13). These forces include ultraviolet (UV) Routine C&D of poultry facilities and related equip- light, heat, and drying. ment and conveyances is essential to preventing or controlling infectious diseases. Inadequate C&D or Ultraviolet Light biosecurity can limit profi tability or lead to eco- In general, UV radiation is not an effective method nomic failure. Cleaning and disinfection are working of destroying microorganisms in poultry production steps in animal husbandry that could not be sepa- environments (19, 18). UV light is not effective for rated from each other without losing effectiveness viral destruction in fecal material as it shields the (3). A thorough cleaning and removal of all organic virus from UV light. Only microbes on a clean material from the environment should precede dis- surface and in the air are killed by UV light (16). infection so that the pathogens come in direct contact UV light positioned close to the surface being treated with the disinfectant (13). The presence of organic may inactivate the pathogen by nucleic acid damage. materials reduces the effi cacy of disinfectants and The surface, however, must be free of dust and renders some disinfectants inactive (19). Effective exposed to direct rays (19). UV radiation from sun- C&D substantially decreases disease transmission light is lethal to most microbes, but it does not pen- by reducing pathogens in the environment below etrate effectively beyond the outer contamination infectious levels (13). The likelihood of an agent layer. Still, UV inactivation can play a minor role in being passed to an uninfected fl ock or house will be poultry house sanitation. Poultry equipment and reduced by reducing the number of surviving patho- breeder slats that have been cleaned and disinfected gens in the environment. may be placed in the direct sunlight and after a suit- Routine C&D of all poultry production units on the able time turned upside down to expose all surfaces premises after fl ock departure from the premises and to natural UV radiation. before the arrival of the next batch of birds will help prevent pathogen build-up. The C&D must be thor- Dry Heat ough and with attention to detail to be effective (12). AI viruses are generally sensitive to heat and drying The choice of C&D method depends not only on the (29). Heat accelerates the inactivation of the AI size and type of construction of the house but also on virus in manure (16). A hot, dry sunny day will the availability of labor and specialized equipment. cause rapid natural inactivation of the AI virus (9). 394 Avian Influenza

Poultry houses should be heated to 100º F (38º C) against the AI virus in the presence of an organic for the 72 hours prior to C&D (30). In a chicken load. It is the user’s decision as to which product(s) house, the temperatures may be raised successfully would be best for the specifi c site. to 40º C (104º F) by ordinary heaters used for chicken The listing of specifi c chemicals in the following brooding. At temperatures of 40º C, the AI virus in summary tables does not mean that only products manure may be killed in a short time (6). Heating of containing these chemicals should be used for AI buildings to 90º F (32º C) for 3 hours or to 100º F virus disinfection. Table 18.1 lists active ingredients (38º C) for 30 minutes has been suggested to inacti- of some EPA-registered AI virus disinfectants. The vate the AI virus (5). Heat produced by appropriate active ingredients are those chemicals directly composting may inactivate the AI virus present in responsible for the disinfection action of the product. litter. Inert ingredients may be included in the disinfectant Flame guns are not recommended as a primary product formulation and are listed as “other ingredi- means of decontamination, but these are a conve- ents” on the label. These are proprietary and may nient method of disinfecting concrete or earthen include antioxidants, carriers, catalysts, diluents, fl oors (1). These are also useful on nonfl ammable emulsifi ers, dispersants, preservatives, propellants, sites where liquid products cannot be used due solvents, stabilizers, surfactants, suspending agents, to inadequate drainage. Flame guns may also be etc. Both active and inert ingredients play a role in useful supplements for drying decontaminated the disinfectant product’s performance. Table 18.2 surfaces but the risk of fi re and injury should be lists some chemicals recommended as AI virus considered. disinfectants by several animal health agencies. The following are brief descriptions of some key Wet Heat (Steam) characteristics of chemicals used for AI virus Steam produced by special equipment by itself may disinfection. Only those disinfectants approved be used for disinfection only if the temperature of by appropriate regulatory authorities should be the treated surface can be raised to 100º C (212º F) used. and held there long enough for the microbe to be inactivated (1, 9, 27). Steam is usually only recom- Soaps and Detergents mended as an adjunct to decontamination because Soapy water and detergents may be the fi rst choice of the uncertainties regarding temperatures and for AI virus decontamination of many items (1). times of contact. Soaps and detergents, in addition to being important for their role in removing organic material (dirt or CHEMICAL METHODS FOR grease from surfaces) are effective AI virus disin- INACTIVATING THE AI VIRUS fectants in their own right due to the presence of the Most poultry producers use chemical disinfectants outer lipid envelope of the AI virus (1). The AI virus as part of their routine C&D procedures. Large is very sensitive to detergents, which destroy the quantities of effective chemical disinfectants would fat-containing outer virus layer necessary for virus likely be used by poultry producers under regulatory penetration of the animal cell (10, 20). Detergents supervision in response to an AI event. Poultry pro- themselves may have a lethal effect on microorgan- ducers would want to know what products are avail- isms and are frequently, if not invariably, used hot able and recommended for use against the specifi c (15). Detergents are formulated to enhance cleans- pathogen causing the disease. The disinfectant ing and contain a combination of surfactants (surface product label must list the specifi c pathogens against active agents) with other organic and inorganic sub- which the disinfectant is effi cacious. The AI disin- stances. Detergents may be classifi ed according to fectants registered by the EPA are listed on the the electrical charge of the surface active moiety, agency’s web site (7). These products are registered i.e., anionic detergents carry a negative charge, cat- for use on farm premises and equipment and may be ionic detergents carry a positive charge, nonionic used in poultry production facilities. All of these detergents have no charge, and amphoteric surfac- products meet EPA’s standards for registration and tants have both a positive and a negative charge. none are recommended as being better than the Washing with soap and water is the principal means other. Some of these products have been tested for disinfecting hands (32). Multiple factors, such as Table 18.1. Active ingredients of some Environmental Protection Agency–registered avian infl uenza virus disinfectants. Chemical Type Example

Acid Octanoic acid Alcohol Ethyl alcohol Aldehyde Formaldehyde Glutaraldehyde Organic iodine Polyoxyethylene polyoxypropylene iodine complex Inorganic chlorine Sodium hypochlorite Organic chlorine Sodium dichlorotriazonetrione Peroxygen compounds Hydrogen peroxide Potassium peroxymonosulfate Peroxyacetic acid Phenols Amyl phenol Benzyl chlorophenol Phenyl phenol Potassium benzylchlorophenate Quaternary ammonium compounds Alkyl dimethylbenzylammonium chloride Alkyl dimethylethylbenzylammonium chloride Didecyldimethylammonium chloride Dioctyldimethylammonium chloride Octyldecyldimethylammonium chloride Other Sodium chloride Potassium salt Hydroxymethylnitropropandiol

Table 18.2. Chemical disinfectants recommended for inactivation of avian inﬂ uenza virus. AusVetPlan European Food Ofﬁ ce Internationale des Epizooties Chemical (1) Safety Authority (9) AI Reference Laboratory (Italy) (24)

Soap and detergent X Sodium hypochlorite X X X Quaternary ammonium salts X X Potassium peroxymonosulfate XX X (in commercial product) Calcium hydroxide X X Cresolic acid X X Synthetic phenols X X Formaldehyde X X X Calcium hypochlorite X X Sodium hydroxide X X Sodium carbonate X X Hydrochloric acid X X Citric acid X X Glutaraldehyde X X Formalin X

395 396 Avian Influenza detergent type, specifi c chemical composition, addi- carcinogenic nor mutagenic; however, concentrated tion of bleaching agents and/or enzymes, water solutions may irritate the eyes, skin, and mucous hardness, environmental pH, temperature, presence membranes. Hydrogen peroxide can be easily of organic matter, may affect the disinfectant effi - destroyed by heat to give the innocuous end prod- cacy of soaps and detergents. AI viruses are gener- ucts water and oxygen. It is an excellent surface ally sensitive to most detergents (29). disinfectant and has been used in the veterinary fi eld to disinfect tools, utensils, and premises (15, 27). Disinfectants Vaporized hydrogen peroxide has been used for low-temperature sterilization. Hydrogen peroxide Inorganic Compounds (Iodine) vaporization resulted in oxidation of uncoated steel Iodine-containing solutions have been recommended and galvanized steel (22). Hydrogen peroxide solu- for killing the AI virus (5). Iodine is listed as an tions are unstable and may require the addition of active ingredient of some U.S. EPA-registered AI benzoic acid or other suitable stabilizers (25). A virus disinfectants. Iodine is available in aqueous newer generation of compounds has increased sta- solution, alcohol solution, and as complexes with bility (27). surface active compounds or polymers that allow both increased solubility and sustained release of Peracetic (peroxyacetic) acid free iodine (25). Iodophors retain much of their Peracetic acid is an active ingredient of EPA-regis- activity in the presence of organic matter and are tered AI virus disinfectants and is an excellent effective at both low and high temperatures (25). surface disinfectant. It is a more potent oxidizer than Iodine when used at recommended levels is not very hydrogen peroxide and remains active in the pres- toxic or irritating. Some iodine products may stain ence of organic matter (31). It is corrosive to plain clothing and porous surfaces. Polyvidone iodine steel, galvanized iron, copper, brass, bronze, and products have been recommended for hand sanitiz- natural and synthetic rubbers. Peracetic acid is very ing (32). attractive because of its antimicrobial effi cacy and its lack of environmental problems. Peracetic acid is Peroxygen Compounds used in commercial poultry operations but may not work reliably on the surfaces of transport vehicles Potassium peroxymonosulfate (KPMS) (3, 13). Peracetic acid may irritate eyes, skin, and Potassium peroxymonosulfate is an active ingredi- mucous membranes (27). ent of AI virus disinfectants registered by the EPA and recommended by some animal health agencies. Ethanol (Ethyl Alcohol) The raw chemical KPMS is a free-fl owing, white Ethanol is an active ingredient of some EPA- odorless granular solid that is soluble in water. The registered AI virus disinfectants. The AI virus is oxidation potential of this chemical is derived from susceptible to 70% ethanol (16). Ethanol has been its peracid chemistry. KPMS provides powerful, used to disinfect small hatchery instruments (e.g., nonchlorine oxidation. The commercial disinfectant vaccination needles) but is not used to disinfect large product containing KPMS should be stored in a machinery or rooms (27). Ethanol has a high rate of cool, well-ventilated area away from all sources of evaporation and may not remain suffi ciently long on ignition and out of direct sunlight and in a dry loca- the object’s surfaces to effectively inactivate the tion away from heat. The disinfectant product may virus. Ethanol provides limited activity in the pres- cause eye, skin, respiratory, and digestive tract irri- ence of organic matter. Ethanol may be incorporated tation and burns. in formulations with other disinfectants. Ethanol is fl ammable and should be used only in well-venti- Hydrogen peroxide lated spaces. Ethanol may also cause discoloration, Hydrogen peroxide is an active ingredient of some swelling, hardening, and cracking of rubber and EPA-registered AI virus disinfectants. It is a strong certain plastics after prolonged and repeated use. oxidizer that produces destructive hydroxyl free Ethanol does not penetrate well into dried organic radicals (31). Hydrogen peroxide has low levels of matter on surfaces. Ethanol should be stored away toxicity to humans and the environment. It is neither from sources of heat. 18 / Methods for Inactivation of Avian Influenza Virus in the Environment 397

Hypochlorites Phenols A large number of EPA-registered phenols are avail- Sodium hypochlorite able with label indications for AI virus disinfection. Sodium hypochlorite comes in a liquid form and is Phenols are commonly known as coal tar deriva- widely used as an AI virus disinfectant because it tives, have a characteristic pine-tar odor, and turn relatively inexpensive and is readily available world- milky in water. One-step phenols may also contain wide (32). Sodium hypochlorite is an oxidizing detergents that clean, dissolve proteins, and disin- agent. It may be purchased in a variety of forms such fect. Phenols are relatively tolerant of anionic and as household bleach [containing approximately organic matter. Phenols may leave a residual fi lm 5.25% (52,500 ppm) sodium hypochlorite] or as a that causes skin irritation (4, 31). Phenols are toxic chemical concentrate containing approximately 12% and are generally hard to break down (4). Phenols, (120,000 ppm) sodium hypochlorite. Some have rec- cresols, and xylenols may be produced synthetically ommended a fi nal dilution of 2% to 3% (i.e., 20,000 or by the destructive distillation of coal tar (26). to 30,000 ppm) available chlorine for a 10- to 30- Phenols are widely used as general disinfectants in minute contact time for many applications (except animal facilities, wheel dips, and foot dips (15). when in the presence of organic material) (1, 10). Phenols are sometimes used for disinfection of farm Sodium hypochlorite loses its stability in warm, buildings but impart a tarry odor (25). Commercial sunny conditions above 15º C (60º F) (1). Hypochlo- disinfectants may contain combinations of different rous acid, released in aqueous bleach solutions, is phenol compounds. Phenols may be scented to mask thought to be the active compound (31). It is effec- the tarry odor (27). tive only on clean surfaces and is quickly inactivated by organic matter. Sodium hypochlorite’s use is Cresylic Acid (Cresols) limited by its corrosive effects, inactivation by Cresylic acid is a commercially available cleaner organic matter, and relative instability. Exposure to that contains cresols and xylenols (15). Cresols are sodium hypochlorite through direct contact with the effective in the presence of organic matter (18). Cre- solution or through inhalation of fumes may result sylic acid must be used very carefully as it is a in injury. hazardous substance that is dangerous both to people and the environment if handled improperly. The use Calcium hypochlorite of cresylic (creosylic) acid at 2.2% solution has been recommended as an AI virus disinfectant for the Calcium hypochlorite is an oxidizing agent that treatment of fl oors (23). Cresylic acid is not affected comes as a solid or powder. It is recommended as by hard water or heat (21). Cresylic acid has been an AI virus disinfectant at a working strength of 20 used on surfaces that could not be completely to 30 g/L giving 2% to 3% available chlorine (i.e., cleaned due to its effectiveness on organic matter. 20,000 to 30,000 ppm) for a 10- to 30-minute contact Cresols have been used in poultry houses, foot baths, time (1, 10). It is not effective in the presence of and as fl oor disinfectants. organic materials and is less stable in warm, sunny conditions above 15º C (60º F). Quaternary Ammonium Compounds (QACs) There are many EPA-registered QACs with a label Sodium Dichlorotriazine Trione claim for AI virus disinfection (7). QACs have been This chemical is an organochloride and is an active recommended at a 4% solution for the treatment of ingredient of some EPA-registered disinfectants. walls, fl oors, ceilings, and equipment (23). QAC are The raw chemical is an off-white granular organo- cationic, odorless, nonirritating clear surface active chloride with a slight chlorine odor. It contains disinfectants (31, 18, 12). QACs are relatively non- about 50% available chlorine and is stable under staining, noncorrosive, and nontoxic. QACs have cool, dry conditions. Organic chlorides may have a deodorizing and detergent action and are very good slower action than hypochlorites, but the disinfect- surface disinfectants in a precleaned environment ing effect of organochlorides decreases less signifi - (18). QACs may be neutralized by soap, residues, cantly than that of hypochlorites in the presence of anionic detergents, and organic matter. Some QACs proteins. are more effective than others when organic material 398 Avian Influenza is present (14). QACs have a highly biodegradable fection (9). Formalin has been used at 1 gallon per pH (27). Their activity may decline in the presence 10 feet2 as a general disinfectant for dirt fl oors (27). of hard water. QACs used in high concentrations Formalin has been used as a general farm disinfec- may cause corrosion of metal and skin irritation tant for the treatment of poultry houses, incubators, (25). QACs may build up over extended use and transport vehicles, and other equipment (15). For- create biofi lms that may be removed with use of an malin poisoning can cause severe abdominal pain, acid cleaner (27). QACs are more effective at alka- central nervous system depression, coma, and death. line than at acid pH (15). The use of QACs on farms may be limited because of the large amount of Acids and Bases organic debris (15). Hydrochloric Acid Aldehydes Hydrochloric acid is a nonorganic mineral acid that has been recommended for AI virus disinfection. Glutaraldehyde It is sometimes used for cleaning and disinfecting Glutaraldehyde is an active ingredient of EPA-reg- farm buildings (25). Hydrochloric acid may come istered AI virus disinfectants (7). It is used in the as a 10 Molar concentrate that should be diluted veterinary fi eld for the disinfection of utensils and 1 : 50 to produce a fi nal concentration of 2% (w/v) of premises (15). Glutaraldehyde is noncorrosive to to be used with a recommended contact time of 10 metal and does not damage rubber and plastic equip- minutes (1). It should be used only when better ment (31). It retains activity in the presence of disinfectants are not available. It is highly corrosive organic matter. Glutaraldehyde is an aqueous solu- and should not be used on metals and concrete (9, tion and does not produce a toxic gas (18). It is not 25). Eye protection and rubber gloves should be corrosive to soft metals and is compatible with non- worn because hydrochloric acid is hazardous (25). ionic, anionic, and cationic surfactants. Glutaralde- Hydrochloric acid may be fatal if swallowed (15). It hyde is biodegradable and does not leave sticky should not come in contact with the skin or eyes, residues. Glutaraldehyde works best at temperatures and it is imperative that the prescribed safety pro- above 10º C (50º F) (3). Glutaraldehyde vapor is irri- cedures are followed when this compound is tating to the eyes, throat, and nose and allergic handled. contact dermatitis, asthma, rhinitis, and epistaxis may occur in workers exposed to glutaraldehyde Citric Acid (31). Glutaraldehyde may be carcinogenic (25). Citric acid has been recommended as an AI virus disinfectant at 2 g/L, fi nal concentration of 0.2% w/ Formaldehyde and formalin v, with a 30-minute contact time (1, 9). Citric acid Formaldehyde is an active ingredient of EPA- is safe for clothes and body decontamination. Citric registered AI virus disinfectants and is recommended acid powder is a white odorless crystalline substance for AI virus disinfection (7, 10). Formaldehyde gas and may cause eye, skin, gastrointestinal, and respi- is toxic and should be used only by authorized indi- ratory tract irritation and injury. viduals only if other disinfectants cannot be used (1). Formaldehyde gas should be left in contact for Sodium Hydroxide 15 to 24 hours. Formaldehyde gas is released from Sodium hydroxide (caustic soda, lye, soda lye) is a either formalin solutions or paraformaldehyde strong alkali that may come as pellets. It has been powder. Formaldehyde for fumigation is effective recommended as an AI disinfectant at a working against surface organisms when both the tempera- strength of 20 g/L, fi nal concentration of 2% (w/v) ture and the humidity of the environment are high (20 g/L) solution with a contact time of 10 minutes (18). Formaldehyde presents serious health and (1). It is a very effective disinfectant but should not safety hazards (19). The gas is very toxic to humans be used in the presence of aluminum and derived and safety precautions must be followed. Formalin alloys. It should be used with hot or boiling water is an aqueous solution of formaldehyde gas (1). For- (15). It should be stored in tightly closed containers malin with the addition of potassium permanganate to prevent conversion to sodium carbonate by carbon is used to produce formaldehyde for AI virus disin- dioxide in the air. Sodium hydroxide has been used 18 / Methods for Inactivation of Avian Influenza Virus in the Environment 399 extensively for cleaning surfaces, especially when safety (19). Some disinfectants are acutely toxic and there is accumulated grease and tissue debris (25). highly concentrated. These should be stored out of Its cleaning activity is related to the concentration the reach of children in closed containers, away and to the temperature at which it is used, having from feed, feed additives, and medication. The marked microbiocidal properties at high concentra- importance of cleaning prior to disinfection cannot tions. Sodium hydroxide is corrosive for metals, be overemphasized because such cleaning is required especially aluminum, has a caustic action, and is to maximize disinfectant effi cacy. Extreme of pH hazardous for workers. Eye protection, rubber and soap residues may inactivate some disinfectants. gloves, and protective clothing are required when Hot disinfectant solutions may penetrate better than using this caustic alkali. cold disinfectant solutions into cracks of crevices. Some disinfectants may carry warnings about cor- Calcium Oxide and Calcium Hydroxide rosiveness to specifi c surfaces. Proper use of disin- Calcium oxide (lime, quicklime) is the anhydrous fectants can greatly improve sanitation at a reasonable form of calcium hydroxide. Calcium oxide may cost. come as powder or pellets. Calcium hydroxide (slaked lime) has been recommended as an AI virus Safety Aspects of Disinfection disinfectant at a 3% solution for the treatment of Many chemicals used as disinfectants are corrosive, walls and fl oors (23). Calcium hydroxide when used toxic, or irritants, and some may be carcinogenic as a 20% suspension provides an effective white- (25). Prolonged occupational contact with toxic washing surface disinfectant (15). Slaked lime itself chemicals should be avoided. Health and safety has been employed in the form of a powder or as a hazards listed on the disinfectant product label thick suspension with water (milk of lime). Lime is should be carefully noted. used to disinfect animal facilities and as a whitewash (15, 24). These alkalis can cause severe burns and Disinfectant Selection Criteria must be handled with care, using prescribed safety It is essential to only use a disinfectant approved by procedures. Alkalis may be fatal if swallowed. appropriate regulatory offi cials for the purpose intended on the specifi c use sites to which it will be Sodium Carbonate applied. A disinfectant approved for use in one juris- Sodium carbonate anhydrous (soda ash, Solvay diction may not be approved for use in another, or soda) powder has been recommended as an AI virus may be approved with a different label and recom- disinfectant at a recommended working strength of mended uses. Some disinfectants may be restricted 40 g/L, fi nal concentration of 4% w/v, with a 10- for use only by certifi ed applicators. A disinfectant minute contact time. Sodium carbonate decahydrate that remains wet for the minimum label-required (sal soda, washing soda) crystals has been recom- contact time in the environment where it is to be mended as an AI virus disinfectant at a working used may be preferable to a disinfectant that needs strength of 100 g/L, fi nal concentration 10% (w/v), to be reapplied to achieve the minimum wet time for a 30-minute contact time (1). These products requirement. A disinfectant available as a concen- may be used in the presence of high concentrations trate may be preferable to a ready-to-use disinfectant of organic material. Sodium carbonate is also used as the concentrate has the advantages of requiring as a cleansing agent, e.g., a 4% w/v solution for less storage space and being easier to transport. The washing vehicles after unloading animals and prior ready-to-use disinfectant is easier to use for rela- to disinfection (25, 15). These alkalis can cause tively small tasks. The label-indicated application severe burns and must be handled with care, using method, e.g., wiping, spraying, fogging, foaming, prescribed safety procedures. Alkalis may be fatal if etc., may infl uence the disinfectant selection. Some swallowed. disinfectants work better than do others in the presence of organic material and hard water. Some dis- General Guidelines for Disinfectant Use infectants contain an added detergent, which helps Disinfectant product label instructions should always to penetrate organic matter. Some disinfectants have be followed when preparing and using disinfectants a faster kill rate that is advantageous when high to ensure economy, effi cacy, and human and fl ock environmental temperatures produce rapid disinfec- 400 Avian Influenza tant evaporation from the applied surfaces. Some such as shoveling, brushing, scraping, sweeping, disinfectants work better at temperature extremes blowing, wiping, and vacuuming, without the use of than do others. Some disinfectants penetrate better water. Dry cleaning should be performed inside the than do others into rough, irregular, cracked, and poultry house, storage rooms, entry rooms, egg porous surfaces. Some disinfectants are more cor- rooms, egg coolers, hallways, stairways, etc., start- rosive than others on certain surfaces. Some disin- ing at the top and proceeding downward to minimize fectants may discolor or leave an offensive odor on contamination of already cleaned surfaces (13, 19). the surfaces to which these applied. Some disinfec- The goal is to remove all visible contamination from tants may discolor the surface to which these are permanent surfaces (1). Commercial vacuum clean- applied. Some disinfectants may have a residual dis- ers, air blowers, wire brushes, and low speed infection action on the surface to which these are mechanical scrapers may be useful (19). Crusted applied. Some disinfectants are user and environ- areas should be hand scraped and wire brushed until mentally friendly and biodegradable. Some disinfec- they are free of visible contamination (1). Scraping tants store better in temperature extremes than do with hand tools may be used to remove concretions others. The shelf life of disinfectants is variable. The and encrustations of material on permanent surfaces. cost of disinfectants varies widely, but cost may be The choice of methods to remove caked on material minor in comparison to the total costs related to will depend on the size of the house, its drainage, depopulation, downtime, and manpower. Some AI and the equipment available (12). Scraping alone virus disinfectants have a relatively broad range of may not remove dried and caked materials. activity and may have label effi cacy claims for many All equipment of the air handling, feed, egg con- diseases in addition to AI. veyance, and drinking systems should be dismantled to facilitate removal of all dust, dirt, and egg debris CLEANING AND DISINFECTION OF THE (13, 3). Egg belts should be removed for cleaning or POULTRY HOUSES AND EQUIPMENT replaced and all debris swept away from the poultry facility (3). Water System Cleaning All old insulation material, and unsound, rotten Water system cleaning should start as soon as the and worn down wooden fi ttings and fl ooring, and last birds are removed from the building before the other structures with unsound pervious surfaces that water fountains and lines are disconnected to mini- cannot be effectively decontaminated should be mize slime deposits forming in the drying system removed for proper disposal (1). All material that (21). The entire water system, including tanks, pipes, cannot be properly cleaned (e.g., surfaces made of drains, nipple drinkers, cups or other water reser- “rough” wood), as well as deteriorated equipment, voirs, and wells should be drained, fl ushed, and should be removed and replaced with new equip- disinfected. ment (19). All broken parts and all soiled items that cannot be cleaned should be removed (3). Precleaning Every part of the poultry house and associated Precleaning begins after the birds, litter, and manure equipment should be repaired (fl oor cracks fi lled, have been removed from the poultry house (25). door frames repaired, damaged panels replaced) and Regulations may determine when the organic mate- made rodent- and bird-proof (19). Waste materials rial is removed from the poultry house and how it is from repair work should be collected frequently and treated (e.g., burial, burning, composting). The use disposed of in an acceptable manner. of water and disinfectant during the precleaning Extreme care is needed for the cages and the egg stage should be minimized to reduce the volume and elevator (3). It is important to check for cleanliness weight of runoff (1). A light misting of the poultry from every possible angle from underneath the pit house internal environment will reduce the presence and from behind rollers. All traces of egg breakage of dust in the air. and spoilage should be removed. Any area that resists earlier cleaning efforts should be manually Dry Cleaning cleaned. Egg conveyance equipment should be Dry cleaning is the fi rst stage of surface cleaning and opened and egg belts removed. All egg debris, dust, involves a variety of mechanical cleaning activities, and dirt should be swept away (19). 18 / Methods for Inactivation of Avian Influenza Virus in the Environment 401

All remaining feed from the previous fl ock should faces working downward towards the fl oor. Clean- be removed from the feed delivery system as resid- ing with warm water is more effective than cleaning ual feed may attract rodents and insects, which can with cold water (3). act as vectors for pathogens (13). Feed storage bins Soaking of heavily soiled areas in about 1.5 L of should be thoroughly cleaned and feed bin boots washing solution per square meter adequately dis- should be disassembled to remove all residual feed. tributed for about 2 to 3 hours may help to loosen All loose and caked feed should be removed from caked-on dirt (3). The soaking solution may be the feed delivery system. Mechanical feeders could applied using a pressure sprayer using a fl at-jet be dry cleaned using air compressors or shop nozzle from a distance of about 2 m from the surface. vacuums. Feed pans should be removed from the Ventilators should be switched off during this pro- feeders and thoroughly dry cleaned. cedure. Soaking in water with detergent may be The power to all electrical devices should be shut useful in removing dried, caked on materials from off prior to dry or wet cleaning (3). An alternate the surfaces of battery cages, fl oors, walls, and tools power source such as a portable generator or elec- (12). tricity delivered through cables connected to an elec- Washing should be performed in such a way that tricity source from an adjacent building may be used all debris and dirt is removed until surfaces are to power electrical equipment for cleaning and dis- visibly clean (3). Work should start at the back and infecting in the facility (25). All delicate electronic proceed toward the front of the building. The ceiling equipment must be protected (1). Electrical equip- should be sprayed fi rst, then the walls, and fi nally ment installed in the building should be either the fl oor. Everything should be cleaned completely, removed or covered with waterproof material after including walkways, steps, crossover platforms, being cleaned prior to manual disinfection at a later cages, troughs, and drinkers. The washing should stage (25). Immovable motors, fan motors, switches, always be directed toward the drain, paying special switch boxes, outlets, and other electrical equipment attention not only to the top but also to the under- should be dry cleaned by vacuum cleaning, dry neath of troughs and obvious and hidden surfaces of brushing blowing with compressed or wiping with a all chains and augers. A detergent compatible with rag before being covered (3, 27, 19, 14). The slots the disinfectant to be later used can be added to the in motor housing should be wiped with a noncor- water to increase the water’s cleansing action (14). rosive disinfectant-soaked cloth prior to covering The use of a detergent may be especially important with duct tape and again after cleaning is completed in controlling or preventing the spread of a highly and the duct tape is removed (3). contagious disease in that it helps to dissolve biofi lm and grease and reduces the possibility of pathogen Wet Cleaning survival and transmission. Wet cleaning follows the completion of dry cleaning Special attention should be paid to the undersides and includes soaking, washing, and rinsing of all of troughs and to obvious and hidden surfaces (19). surfaces in the house (19, 25). Wet cleaning serves After soaking is completed, hosing with a high pres- to further reduce the number of microorganisms on sure jet by wet scrubbing or by live steam may be surfaces begun with dry cleaning. Detergents and useful in removing caked on material from surfaces alkaline surfactants (compatible with the disinfec- such as cages, fl oors, walls, tools, and related items tant planned for later use) may be added to the (12). Steam cleaning may be used in houses with washing solution to help loosen debris and biofi lms inadequate drainage but that can be well ventilated. and to improve the penetration of cleaning agents Steam is a convenient method of stripping dirt off (19, 27). Simply cleaning surfaces by brushing with surfaces without the production or use of large a detergent solution may be effective in removing amounts of water. contaminating viruses (9). A fi nal rinse of all washed surfaces with cold or The detergent solution may be applied using a warm water at lower pressure will reduce cleaning high pressure sprayer to help dislodge any remaining chemical residues that might interfere with the dis- debris. The high pressure spray may cause aerosol- infectant (3). The building should be allowed to ization of pathogenic organisms from contaminated dry before the application of a disinfectant (25). If surfaces. Washing should start with the upper sur- possible, heating and ventilation should be switched 402 Avian Influenza on to support the evaporation of water (3). Moisture Animal health regulatory authorities may require remaining on rough or porous surfaces like wood or multiple applications of disinfectants following a concrete will reduce the amount of disinfectant that highly contagious disease outbreak to increase con- these surfaces can absorb. fi dence that the facilities may be repopulated.

Disinfection Postdisinfection Inspection Disinfection should begin within a day after wet The aim is to ensure that all tasks related to C&D cleaning is completed. Surfaces should be clean and were performed satisfactorily (1). A second full dis- dry (19). The use of pressure sprays is advisable to infection may be required if the fi rst full C&D was help force disinfectant into wood pores, cracks, and unsatisfactory. A fi nal inspection of the premises, crevices (19). A second application of the disinfec- including visual inspection, surface sampling, and/ tant may be needed after the fi rst disinfectant appli- or sentinel animal placement, is best done by a gov- cation has had a chance to dry. All label use directions ernment authority within a disease eradication and safety precautions must be followed. Increasing program. the concentration of a disinfectant should not be a substitute for thorough cleaning (13). Postdisinfection Actions Label directions should be used to estimate disin- After the required exposure time for the disinfectant, fectant volumes required. The volume of disinfectant the building should be allowed to dry completely. solution required to treat rough or porous surfaces (3) Any residual disinfectant should be removed (e.g., concrete or wood) may be two or three times from drinkers and feed troughs as should the cover- the volume of disinfectant required to treat a pol- ings and tape used to protect electrical equipment. ished, nonporous fl oor (9). It is even more diffi cult The house should remain empty for 4 to 5 days, if to estimate the amount of disinfectant needed for possible. After the ventilation is turned on, as much ceilings and vertical walls. Estimates of 0.4 L/m2 or light and air should be allowed into the house as 1 gallon/100 ft2 of disinfectant solution have been possible during the down time (14). Wild birds recommended for ceiling, fl oor, and wall surfaces, or other animals should not be allowed to enter with an additional 30% for equipment and 100% the house at any time but especially after it has more for layer houses with multiple stages (3, 19). been disinfected. It is necessary to observe the post- Disinfectant should be sprayed on every surface so disinfection downtime indicated by regulatory that the small drops reach the lower parts of the walls authorities. and the concrete fl oor is wet. It is important to apply a suffi cient amount of disinfectant as the mechanical Special Considerations action of excess liquid runoff will facilitate the penetration of disinfectant into uneven surfaces (3). Equipment Disinfection is performed in the same order as wet Small equipment and equipment that can be dis- cleaning by moving from the back to the front of the mantled may be placed in special plastic or stainless house and from top to bottom (19). Disinfection steel baths or containers (containing a disinfectant should be carried out in a systematic fashion to for up to a couple of minutes) (19). In tropical coun- ensure that areas that have been disinfected are not tries, poultry house equipment may be dismantled, recontaminated by people or machinery (1). Each cleaned, and brought outside to be placed in the sun building or area that has been disinfected should be for further disinfection. cordoned off with marking tape because it will be Electronic equipment (waterproofed), egg- diffi cult to know otherwise where the disinfected handling equipment, and other large equipment area begins and ends once the disinfectant dries. should be disinfected in accordance with the recom- Appropriate warning signs should be in place mendations provided by the manufacturers of the during disinfection and nobody should be allowed equipment and the disinfectant (19). Fuse boxes entry during the disinfection processes other than the should be disinfected by hand, using a cloth soaked personnel performing the work (27). The ventilation in disinfectant, but only after electrical power has system should be switched off during disinfection to been switched off. All fuses should be removed reduce the evaporation rate (3). before disinfection. All accessory decontamination 18 / Methods for Inactivation of Avian Influenza Virus in the Environment 403 equipment (e.g., rakes, shovels, scrapers, brushes, into the eyes, nose, and mouth (29). Workers engaged trucks, tractors, manure spreaders and bucket in C&D of contaminated premises must follow loaders) should be cleaned and disinfected after use health and safety regulations, be enrolled in a worker and stored in a secure location. health and safety program, and comply with the site- specifi c Health and Safety Plan (HASP). Feed System All previously cleaned and dried components of the Respiratory Protection feed system should be disinfected. After the applica- Poultry workers should wear respiratory protection tion of disinfectant, the feed pans may be placed because AI virus may be transmitted by breathing outside in the sun to dry and to allow the sun’s UV contaminated dust. Air purifying masks and respira- rays to aid in killing pathogens (13). The pan feeders tors that have fi lters or cartridges are the most prac- should not be returned to the poultry house until the tical and appropriate choices for poultry workers to entire house C&D has been completed. Silos may wear. Poultry workers at risk of prolonged direct or be disinfected using formaldehyde fumigation (19). indirect exposure to any AI virus in an enclosed setting should be in a worker health and safety Temperature program that includes respirator training and fi t During cold weather, buildings should be heated to testing. approximately 20º C (68º F) because many disinfectants are ineffective at low temperatures (25). Eye Protection Eye protection will reduce direct exposure of the Fumigation eyes to contaminated dust and aerosols and help Chemical fumigants are sometimes used to disinfect keep workers from touching their eyes with con- poultry houses (14). However, safety concerns taminated fi ngers. To prevent the mucous mem- related to the potential toxicity and carcinogenicity branes of the eyes from being exposed to the AI of chemical fumigants and the construction charac- virus, poultry workers, including those with pre- teristics of many poultry houses have limited the use scription glasses or contact lenses, should wear of chemical fumigants for disinfection. safety goggles or a respirator that has a full face piece, hood, helmet, or loose-fi tting face piece. If Vehicles entering and leaving safety goggles are worn, they should be nonvented contaminated premises (eyecup goggles, for example) or, at a minimum, Cleaning and disinfection of vehicles, including indirectly vented. transport vehicles, is best performed using special Caution should be used when removing eye pro- equipment in a separate building at temperatures tection to ensure that contaminated equipment does above 10º C (50º F) (3). If this cannot be done, sur- not come in contact with the eyes or other mucous faces should be soaked with disinfectant brought to membranes. a temperature of 40º C (104º F) or the vehicles should be high-pressure washed using water temperatures Additional Protective Clothing as high as possible until surfaces are visually clean. PPE, which includes gloves, aprons, outer garments The underside of fender wells and vehicle frames of or coveralls, and boots or boot covers, should be all vehicles leaving or entering contaminated prem- used to prevent direct skin contact with contami- ises should be pressure-washed to ensure that all nated materials and surfaces, and to reduce the like- dirt, manure, and other materials are completely lihood of transferring contaminated material outside removed prior to applying the approved disinfectant a poultry barn or work site. Disposable PPE is pre- with hand-held sprayers or low-pressure motorized ferred. Because PPE is more insulating than regular sprayers (11). work clothing, precautions should be taken to protect workers from the effects of heat stress. WORKER HEALTH AND SAFETY AI infections in humans are thought to have resulted CONCLUSIONS from contact with infected poultry or contaminated Cleaning and disinfection of limited areas in house surfaces followed by self-inoculation of the virus while poultry are present and C&D of the entire 404 Avian Influenza house after poultry have been removed are essential 2. Block, S.S. 2001. Defi nition of terms. In: S.S. operations to reduce the AI viral load in the house Block (ed.). Disinfection, Sterilization, and Preser- environment and to maintain poultry health. Adher- vation, 5th ed. Lippincott, Williams and Wilkins: ence to strict biosecurity procedures limits the entry Philadelphia, PA, pp. 19–28. onto and departure from the poultry house of AI 3. Bohm, R. 1998. Disinfection and hygiene in the veterinary field and disinfection of animal virus as a contaminate on people, animals, and houses and transport vehicles. International objects. A C&D plan is important for all farms as Biodeterioration and Biodegradation 41:217– well as having C&D equipment and products avail- 224. able on site for emergency use. In a C&D strategy, 4. Bruins, G., and A.J. Dyer. 1995. Environmental fi rst thoroughly remove all organic contaminants con siderations of disinfectants used in agriculture. from a surface before applying a disinfectant because OIE Review of Science and Technology 14(1):81– organic material will reduce disinfectant effi cacy. 94. Therefore, cleaning often begins with a dry stage 5. Cardona, C. 2007. Recommendations to prevent that may include operations such as dusting, sweep- the spread and/or introduction of avian infl uenza ing, raking, shoveling, etc., and is followed by a wet virus. Available at www.vetmed.ucdavis.edu/ stage that may include operations such as soaking vetext/INF-PO_AI-Recommendations.pdf. Uni- versity of California Davis, Veterinary Medicine. and high pressure spraying with a warm detergent Accessed April 10, 2007. solution. A thorough rinse with clean water is impor- 6. Chumpolbanchorn, K., N. Suemanotham, N. Sirip- tant to remove residual detergent chemicals that ara, B. Puyati, and K. Chaichoune. 2006. The might diminish the activity of certain disinfectants. effect of temperature and UV light on infectivity The rinse water should be allowed to dry to prevent of avian infl uenza virus. Southeast Asian Journal further dilution of the disinfectant’s active ingredi- of Tropical Medicine and Public Health 37(1):102– ents. Second, disinfectants should only be applied in 105. accordance with label instructions using appropriate 7. Environmental Protection Agency. 2006. Regis- personal protection equipment. Federal, State, and tered antimicrobial products with label claims for local authorities as well as industry groups may avian (bird) fl u disinfectants. Available at http:// defi ne procedures for C&D. www.epa.gov/pesticides/factsheets/avian_flu_ products.htm. Accessed April 12, 2007. A wide variety of commercially available disin- 8. Environmental Protection Agency. Undated. Pes- fectants are available that can inactivate AI virus. ticide product label system (PPLS)—search. Avail- These vary considerably by active ingredients, inert able at http://oaspub.epa.gov/pestlabl/ppls.home. ingredients, application methods, etc., all of which Accessed April 12, 2007. are indicated on the product label. A disinfectant 9. European Food Safety Authority. 2005. Animal should be selected that is approved for use on the health and welfare scientifi c report. Animal health site and surfaces to which it will be applied. Disin- and welfare aspects of avian infl uenza. Annex to fectant labels should be carefully reviewed for infor- the EFSA Journal. Available at http://www.efsa. mation regarding worker health and safety and europa.eu/en/science/ahaw/ahaw_opinions/1145. environmental hazards. Disinfectants may be cor- html. Accessed April 17, 2007. rosive and their use should follow the original equip- 10. Food and Agriculture Organization. Undated. Animal production and health division. Avian ment manufacturer’s recommendations. Care should infl uenza fact sheet. Disinfection. Available at be taken to dispose of any unused disinfectant in http://www.fao.org/docs/eims/upload//224887/ accordance with label instructions. factsheet_disinfection_en.pdf. Accessed April 17, 2007. REFERENCES 11. Ford, W.B. 1995. Disinfection procedures for per- 1. Australian Veterinary Emergency Plan. 2000. sonnel and vehicles entering and leaving contami- AusVetPlan. Decontamination Operational Pro- nated premises. Disinfectants: Actions and cedures Manual. Available at http://www. Applications OIE Review of Science and Technol- animalhealthaustralia.com.au/fms/Animal% ogy 14(2):393–398. 20Health%20Australia/AUSVETPLAN/decfnl2. 12. Great Britain Ministry of Agriculture, Fisheries pdf Animal Health Australia, Canberra, Australia. and Food. 1982. Disinfection and Disinfestation of Accessed April 9, 2007. Poultry Houses. Leafl et 514, pp. 1–8. 18 / Methods for Inactivation of Avian Influenza Virus in the Environment 405

13. Kuney, D.R., and J.S. Jeffrey. 2002. Cleaning and A_Fiches_IA.pdf. Offi ce Internationale des Epizo- disinfecting poultry facilities. In: D.D Bell and oties: Paris. Accessed April 17, 2007. W.D. Weaver. Commercial Chicken Meat and Egg 25. Quinn, P.J., and B.K. Markey. 2001. Disinfection Production, 5th ed. Kluwer Academic Publishers: and disease prevention in veterinary medicine. In Norwell, MA, pp. 557–564. S.S. Block (ed.). Disinfection, Sterilization, and 14. Lacy, M.P. 1989. Effective broiler house clean out Preservation, 5th ed. Lippincott, Williams and and disinfection techniques. Circ Cooperative Wilkins: Philadelphia, PA, pp. 1069–1103. Extension Service of the University of Georgia, 26. Russel, A.D., W.B. Hugo, and G.A.J. Ayliffe. No. 813, p. 12. 1999. Viricidal activity of biocides. In: Principles 15. Linton, A.H., et al. (eds.). 1987. Disinfection in and Practice of Disinfection, Preservation, and Veterinary and Farm Animal Practice. Blackwell Sterilization, 3rd ed. Blackwell Science: Oxford, Scientifi c Publications: Oxford, England. England, pp. 207–222. 16. Lu, H., A.E. Castro, K. Pennick, J. Liu, Q. Yang, 27. U.S. Poultry and Egg Association. Undated. Infec- P. Dunn, D. Weinstock, and D. Henzler. 2003. tious Disease Risk Management. Practical Biose- Survival of avian infl uenza virus H7N2 in SPF curity Resources for Commercial Poultry Producers chickens and their environments. Avian Diseases [CD]. U.S. Poultry and Egg Association: Tucker, 47:1015–1021. GA. 17. Martin, V., A. Forman, and J. Lubroth. 2006. Pre- 28. U.S. Department of Agriculture. 2007. Biose- paring for highly pathogenic avian infl uenza. A curity Factsheet: Protecting Your Livestock and manual for countries at risk. Available at http:// Poultry. Available at http://www.aphis.usda.gov/ www.fao.org.ec/archivos/informes/ag061e00.pdf. publications/animal_health/content/printable_ FAO and OIE: Rome and Paris. Accessed April 17, version/fs_bio_sec_07.pdf. U.S. Department of 2007. Agriculture–Animal and Plant Health Inspection 18. Mauldin, J.M. 1984. Sanitation, disinfection agents: Service (USDA/APHIS)/VS: Riverdale, MD. how they work. Broiler Industry 47(6):78–82. Accessed April 10, 2007. 19. Meroz, M., and Y. Samberg. 1995. Disinfecting 29. U.S. Department of Labor. 2004. Avian Infl uenza poultry production premises. OIE Review of Protecting Poultry Workers at Risk. Available at Science and Technology 14(2):273–291. http://www.osha.gov/dts/shib/shib121304.html. 20. Muhmmad, K, P. Das, T. Yaqoob, A. Riaz, and R. Accessed April 15, 2007. Manzoor. 2001. Effect of physico-chemical factors 30. U.S. Federal Register. 2006. Low pathogenic avian on survival of avian infl uenza virus (H7N3 Type). infl uenza; Voluntary control program and payment International Journal of Agriculture and Biology of indemnity; Final rule. 9 CFR Parts 53, 56, 3(4):416–418. 145, 146 and 147. Available at http://www. 21. National Turkey Federation. 2004. Animal Care regulations.gov/fdmspublic/component/main?main= Best Management Practices for the Production DocketDetail&d=APHIS-2005-0109. U.S. Depart- of Turkeys. National Turkey Federation: ment of Agriculture–Animal and Plant Health Washington, D.C. Inspection Service (USDA/APHIS): Riverdale, 22. Neighbor, N.K., L.A. Newberry, G.R. Bayyard, MD. Accessed April 19, 2007. J.K. Skeeles, J.N. Beasley, and R.W. McNew. 31. Widmer, A.F., and R. Frei. 2003. Decontamina- 1994. The effect of microaerosolized hydrogen tion, disinfection, and sterilization. In: P.R. Murray, peroxide on bacterial and viral poultry pathogens. E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R. Poultry Science 73(10):1511–1516. Yolken (eds.). Manual of Clinical Microbiology, 23. Offi ce Internationale des Epizooties. Undated. 8th ed. ASM Press: Washington, D.C., pp. 77– Contingency Manual for Avian Infl uenza. Avail- 108. able at http://www.oie.int/eng/AVIAN_INFLU- 32. World Health Organization. 2006. Collecting, Pre- ENZA/Contingency%20Manual.pdf.Office serving and Shipping Specimens for the Diagnosis Internationale des Epizooties: Paris. Accessed of Avian Infl uenza A (H6N1) Virus Infection. April 09, 2007. Guide for Field Operations. Annex 7. Disinfection. 24. Offi ce Internationale des Epizooties. Undated. Available at www.who.int/entity/csr/resources/ Avian Infl uenza. Disinfecting Methods. Available publications/surveillance/Annex7.pdf. World Health at http://www.oie.int/eng/AVIAN_INFLUENZA/ Organization: Geneva. Accessed April 10, 2007. 19 Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry

David E. Swayne and Darrell R. Kapczynski

INTRODUCTION to produce vaccines were unsuccessful or the vac- Avian infl uenza (AI) vaccines and their fi eld appli- cines were inconsistent in producing immunity (178, cation can be an effective tool within a comprehen- 252). The fi rst vaccines were derived by drying sive control program which should include the spinal cord from fowl plague cases, an attempt to following additional components: (1) biosecurity reproduce the successful rabies vaccine research of (bioexclusion and biocontainment, including quar- Pasteur, or they were produced by using heat, light antine, limiting human access to affected premises, and various chemicals (e.g., formalin, phenol glyc- cleaning and disinfection, and movement controls erine, tricresol glycerine, etc.) to inactivate the virus for poultry) (2) education on how to prevent AI (3) in blood or liver of chickens that died from fowl diagnostics and surveillance to detect the disease plague. Vaccine failures usually resulted from and infection, and (4) elimination of AI virus infected incomplete inactivation of the HPAI virus contained poultry through humane euthanasia and environ- in the injected vaccine that produced fowl plague, mentally sound disposal of carcasses or controlled or the vaccine did not provide adequate protection marketing (231, 234). Usage of these components in because of insuffi cient quantity of inactivated virus various combinations within a control strategy can in the vaccine such that vaccinated birds succumbed prevent, manage, or eradicate AI. However, the use following challenge. However, when a successful of AI vaccine alone can severely limit the effective- vaccine was produced, it could maintain effi cacy for ness of any control strategy. Use of AI vaccine can at least 120 days if stored at −3º C. Early immuniza- manage the disease, but addition of the other four tion and challenge studies indicated the European components of a comprehensive control program is fowl plague viruses were all cross-protective; i.e., needed to prevent or eradicate the disease and the homosubtypic H7 HPAI virus protection. However, infection. because of the success of stamping-out programs and inconsistency of vaccines, vaccines were not HISTORY OF AVIAN used in HPAI control programs until the mid- INFLUENZA VACCINES 1990s. In the early part of the 1900s, some chickens infected The development of vaccines to control low with fowl plague virus (i.e., H7 high pathogenicity pathogenicity avian infl uenza (LPAI) arose after the avian infl uenza [HPAI] virus) were observed to mid-1960s and was based on economic need. As one recover from the disease and were refractory to fowl of the fi rst observations on the potential for immu- plague upon reexposure (reviewed in Refs. 22 and nity to control LPAI, some fl ocks of turkey pullet 252). Their blood contained virus-neutralizing sub- breeders raised on range in California would develop stance, i.e., neutralizing antibodies. Initial attempts LPAI infections and mild clinical disease, but these

Avian Influenza Edited by David E. Swayne 407 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 408 Avian Influenza recovered birds were protected from LPAI-induced (humoral immunity) and thymus-derived T (cellular egg production drops after being moved to the immunity) lymphocytes that are generated by breeder production houses (R. McCapes, personal random processes in gene rearrangement. The adap- communication, May 23, 2007). The occurrence of tive immune responses to vaccines results in virus- severe losses in Minnesota breeder turkeys during neutralizing antibodies and AI virus-specifi c the fall of 1978 from LPAI resulted in special U.S. cytotoxic lymphocytes, which are responsible for Department of Agriculture (USDA) approval of the virus recognition and clearance in the host. These fi rst commercial inactivated AI vaccines through a two immunological systems work in concert follow- new special conditional license. These fi rst vaccines ing vaccination to establish protection of a host were produced in late 1978 and initially used in against disease. 1979 to control LPAI in Minnesota and California turkeys (146, 177). Some AI vaccine was used in Overview of Antigen Recognition Minnesota meat turkeys, but in California, the AI and Processing vaccines were only used in turkey breeders, each The avian immune system appears to be similar in bird receiving two vaccine doses given 4 to 6 weeks many ways to the mammalian immune system (27, apart. In 1980, a polyvalent H5N2, H6N2, and 112, 183, 205). Traditionally, the adaptive immune H10N2 inactivated AI vaccine along with Newcastle response to a pathogen has been divided into humoral disease virus (NDV) was reported to have been used immunity and cell-mediated immunity. Humoral in Italy to control multiple subtypes of LPAI virus immunity is mediated by antibodies found in the (279). blood that are secreted by bursal-derived (B) lymphocytes. Antibodies specifi cally target and neutral- IMMUNOLOGICAL BASIS FOR VACCINES ize the infectivity, as well as label infectious agents AND VACCINATION OF POULTRY for other effector immune cells. B lymphocytes recognize and process antigen (protein, carbohydrate, Introduction lipid, and nucleic acids) and interact with CD4+ T- This section provides an overview of the basic helper cells through the major histocompatibility immunology involved in the host response to AI complex (MHC) class II to promote cell differentia- vaccines. Various factors including species and age tion and proliferation. Other professional antigen- of bird, type of vaccine (inactivated versus live), presenting cells (APC), including dendritic cells and dose, and vaccination route will affect presentation macrophages, also process exogenous antigen for and processing of antigen by host immune cells. presentation to T-helper cells via MHC class II mol- While many different cell types are involved with ecules. Cell-mediated immunity (CMI) is specifi c establishing an immune response, vaccination may immunity mediated by T lymphocytes (T cells) and or may not produce protective immunity. Factors has been suggested to be an important factor in the that can contribute to immunity failure are numerous development of protection in chickens vaccinated and include a lack of vaccine antigenic similarity to against viral diseases (204, 205). This type of the fi eld strain, overwhelming dose of challenge, or response functions to destroy intracellular patho- insuffi cient vaccine antigen content to induce a pro- gens, including viruses, found in infected cells. The tective immunological response. subsets of T cells (CD4+ helper cells and CD8+ cyto- Following vaccine administration, the host toxic cells) constitute the principal cells of the CMI immune system directs uptake and processing of the response. For stimulation of CD8+ lymphocytes, antigen. The immune system of vertebrates is made MHC class I molecules expressed on infected-cell up of two functional elements, the innate and adap- surfaces present peptide antigen that results in target- tive, which contrast by their time of response and cell lysis. mechanisms of pathogen recognition. The early Immunity to different infectious diseases requires reactions of the innate immune system use germ-line distinct types of immune reactions, which have to encoded receptors, which recognize evolutionarily be evoked by differently designed vaccines. Criteria conserved molecular markers of infectious microbes. for effi cacious AI vaccines include an early onset The latter adaptive immune responses use highly and long duration of immunity. Because infected specifi c antigen receptors on bursal-derived B birds can succumb to HPAI before mobilization of 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 409

CMI responses, neutralizing antibodies form the key centers are incompletely capsulated, whereas those immunological parameter relevant for AI vaccine– closer to the surface are fully capsulated. Peyer’s induced immunity. Most antibody responses aimed patches are primarily located along the distal ileum to evoke specifi c immunoglobulin G (IgG) of high and consist of germinal centers and diffuse lym- affi nity are dependent on assistance from CD4+ T- phoid tissue (38). Intestinal antigens are absorbed helper cells, which receive their activation signals and processed by germinal center macrophages and from APCs. epithelial cells. Meckel’s diverticulum contains epithelial secretary cells in the germinal center and Physiology of Avian Immune System produce large numbers of plasma cells (167). In terms of the anatomy and physiology, the avian Several accumulations of lymphoid tissue have and mammalian immune systems have similarities been described in the paranasal area. The most and differences in structure and distribution of lym- important of these is the Harderian gland, which is phoid tissue. One of the major differences is the considered a secondary lymphoid organ (11). In lymphatic system, which facilitates migration of chickens, the Harderian gland is the major site of lymphocytes to and from sites of vaccination and antiviral IgA–antibody–forming cells, whereas free challenge. The mammalian immune system contains IgA is found in bile and mucosal washes and can lymph nodes, highly organized sites for interactions exist in both monomeric and multimeric forms between B cells, T cells, macrophages, and other (104). The Harderian gland is located medial to the APCs important for activation of adaptive immu- eye in the orbit and is heavily populated by plasma nity. However, lymph nodes are absent in most cells (276). The Harderian gland is believed to be avian species, including chickens (170, 275). Instead critical for local immune responses of the eye, nasal of lymph nodes, avian species have concentrations turbinate and upper respiratory tract areas because it of lymphoid tissue throughout the body that are discharges into the conjunctival sac, which is drained unencapsulated and contain small lymphocytes by the lacrimal duct to the nasal cavity. The produc- (170). Like lymph nodes, these small lymphocyte tion of germinal centers in the Harderian gland can aggregates form germinal centers in response to be observed as early as 3 weeks of age in chickens antigen (253). Avian species do possess lymphatic (3). Vaccinations of poultry via eyedrop method are vessels, which are believed to be involved in traf- believed to stimulate mucosal immune responses fi cking of mesenchymal stem cells to central lym- through the Harderian gland. phoid organs (170). In chickens, lymphoid Despite the physiological differences in structure accumulations have been observed along the poste- and organization between mammalian and avian rior tibial, popliteal, and lower femoral veins (167). species, the functional aspects of lymphoid cells and In contrast to chickens, ducks do contain lymph peripheral organs are similar. This includes lym- nodes that are formed as swellings along the lymphoid cell functions, division, classes, interactions, phatic ducts, and contain both efferent and afferent specifi city, and net effect, which are highly similar lymphatic vessels (275). between mammalian and avian species. A number of immunologically relevant tissues used for antigen processing have been identifi ed in The Immunological Response to Antigen chickens. Intestinal avian lymphoid tissue includes The major function of an immune response is to the cloacal bursa, cecal tonsils, Meckel’s diverticu- recognize and eliminate the infectious agent. The lum, Peyer’s patches, and diffuse mucosal lymphoid immune system of vertebrates include the innate and infi ltrates. The bursa is the main organ responsible adaptive, which contrast by their time of response for B-cell production and differentiation and is and pathogen recognition (3, 152). The innate and found dorsal to the distal end of the cloaca. The adaptive immune responses are mechanisms of an cecal tonsil is the most concentrated lymphoid tissue integrated system of host defense in which numer- in the intestine and is observed as two oval areas on ous cells and molecules function cooperatively. The the facing walls of the ceca at the junction between early reactions of the innate immune system use the ileum, the rectum, and the two ceca. There are germ-line encoded receptors, known as pattern rec- two types of germinal centers found in the cecal ognition receptors (PRRs), which recognize evolu- tonsil (166). Deep in the mucosa, the germinal tionarily conserved molecular markers of infectious 410 Avian Influenza microbes, known as PAMPs (pathogen-associated review, see Ref, 28). Homologues of the TLRs pre- molecular patterns) (83, 151, 152). The later adap- sented above have been described in avian species, tive immune responses use highly specifi c antigen with the notable exception of TLR9, although birds receptors on T- (cellular immunity) and B-lympho- have been shown to respond to CpG motifs (100, cytes (humoral immunity) that are generated by 121, 172). random processes via gene rearrangement (83, 108). Recently, the preliminary phylogenetic character- The innate immune response stimulates the adaptive ization of 414 base pairs of TLR7 from different immune response and infl uences the nature of the avian species was determined and compared with response. The response outcome can be determined sequences to TLR7 from mammalian sources (113, based on the type of cytokine response generated. A 140, 172). Preliminary results indicate human TLR7 + CD4 T-helper type 1 (Th1) (cellular pathway) has approximately 83% similarity to rat or mouse response profi le includes γ interferon (IFN), inter- TLR7. By comparison, chicken TLR7 displays 80% leukin (IL)-2, IL-12, IL-15, and IL-18, and is associ- and 83% similarity to TLR7 from turkey and duck, ated with vigorous CD8+ T-cell–specifi c responses, respectively. Between avian and mammalian species, + while a CD4 T-helper type 2 (Th2) (humoral only 56% similarity was determined between chicken pathway) response profi le includes IL-4, IL-5, and and mammalian species. While mammalian TLRs 7 IL-10 to stimulate antibody production. and 8 have been reported to respond to AI virus The innate immune response depends on factors infection, gallinaceous bird species appear to contain that exist prior to the advent of infection and are an insertion element at the TLR8 genomic locus, capable of a rapid response to microbes. The primary which results in a lack of TLR8 expression (172). components of innate immunity are (1) physical and The relevance of genetic differences in TLR7 and chemical barriers, such as skin and epithelia, and the lack of TLR8, in terms of receptor binding production of mucus; (2) phagocytic cells, including and downstream cytokine signaling, remain to be granulocytes (heterophils, basophils and eosino- determined, but may play a role in innate im- phils), macrophages and natural killer (NK) cells; munity against AI virus and disease resistance or (3) complement proteins and mediators of infl am- susceptibility. mation; and (4) cytokines. In terms of viral infec- The adaptive immune response is an inducible tions, our understanding of how the innate immune response found only in vertebrates. The adaptive system responds and initiates an appropriate response host defense, mediated by T- (thymus-derived) and has increased with the discovery and characteriza- B- (cloacal bursa–derived) lymphocytes are adjust- tion of the toll-like receptor (TLR) family (247). able to antigenic response because of the somatic TLRs are well recognized as PRRs that detect rearrangement of T-cell receptor genes and immu- PAMPs. Engagement of TLRs by PAMPs on cells, noglobulin, respectively. This results in the creation such as macrophages and heterophils, drives innate of individual lymphocyte clones that express distinct immune effector function, such as production of antigen receptors. The receptors on lymphocytes proinfl ammatory cytokines, while stimulation of are generated by somatic mechanisms during the TLRs on dendritic cells induces T-cell activation ontogeny of each individual and thus generate a cytokines (e.g., IL-12) (169, 184). Although knowl- diverse repertoire of antigen receptors with random edge of avian TLRs is primarily derived from mam- specifi cities on the lymphocytes. For infl uenza, malian systems, it is apparent that that the immune CD8+ CTL play a crucial role in controlling infec- system of avian species is close enough to use mam- tious virus from the lungs of mice. Previous studies malian immunology as a model for avian immunol- have provided evidence that CD8+ CTL directed ogy. Several TLRs have been identifi ed that against viral epitopes conserved among infl uenza A recognize viral PAMPs and include, but are not viruses, such as those within the hemagglutinin limited to, TLR2 (cytomegalovirus proteins), TLR3 (HA) and nucleoprotein (NP), contribute to protec- (double-stranded RNA, reovirus), TLR4 (RSV tion against infl uenza viruses (7, 8). Infl uenza virus fusion-protein), TLR7 (single-stranded RNA, infl u- NP–specifi c CTL generated through vaccination enza), TLR8 (single-stranded RNA, HIV), and or introduced by adoptive transfer lead to a more TLR9 (DNA, herpes simplex virus, CpG motifs) (for rapid viral clearance and recovery of the host and 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 411 protection from death (2, 9). The MHC plays a provide a more rapid response to secondary expo- central role in the presentation of antigens by APCs sure to the antigen. to CTLs and optimal activation of CTLs depends on identical MHC class I antigens. There are limited Mucosal Immunity data on CTL responses to infl uenza viruses in Mucosal surfaces of the respiratory and gastrointes- poultry, which requires the availability of inbred tinal tracts serve as portals of entry for many infec- chickens. In 2001, Seo and Webster demonstrated tious agents that affect poultry (156, 280). Mucosal splenic-derived T-lymphocytes or CD8+ cells pro- immunity is the fi rst line of defense for the host and duced in H9N2 AI virus–infected chickens could extensive efforts are currently underway in rodent provide some protection against lethal H5N1 HPAI animal models and humans to design vaccines able virus challenge when adaptively transferred into to confer protection at the mucosal site (210). In naïve MHC-matched birds (204). How the CTL mammals, both mucosal sites can respond individu- response contributes to fi eld protection of vacci- ally to antigenic stimulation and the induction of an nated poultry is unknown. immune response in one region results in subsequent In terms of AI virus infection and host immune immunity at other mucosal sites, a phenomenon interactions, attachment of the virus occurs primar- known as “common mucosal immune system” (147, ily through interactions of the HA protein with host 150). A similar immune mechanism has been sialic acid residues found on cells lining the mucosal described in chickens (156). Immune lymphocytes surface, and results in internalization and infection can migrate from one mucosal site to repopulate and of the virus within the host cell. Endosomal enclo- provide protective immunity at distant mucosal sites sure and maturation around the virus result in a drop (103). Because respiratory pathogens, like Al virus in pH that is necessary for uncoating of the virus. invade at mucosal surfaces, vaccines that can induce During this stage, the host innate immune response strong mucosal immunity would be superior to other is stimulated through interactions of the endosomally types. Current parenterally administered inactivated located TLR with the single stranded viral RNA Al vaccines are not optimal inducers of mucosal genome. Activation of TLR7 with the RNA agonist immunity and therefore some mucosal replication results in a cascade of cytokine and interferon pro- may occur before the systemic immunity stops the duction designed to limit or suppress viral replica- infection (64). Mucosal immunization has the major tion and recruit effector cells for stimulation of advantage of inducing both mucosal and systemic adaptive immunity. Interestingly, activation of TLRs immunity (210). Al viruses that infect poultry invade in endosomal compartments also appears to require two primary mucosal regions: the respiratory tract endosomal maturation with subsequent pH change, and the gastrointestinal tract. Early work in mucosal as pretreatment of cells with chloroquine results in immunology indicated that a vigorous T-cell decreased or no detection of cytokine induction fol- response could be observed in the respiratory tract lowing AI virus infection (76, 137). As the virus after immunization of the respiratory tract, indicat- replicates in the cytoplasm, viral proteins are pro- ing that a local response was capable of being gener- cessed and expressed by MHC class I molecules for ated at the site of immunization, a critical element presentation to CD8+ lymphocytes, resulting in for mucosal immunization (61, 268). The impor- development of cytotoxic T lymphocytes capable of tance of the specifi city for local immune protection lysing virus infected cells. At the same time, newly is underscored by recent observations that exposure released virions are taken up by professional APCs of the lung to aerosol formulations for protection including macrophages, dendritic cells, and B cells. against infl uenza was more effective than either The processed viral peptides are expressed through intranasal or parenteral vaccination (208). MHC class II molecules that are recognized by The concept of mucosal priming at one site pro- CD4+ T-helper lymphocytes. These activated CD4+ viding sensitized cells to other mucosal sites has the lymphocytes present viral antigen to B cells result- potential for an oral administered vaccine to provide ing in antibody production. Following recovery protection against a respiratory challenge (62). from infection, the antigen-specifi c clones remain as Adaptive transfer of infl uenza specifi c T-cell clones memory lymphocytes (both T and B cells) that can migrate to mucosal sites (25). More recently, 412 Avian Influenza immune lymphocytes were demonstrated to migrate immune response occurs (31, 270). At this time, from one mucosal site to repopulate and provide there is no universal vaccine which will protect protective immunity at distant mucosal sites (103). against all AI viruses, but vaccines must be tailored This “mucosal traffi cking” has been demonstrated against the specifi c HA and/or NA subtypes, and in with B and T cells, and as a result, the mechanisms some cases against specifi c lineages of virus within of immune protection at mucosal sites may involve an HA subtype. In practice, protection is provided both humoral and cell-mediated components (148). against the individual HA subtype(s) included within Taken together, these observations are consistent each vaccine. with the known circulation pathway of lymphoblasts The principal contribution to protective immunity from lymph nodes through the thoracic lymph to against AI viruses is an antibody response because distant mucosal surfaces. removal of the bird’s ability to mount a humoral The primary antibody to mediate protection at response by chemical cloacal bursectomy prevented mucosal surfaces in birds is immunoglobulin A protection against HPAI virus challenge (56). Neu- (IgA), although IgG and IgM can also be found tralizing IgG antibodies produced against the HA (195). Resistance to infl uenza infection in rodent can block viral attachment to host cells, are neutral- models and humans correlates with the induction of izing, and can persist for extended periods of time. IgA antibody in the respiratory tract (124). In birds, Such neutralizing anti-HA antibodies can be mea- IgA is found in bile, crop, and mucosal washes and sured using a neutralization or plaque-reduction can exist in both monomeric and multimeric forms assay or a more standard, easier-to-perform hemag- (132, 175, 203). Similar to its mammalian counter- glutinin-inhibition (HI) test using serum from vac- part, avian IgA possesses J chain and secretory com- cinated or infected birds. In addition, the immune ponent (155). Although IgA does not fi x complement, response against AI viruses and vaccines is gener- the immunoglobulin does possess a number of effec- ally the strongest against the specifi c strain or closely tor functions including viral neutralization, inhibi- related strains. However, cross-reactivity and neu- tion of bacterial adherence and acting as an opsonin tralization of other strains within the same subtype for mucosal phagocytes (117, 148). Mucosal IgA of AI virus do occur and are dependent on the degree antibody has also been shown in some cases to of antigenic relatedness between the vaccine strain possess more cross-reactivity than serum IgG anti- and the fi eld strain (i.e., homosubtypic cross- body and thus may contribute to cross-protection reactive immunity). observed in mucosal-vaccinated animals (185, 186). CRITERIA FOR VACCINE PROTECTION The goals of AI vaccination are the production of an Immunological Basis for Protection Against immune response that is protective against the Avian Infl uenza Viruses disease and prevention of infection. Assessment of Vaccine-induced protection of poultry against AI is protection conferred by the vaccine is important to the result of immune response primarily against the national regulatory authorities for licensing vaccines HA, of which there are 16 different HA subtypes, that are effi cacious and potent and for assessment of and the neuraminidase protein (NA), of which there vaccines for practical use in the fi eld. Protection can are 9 different NA subtypes (240, 241). However, be directly measured in in vivo laboratory studies the NA-induced response is less protective. For using a variety of avian models and measurable cri- example, in four live LPAI vaccine studies, the NA teria. Laboratory models can be useful for directly provided mostly partial and, occasionally, complete measuring protection in the target or surrogate protection in chickens from mortality following species when variables such as virus strain and HPAI virus challenge (4, 5, 149, 191), but immuni- antigen content are standardized. In addition, a zation of chickens with NA protein alone in an inac- variety of indirect measures can be used to assess tivated vaccine only provided partial protection after protection when compared to the in vivo protection two or three vaccinations (246). Immune responses data. These measures can include assays for immu- to the internal proteins, such as nucleoprotein or nological response such as antibody titers or assays matrix protein, have been insuffi cient to provide sig- to quantify the amount of the immunologically pro- nifi cant protection in poultry although a measurable tective protein in the vaccine. In this section, the 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 413 term effi cacy will be used to indicate the vaccine is contributor to the maintenance and spread of the protective in defi ned, standardized experimental H5N1 HPAI virus and to economic losses from studies, while the term potency indicates the vaccine H5N1 HPAI. Thus, evaluation of protection in addi- has passed quality control tests that ensure adequate tional host species may be needed such as ducks, antigenic mass of the protective immunogenic geese, Japanese quail, pheasants, partridges, various protein to produce a consistent immunological ratites, and other species of birds. All in vivo studies response that should be protective under a variety of should include a group of birds vaccinated with a fi eld conditions. placebo control (sham) to ensure proper challenge was accomplished. Studies should be properly Direct Assessment of Protection designed and evaluated with statistical methods to The “gold standard” for assessing protective immu- establish unbiased treatment effects for the vaccine. nity of AI vaccines is with the use of LPAI or HPAI Simple numeric differences in small numbers of virus challenge models. Historically, most AI birds that are not statistically tested should not be vaccine studies and subsequent fi eld use of vaccine interpreted as signifi cant. has focused on chickens and, to a lesser degree, Criteria used to assess protection can vary depend- turkeys, because they have been the major poultry ing on whether the challenge is an LPAI or HPAI species raised in developed countries and impacted virus. For HPAI virus challenge, prevention of respi- by both LPAI and HPAI. In addition, these species ratory and general clinical signs (morbidity) and have experienced the highest death rates from HPAI death (mortality) has been the most frequent used virus exposure and, when infected, excreted high criteria to assess protection (Table 19.1) (32, 214, concentrations of virus in the environment, resulting 215, 277, 278), but because most experimental LPAI in effi cient transmission between individuals and virus challenges typically do not produce clinical premises. With the changing epidemiology of the signs or death, such criteria cannot be used in assess- H5N1 HPAI virus in Asia, domestic ducks and ing LPAI vaccine protection. However, both LPAI geese have emerged to become a very important and HPAI viruses can affect the reproductive health

Table 19.1. AI vaccine protection as measured by prevention of clinical signs (Morbidity) and death (Mortality) of vaccinated chickens following challenge with different doses of HPAI virus (mean embryo infectious [EID50] and mean chicken lethal doses [CLD50]). Challenge Dose

Vaccine EID50(log10) CLD50 Morbidity Mortality rFP-AIV-H5 0.5 0.003 0/10 0/10 2.0 0.1 0/10 0/10 3.5 3.2 0/10 0/10 5.0 100 0/10 0/10 6.5 3,200 0/10 0/10 8.0 100,000 2/10 2/10 (4.5) Sham 0.5 0.003 0/10 0/10 2.0 0.1 0/10 0/10 3.5 3.2 8/10 8/10 (2.75) 5.0 100 10/10 10/10 (2.4) 6.5 3,200 10/10 10/10 (2.0) 8.0 100,000 10/10 10/10 (2.0) Chickens were vaccinated subcutaneously at 1 day of age with recombinant fowl poxvirus containing H5 AI virus gene insert (reFP-AIV-H5) and intranasally challenged at 3 weeks of age with various challenge 0.5−8.0 doses of HPAIV (10 EID50, A/chicken/South Korea/2003 [H5N1]) (35). 414 Avian Influenza

Table 19.2. AI vaccine protection as measured by reduction in number of vaccinated AI Challenge Virus Shedding chickens shedding HPAI virus from orophar- 7 6 ynx and cloaca. 5 Sham Virus isolation, 2 days 4 Mex/94 3 EU/86

postchallenge EID50/ml) 2

Virus Titer (log10 1 Inactivated AI virus Oropharyngeal Cloacal 0 a a vaccine group swab swab Oropharynx Cloacal Swabs

Sham Control 10/10A 10/10A Mexican/94 H5N2 5/10B 3/10B Figure 19.1. Reduction in titer of HPAI virus Vaccine shed from the oropharynx and cloaca of European/86 H5N2 6/10AB 3/10B vaccinated chickens 2 days postchallenge. Vaccine See Table 19.2 for details. Dashed line indicates minimal detection limit for virus a Number positive/total tested. Different uppercase isolation assay. superscript letters indicate signifi cant differences in frequency of positives between different vaccine groups. Chickens were vaccinated subcutaneously at 3 weeks of age with inactivated whole AI virus the difference should be statistically signifi cant vaccines and intranasally challenged at 6 weeks of (238). National veterinary biologic regulatory agen- 6.0 cies may require demonstration that reduction in age with high dose (10 EID50) of A/chicken/ Indonesia/7/2003 (H5N1) HPAI virus (242). shedding is clinically relevant (i.e., reduces both Chickens were swabbed on 2 days postinoculation. shed and spread). In addition, immunized birds have a quantifi able resistance to infection as measured by 2 5 requiring a 10 to 10 EID50 greater challenge dose to produce infection in vaccinated compared to non- of laying chickens and turkeys, and prevention of vaccinated birds (35, 48). For protection, the easiest drops in egg production can be an quantifi able indi- to elicit by vaccination is the prevention of mortality cator of protection (32, 214). In addition to preven- followed by prevention of morbidity, then reduction tion of disease and death, the prevention of infection in replication and shedding from alimentary tract, or the qualitative and/or quantitative reduction in and most diffi cult to produce is the reduction in virus virus replication in respiratory and digestive tracts replication and shedding from respiratory tract. Pre- are essential protective criteria that indirectly assess vention of egg productions drops is the most diffi cult the role of the vaccine to limit fi eld virus spread and to prevent of clinical syndromes. are critical for control (Table 19.2 and Fig. 19.1) The prevention of contact transmission is a more (16, 48, 230, 236, 238). The reduction in challenge direct method for direct laboratory assessment of the virus replication can be quantifi ed using classical protective capacity of the vaccine to limit fi eld virus isolation and titration methods in embryonat- spread (Table 19.3) (238). Prevention of contact ing chicken eggs (ECE) or tissue culture systems transmission has been used as an epidemiological (214, 238), or by assaying for AI virus specifi c evaluation tool to determine if vaccine use could nucleic acids such as with real-time reverse tran- stop HPAI epidemics (260). The prevention of scriptase–polymerase chain reaction (RRT-PCR) contact transmission is a desirable goal, but routine (127), or by demonstration of AI viral proteins such laboratory assessment is typically not standardized. as in an ELISA test (119). Demonstration of a reduc- Most studies simply place nonchallenged birds in tion in replication and shedding titers of virus from cages containing challenged vaccinated birds, but respiratory and intestinal tracts should be at a prevention of contact transmission is impacted by 2 minimum of 10 EID50 (100-fold) less virus in vac- multiple variables including bird density and sanita- cinated compared to nonvaccinated birds (223), or tion and ventilation features. Recently, a quantita- 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 415

Table 19.3. AI vaccine protection as measured by interruption of contact transmission. Virus shedding from contact groups (log10 EID /ml) Mortality in 50 IN challenge group Contact group contact group Oropharynx Cloaca

WI/68 vaccine WI/68 vaccine 0/10 0/10 (≤0.9)aA 1/10 (≤0.9)A Sham vaccine 10/10 2/10 (≤1.5)A 1/10 (≤1.3)A Italy/98 vaccine Italy/98 vaccine 0/10 0/10 (≤0.9)A 0/10 (≤0.9)A Sham vaccine 5/10 1/10 (≤1.0)A 1/10 (≤1.0)A Sham vaccine WI/68 vaccine 0/10 8/10 (≤2.0)B 0/10 (≤0.9)A Sham vaccine 10/10 10/10 (5.7)C 10/10 (5.0)B a Number positive/total tested. Different uppercase superscript letters indicate signiﬁ cant differences in frequency of positives between different vaccine groups. Chickens were vaccinated subcutaneously at 3 weeks of age with inactivated whole H5N9 AI virus vaccines (WI/68 and Italy/98) and intranasally challenged at 6 weeks of age with high challenge dose (106

EID50) of HPAI virus A/chicken/Supranburi/2/2004 (H5N1) isolated in Thailand. Vaccinated (WI/68 and Italy/98) and nonvaccinated chickens (Sham) were put in contact with intranasally challenged chickens 18 hours postchallenge to determine the impact of vaccination in reducing AI virus transmission (34).

tive standardized model was developed for assessing while poor-quality vaccines may only protect the impact of vaccination on reducing transmission against low challenge doses (238). of HPAI virus in caged chickens (259, 260). This 2. Content of the HA in the inactivated vaccine or model demonstrated that two different inactivated titer of the live vaccine—High HA content (inac- H7 AI vaccines completely blocked HPAI virus tivated) or high titer (live) vaccines provide the transmission 2 weeks after vaccination, while at 1 best protection against AI virus replication in the week, the blocking of transmission was only partial. respiratory and digestive tracts, while lower A high percentage of immunized birds within the content vaccines may protect from morbidity and population are critical to stop contact transmission. mortality but not reduce replication and shedding To prevent major outbreaks, 60% of the birds in the from respiratory and digestive tracts (236, 238). population should be immunized but using the upper 3. Adjuvants—the use of oil adjuvants are common limits of the confi dence interval, 90% (experimental in inactivated poultry vaccines, including AI vac- data) and 80% (observational data) of chickens in cines, and such usage produces robust, broad and the population should be vaccinated to prevent trans- long lasting protective immune responses but mission (26). Effective immunity may be achieved may require two or more doses to produce suf- in chickens following a single vaccination, but to fi cient long-term protection (77, 218). prevent AI virus transmission in ducks and geese, 4. HA match—The greater the genetic similarity some studies have suggested more than one vaccina- between the HA of vaccine and fi eld viruses, the tion is needed to produce protective immunity (261). greater is the reduction in challenge virus replica- Another variable in transmission is the type of tion and shedding from the respiratory tract housing. For example, it may be more diffi cult to (240). block transmission in birds housed on litter than in 5. Length of protection—The best vaccines produce cages. protection beginning in 7 to 10 days after vac- Multiple factors will impact the protection of AI cination with the peak protection at 3 to 4 weeks vaccines including the following: and protection may last up to 6 to 12 months, but the length of protection is directly associated 1. Challenge virus dose—High-quality vaccines with the quantity of protective antibodies pro- protection against high virus challenge exposure, duced (i.e., titers) following the immunization, 416 Avian Influenza

but in some species long periods of protection sota/81 (H5N2), A/duck/Potsdam/1402/86 (H5N2), may require multiple vaccinations (238). A/chicken/Mexico/232/1994 (H5N2), and A/duck/ 6. Route of administration—Inactivated AI virus Singapore/F119/1997 provided protection against and recombinant fowl poxviruses (rFPV) with A/Hong Kong/156/1997 and A/chicken/Indone- H5 AI virus gene insert (rFP-AIV-H5) vaccines sia/7/2003 H5N1 HPAI viruses (239, 242). However, require parenteral administration while some live the loss of protection following drift of fi eld viruses vectored vaccines such as recombinant Newcas- has been suggested for the 1994 H5N2 AI vaccine tle disease virus with H5 AI virus gene insert strain used in Mexico against the emerged 1998 (rND-AIV-H5) can be administered by mass Mexican and 2003 Guatemalan LPAI fi eld strain topical routes such as spray or drinking water (125). As a result, vaccines should be constantly administration to achieve protection while other evaluated for protection against emergent drift fi eld vaccines have the potential for in ovo application strains. At a minimum, in vivo protection against (19, 168, 238, 239). current circulating fi eld viruses should be done 7. Species of bird and number of vaccinations— every 2 years. Short-lived meat chickens may be protected for their entire production life following a single Indirect Assessment of Protection vaccination, but some species of meat poultry Direct assessment of effi cacy is time consuming and (e.g., turkeys, ducks and geese) and long-lived expensive but necessary to initially demonstrate the birds (e.g., layers and breeders) may require mul- vaccine strain is protective against the specifi c fi eld tiple vaccinations to achieve protection that will strain. However, indirect assessment can be a viable last through the longer production cycle (15, option in some situations to assess protection, espe- 238). cially when determining the consistency of vaccine 8. Age of vaccination—Optimal immune response batches as a means to ensure a minimal protective with inactivated AI vaccines is achieved in most level. Such indirect assessment of protection can be birds after 2 weeks of age and before puberty; derived by several methods: (1) protective serologi- that is, suboptimal protection may be seen in cal responses such as neutralization or HI titers in birds vaccinated before 2 weeks of age and in vaccinated birds; or (2) quantifi cation of the primary adults during the stress of the laying cycle, but protective protein, the HA, for inactivated AI vaccine such suboptimal vaccination timing may be nec- or the infectious titer of live vaccines (139). Quan- essary as a priming or boost vaccination in a tifi cation of antigen in inactivated vaccines has been multidose vaccine program within some produc- accomplished by the radial immunodiffusion assay tion systems. (277), quantitative RRT-PCR assay (242), infectious 9. Field versus laboratory protection—Field pro- titer prior to inactivation (236), hemagglutinating tection is less than achievable in the laboratory titer (236), and potentially other methods that could because of immunosuppressive viruses, vaccine quantify HA protein. For recombinant vectored or storage and transport problems, incomplete or live virus vaccines, virus titer in ECE or cell cultures missed vaccination of poultry on a farm or within are appropriate indirect measures. a region, and failure to follow manufacturer label including usage of reduced vaccine dose admin- Potency istration (230, 231). Potency measurements provide quantitative assessment of protection that ensures effi cacy under a Historically, poultry AI vaccines have had a variety of fi eld conditions and not just to a minimal longer life of fi eld usage without changing vaccine level of protection. Theoretically, potency testing strains as compared to human infl uenza vaccines. assures suffi cient antigen mass or virus titer to be The rFPV vaccine with a 1983 H5 AI virus gene effi cacious under fi eld usage. Potency for some insert protected chickens against diverse H5 HPAI poultry vaccines including NDV and AI vaccines

North American and Eurasian challenge viruses iso- has been quantiﬁ ed in a mean protective dose (PD50) lated between 1959 to 2004 (35, 37, 240). Similarly, test in speciﬁ c pathogen–free (SPF) chickens under inactivated H5 AI vaccines based on A/turkey/Wis- laboratory conditions with challenge occurring at 21 conson/68 (H5N9), A/turkey/3689-1551/Minne- days after vaccination; i.e., the PD50 is the dose of 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 417 vaccine that provides protection in 50% of the birds enteral (subcutaneous or intramuscular) administra-

(236, 250). In experimental studies, PD50 for AI vac- tion (Table 19.4). Such nonreplicating vaccines are cines have measured prevention of mortality in a preferred because of their safety in conventional and series of reduced vaccine doses, such as 1×, 0.1×, immunocompromised hosts. However, to induce 0.01×, and 0.001×, followed by intranasal challenge protective immunity, inactivated vaccines require with a defi ned dose of HPAI virus (60, 236). Using injection of high antigen quantities, and the inclu- these data, the PD50 becomes a simple mathematical sion of adjuvants greatly enhances immunogenicity calculation. Once the PD50 has been calculated, the (196–199). The route and timing of vaccination will minimum number of PD50/dose for a potent vaccine affect the immunogenicity and effi cacy of the must be decided. For avian paramyxovirus type 1 immune response to the vaccine. Most often, AI infections (i.e., Newcastle disease [ND]), potent vaccine programs for poultry are combined with vaccines have been proposed to contain an average other viral and bacterial vaccines and administered of 50 PD50/dose with a minimum deviation to no less concurrently at specifi c periods in the bird’s life and than 35 PD50 (250). However, potency can also be must be considered when developing a program for determined indirectly by serological response of the AI control. Numerous experimental studies have birds to different vaccine doses or by quantifi cation described the effi cacy of inactivated AI vaccines in of the HA protein in the vaccine. In one study, an avian species (see Table 19.4). HI titer of 139 or greater in immunized chickens was Inactivated AI vaccines have primarily utilized associated with protection from illness and death seed stock of LPAI viruses obtained from fi eld out- and absence of recovery of the challenge virus from breaks, and occasionally HPAI viruses when manu- the respiratory and alimentary tracts (277), but the factured in high biocontainment facilities (231, 232). experiment was not designed to determine minimal With the recent development of infectious clone protective dose. Potency test based on 90% protec- systems, experimental and licensed vaccine strains tion at the recommended dose has been used but is have been developed that incorporated the HA and a much less stringent measure of potency (250). NA of recent fi eld AI viruses and remaining six gene Live NDV vaccine potency in the United States is segments from a high growth infl uenza A vaccine based on virus titer per dose (9 CFR 113.329), which virus (126, 133, 251, 273). The reverse genetic gen- could potentially be used to determine potency of erated seed strains usually have the HA proteolytic live virus vectored AI vaccines. cleavage site altered from HPAI to LPAI virus. Regardless of the source, the seed viruses are grown TYPES OF AVIAN INFLUENZA VACCINES in ECE, and the infective allantoic fl uid collected, chemically inactivated, and emulsifi ed in mineral oil Categories of Avian Infl uenza Vaccines adjuvant. The degree of purifi cation of the allantoic Historically, the worldwide use of vaccines has pro- fl uid can affect the overall response following vac- foundly reduced infectious diseases in poultry over cination, but most AI vaccines in poultry use crude the past 50 years. High quality vaccines can elicit allantoic fl uid without purifi cation. Typically, inac- immune effector elements such as circulating anti- tivation of the virus is achieved with formalin, which bodies and various antigen-specifi c memory lym- cross-links the viral proteins such that the viral rep- phocytes. AI vaccines fall into four broad categories lication cannot occur. Other chemicals such as β- (231): (1) inactivated whole AI virus (2) in vitro propiolactone or binary ethyleneimine can be used expressed HA protein, and potentially other AI viral as inactivants (116). proteins (3) in vivo expressed HA protein, and potentially other AI viral proteins, and (4) nucleic Adjuvants acids (Table 19.4). Vaccines in each of these Inactivated AI vaccines are formulated with adju- categories have specifi c advantages as well as vants prior to application. Vaccine adjuvants are disadvantages. chemicals, microbial components, or mammalian proteins that enhance immune responses to vaccine Inactivated Whole Avian Infl uenza Virus Vaccines antigens (267). Adjuvants are necessary to activate The majority of AI vaccines used in the fi eld are and direct the innate and adaptive immune responses inactivated whole AI virus vaccines licensed for par- to the rather poorly immunogenic inactivated vaccine Table 19.4. Compiled information on AI experimental and fi eld vaccine studies in specifi c-pathogen-free (SPF) or commercial (c) poultry and other birds. Protection criteria

Prevent Prevent Vaccine morbidity/ Decreased contact category Vaccine Species Challenge mortality shedding transmission Comments References

Inactivated Oil emulsion Chicken: Field, Yes Yes Yes Experimental and commercial vaccines tested; SQ, 5, 26, 32–34, AI virus layer LPAIV, IM, and in ovo routes protective; better 52, 67, 75, (SPF, C) HPAIV protection produced if vaccinated at 4 weeks of 80, 81, 90, and age versus 1 day of age; DIVA strategies 94, 94, 130, broiler included nonvaccinated sentinels, and 133, 161, heterologous NA and NS1 serological tests; HA (SPF, C) content of vaccine correlated with protection and 187, 213– gives broad subhomotypic protection; 215, 218, commercial vaccines varied in antigen content 220, 236, and quality; PD50 good measure of vaccine 239, 242, protection; transmission completely blocked at 2 249, 251, weeks postvaccination but only partially blocked 257, 260, as 1 week; infectious clone vaccine strains 418 273, 277, available; protection to 43 weeks with 1 SQ 278 vaccination; 9 doses given orally protected chickens from HPAIV challenge; can protect from egg production drops; metabolizable oils can be used in place of mineral oil as adjuvants; 2 doses of ISCOM used to get serological response and protection Turkey (C) Field, Yes Yes Yes Experimental and commercial vaccines tested; 1, 48, 82, 85, LPAIV, DIVA strategies included nonvaccinated 115, 120, HPAIV sentinels, and heterologous NA and NS1 146, 161, serological tests; increased reduction in challenge 256, 257 virus shedding with 2 vaccinations; vaccination increased resistance to infection by 102 virus; NP/HA ISCOM vaccine produced partial reduction in challenge virus shedding from respiratory tract; superior protection with positively liposomal-acridine adjuvant vaccine Duck (C) HPAIV. Yes Yes Two vaccine doses produced best protection with 21, 153, 176, LPAIV protection up to 52 weeks; bivalent AI vaccine 212, 251, did not interfere with protection; infectious clone 273 vaccine strains available; 2 doses protected Muscovy ducks from HPAIV; 2 doses prevented viremia and visceral organ infection in Pekin ducks Geese (C) HPAIV Yes Yes Infectious clone vaccine strains available, 3 doses 251 required to provide 34 weeks protection Other No Houbara bustards produced HI antibodies 274 poultry challenge Zoo birds No Serological response to H5 and H7 inactivated AI 24, 173, 174 challenge vaccines, 2 doses—76%, 80.5%, and 81.5% had HI titers ≥32 In vitro Baculovirus in Chicken: HPAIV Yes Yes HA content and virus strain infl uenced protection; 66, 88, 176, express insect cell culture layer LPAIV NA insert only partially protective; HA/NA/M2 239, 246 HA (SPF); combined oil emulsion vaccine protective in Muscovy Muscovy ducks against LPAIV duck (C) Vaccinia in TK–143 Chicken: HPAIV Yes Good protection from intraperitoneal, IM or 56, 73, 271 or chicken layer subdermal routes; partial protection from embryo fi broblast (SPF, C) scarifi ed skin route; bursectomized chickens not 419 cultures protected; live virus noninfectious or poorly infectious to chickens; oil emulsifi ed vaccine protective; partial protection with NA based vaccines Nonreplicating Chicken: HPAIV Yes Yes Complete protection when administered by SQ or in 89, 254 adenovirus in layer ovo routes but only partial protection by IN PER.C6 cells (SPF) route Alphavirus-based Chicken: HPAIV Yes Yes NA insert only partially protective after 2 or 3 201, 246 virus-like layer vaccinations replicon particles (SPF) In vivo Live AI virus Chicken (C) HPAIV Yes Homologous NA partial to occasionally complete 4, 5, 17, 39, expressed protective from mortality when challenged with 67, 149, HA HPAIV; best HI response with spray or cloacal 191, 209, vaccination routes; wild-type LPAIV vaccine 211 transmitted by contact and not recommended as AI fi eld vaccine; AI-NDV chimera poor replication in posthatch chickens but in ovo vaccination protects from HPAIV and velogenic NDV challenge Table 19.4. Continued Protection criteria

Prevent Prevent Vaccine morbidity/ Decreased contact category Vaccine Species Challenge mortality shedding transmission Comments References

Fowl poxvirus Chicken: HPAIV, Yes Yes Yes H5 and H7 vaccines developed; broad protection by 18, 19, 30, 35, vectored layer LPAIV AI-H5 insert vaccine against 1959–2004 H5 HPAIV 40, 91, 110, (SPF, C) given in low to very high challenge doses in chickens; 154, 176, and effi cacious if give by SQ or WW routes but not by 180, 212, broiler comb scarifi cation, eyedrop, IN or drinking water 231, 237, (SPF, C); routes; duration of immunity over 20 weeks from 238, 240, Goose single vaccination; pre-existing immunity to vector 248, 255, (C); inhibits vaccine effi cacy; greatest reduction in 270, 272 Muscovy respiratory shedding of challenge virus if vaccine and ducks (C) challenge viruses were more genetically similar; protection 40–45% at 1 week, 95–100% after 2 weeks postvaccination; NP express vaccine not protective; maternal immunity against fowl poxvirus does not inhibit posthatch immunization at 1 day; SQ immunization of geese at 21d protected 80% from mortality, reduced viral shedding and 420 shortened infection; WW immunization only gave partial protection in turkeys; 2 doses provided protection in Muscovy ducks from HPAIV, but slightly less effective than inactivated vaccine Avian leukosis virus Chicken HPAIV Yes In chickens, vaccine with AI HA insert protected 31, 106 vectored (SPF) but with AI NP was not protective Paramyxovirus type Chicken HPAIV Yes Yes Mass immunization vector (eyedrop or spray); 92, 168, 245, 1 vectored (SPF) protects from both velogenic NDV and HA 264 (lentogenic subtype specifi c HPAIV Newcastle disease virus) Gallid herpesvirus-1 Chicken: HPAIV Yes Yes ILTV vector was attenuated by ΔUL0 or ΔUL50 138, 263 (infectious layer insertions of AIV-H5 or H7 HA; mass laryngotracheitis (SPF) immunization potential (eyedrop or IT routes) virus [ILTV] vector) DNA Naked DNA Chicken: HPAIV, Yes Yes Requires high doses of DNA and multiple immunization 59, 86–88, broiler LPAIV if given IM, intraperitoneal or IV routes to produce 117, 118, (SPF), partial protection; response improved when using 225, 246 layer pCI-neo HA plasmid and lipofectin or lipotaxi (SPF) adjuvants; gene gun administration reduced quantity of DNA after 2 vaccinations to produce protection; NA only partially protective; NP gene not protective; DNA vaccine followed by HA protein prime boost in chickens gave the best response versus DNA/DNA or HA protein/HA protein primary and boost vaccinations 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 421 antigens. In general, although it is dependent on tory tract in mice and also protected them against purity and quantity, the antigen is a passive element, challenge with infl uenza virus. while the adjuvant represents the activating and modulating intermediate operating at the interface Liposomes between the immune system of the host and the A variety of liposomes have been used as adjuvants administered vaccine inoculum. A single adjuvant in mammalian and avian experimental vaccine may have more than one mechanism of action. studies including AI vaccines (82), but currently, the Interest in reducing vaccine-related side effects technology has not been applied to licensed AI vac- and inducing specifi c types of immunity has led to cines for poultry use in the fi eld. Liposomes are the development of numerous new adjuvants. Adju- vesicles of cholesterol and phospholipids that resem- vants in development or in experimental and ble crude cell membranes. As adjuvants, liposomes commercial vaccines include aluminum salts, oil can incorporate the desired antigens either within the emulsions, saponins, immune-stimulating com- center of the vesicle or the cell membrane. They can plexes (ISCOMS), liposomes, microparticles, non- induce humoral immunity and, in some cases, acti- ionic block copolymers, polysaccharides derivatives, vate CTLs (267). Nanoparticles and microparticles cytokines and a wide variety of bacterial derivates are tiny solid particles made from biodegradable (10, 70, 82, 95, 101, 105, 189, 193). Less purifi ed polymers, particular cyanoacrylates and poly inactivated vaccines sometimes contain bacterial or (lactide-co-glycolide) copolymers. Nanoparticles viral components that can serve as “built-in” adju- (10–1000 nm) differ from microparticles (1–100 μm) vants, whereas more purifi ed antigens do not usually only in their size. Microparticles can induce CMI, stimulate a strong and lasting immune response including CTLs and humoral immunity (163). (163). Aluminum and calcium salts are relatively Calcium phosphate nanoparticles induced mucosal weak adjuvants that mainly induce Th2 responses immunity and protection against herpes simplex and few, if any, antigen-specifi c CTLs (163). virus type 2 in mice (101). Chitosan easily forms microparticles and nanoparticles that encapsulate Oil adjuvants large amounts of antigens such as ovalbumin, diph- Oil emulsion adjuvants contain a mixture of oil and theria toxoid, or tetanus toxoid. Chitosan particulate aqueous phase stabilized by a surfactant and have drug carrier systems are promising candidates for been commonly used in experimental and licensed oral vaccination. After co-administration of chitosan inactivated AI vaccines for poultry. These emulsions with antigens in nasal vaccination studies, a strong contain the antigen in either “oil-in-water” or “water- enhancement of both mucosal and systemic immune in-oil-in-water” formulations. Without other compo- responses was observed in mice (107, 262). Sapo- nents, oil-based adjuvants stimulate mainly antibody nins are complex chemical adjuvants extracted from responses although under some circumstances water- plants, most often the tree Quillaia saponaria. Sapo- in-oil emulsions may be able to activate CTLs (10, nins are immunomodulators and can induce strong

23, 84, 213, 214, 216–219, 221, 278). Most licensed Th1 and Th2 responses as well as CTLs in animals. and experimental poultry inactivated vaccines use a Saponins may stimulate CMI to an antigen that mineral oil base, including AI vaccines, but metabo- would normally induce only antibodies (267). lizable oils have been shown to be effective (219). ISCOMS are cage-like structures that contain sapo- The advantages of using emulsions include enhanced nins, cholesterol, and phospholipids. They can antibody production, as well as extended release of induce Th1 reactions and CTLs as well as concurrent the antigen, which results in an overall higher Th2 responses under some circumstances (162, 193, immune response of the birds to the vaccines. 196). Nonionic block copolymers are synthetic adju- Experimentally, nanoemulsion formulations made vants composed of hydrophobic polyoxypropylene from soybean oil, tributyl phosphate, and Triton X- ﬂ anked by blocks of polyoxyethylene. As adjuvants, 100 provided early protection of mice against an these chemicals can enhance humoral immunity to intranasal lethal challenge with inﬂ uenza virus (78). many antigens, but most often they are used in an The nanoemulsion was a mixture of nonionic deter- aqueous buffer, oil-in-water, or water-in-oil emul- gents, GRAS list solvents, and soybean oil. The sions (6). Muramyl dipeptide (MDP) is the active nanoemulsion had no toxicity in the upper respira- component of an immunomodulatory peptidoglycan 422 Avian Influenza

from mycobacteria. The MDP induces mainly Th1 19.4). In theory, the method requires less manufac- and Th2 responses. MDP derivates are often incor- turing expense because the process uses the bird’s porated into liposomes, water-in-oil, and oil-in- own cells to produce the immunogen rather than water emulsions (65, 163). Bacterial toxins, cholera expensive in vitro expression systems. The advan- toxin (CT) and Escherichia coli heat-labile exotoxin tage of this type of vaccine is they can stimulate (LT) have been tested most extensively as mucosal humoral and cellular immunity when given paren- adjuvants in animal models. They appear to induce terally and, if they replicate at a mucosal site, induce strong humoral responses as well as CTLs (65, 84, mucosal immunity. Live virus vaccines are usually 93, 182). Cytokine protein and genes are themselves superior to inactivated vaccines in inducing mucosal being considered as vaccine adjuvants (131). The immunity and thus reducing shedding. specifi c effects vary with the cytokine; some enhance the activity of defi ned immune cells while others act Live Avian Infl uenza Virus Vaccines as general activators. Cytokines also induce other Live LPAI virus vaccines have been studied experi- cytokines; this property can make the effects of a mentally in poultry (Table 19.4). They provide specifi c cytokine diffi cult to predict (102, 148). advantages of good protection against HPAI viruses, can be mass applied by spray vaccination or in the In Vitro Expressed Hemagglutinin drinking water, are economical, and provide more Various in vitro expression systems have been used rapid protection than inactivated vaccines (13, 17, to produce experimental AI vaccines, but none of 149). Yet, live LPAI virus strains are not recom- the technologies have been commercialized (Table mended for use as poultry vaccines because of the 19.4). The specifi c expression methodology varies following (13, 14, 16, 128): (1) they can produce but the total amount of immunogen (e.g., HA) to economically important losses associated with respi- be administered is produced in eukaryotic tissue ratory disease or drops in egg production (2) they cultures (plant and animal cells), plants, yeast, bac- can easily spread from bird-to-bird and farm-to-farm teria, or viruses [e.g., vaccinia (12, 56, 73), some potentially creating endemic infection and disease repli cation-defi cient adenovirus, Venezuelan equine with the need for eradication of the vaccine strain, encephalitis virus (201, 246) and baculovirus (66)]. and (3) some LPAI viruses have a potential for Yeast-expressed infl uenza A HA and NA genes pro- mutation or reassortment creating more pathogenic tected mice from lethal challenge (144, 192). viruses such has been reported with some H5 and However, the most frequently used system has been H7 LPAI viruses becoming HPAI viruses in the to insert the AI viral HA into a baculovirus (insect fi eld. virus) vector and infecting insect cell cultures with However, various genetically altered infl uenza A expression and production of the HA (66, 239). The viruses have been developed and investigated in HA is recovered from the supernatant or cell lysates, mammals that allow regulated viral replication and inactivated with chemicals and emulsifi ed similarly the induction of immunity without negatively affect- to inactivated whole AI virus vaccines. Therefore, ing growth or immunocompromising the individual. the entire quantity of the immunogenic protein (i.e., This strategy for developing a genetically altered HA) is produced external to the bird host. live infl uenza A virus vaccine was to attenuate the virus to a lower pathogenicity level either through In Vivo Expressed Hemagglutinin laboratory passage to generate cold-adapted, tem- With in vivo expression systems, the immunogen is perature-sensitive phenotypes or through biotech- produced within the bird host by use of a live bacte- nology to alter the viral genome directly. The rial or viral vector such as recombinant fowl poxvi- majority of these types of vaccines have been devel- rus (rFPV), some adenoviruses, replication-defi cient oped and tested in mammalian models (57, 74, 135, Venezuelan equine encephalitis virus (rdVEE), 228). At issue is the potential of the vaccine virus to recombinant avian leukosis virus (rALV), recombi- revert or recombine with fi eld viruses resulting in a nant infectious laryngotracheitis virus (rILT), virus with enhanced pathogenicity. The reassortant, recombinant NDV (rNDV), AI-NDV chimera virus, cold-adapted phenotype has been applied to infl u- Salmonella sp., or other organisms (233) (Table enza A vaccines for humans with no serious side 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 423 effects and without evidence for reversion to viru- AIV-H5 vaccine has been licensed in the United lent virus (269). However, given the documented States for emergency use in HPAI outbreak situa- ability of AI viruses to mutate through viral recom- tions but has not been used to date in the fi eld (157). bination or gene segment reassortment, live AI virus However, this vaccine has been used extensively in vaccines are currently not approved for use in com- Mexico, El Salvador, and Guatemala against mercial poultry. However, laboratory-passaged, endemic H5N2 LPAI viruses (1998–2007) (265, attenuated AI viruses have been described. For 266). Other recombinant live vectored viral vaccines example, truncation of the NS1 gene in A/turkey/ have been engineered to express AI genes and dem- Oregon/1971 (H7N3) LPAI virus resulted in onstrated experimental protection in chickens; they decreased replication in chickens and attenuation of include rILT virus (herpesvirus), rNDV (paramxy- virulence (55). This virus has the potential for virus type (1), rALV, and recombinant adenoviruses vaccine use, but safety must be determined before (31, 56, 92, 134, 138, 245, 263, 264). The rNDV, licensure and use in the fi eld. rILT, and some recombinant adenovirus vectors The development of infectious clones for infl u- could be applied by mass application via drinking enza A viruses has created a system that allows water or sprays to reduce costs because they repli- directed mutations in the infl uenza genome that can cate in the mucus membranes. However, some result in attenuation. Recently, an AI infl uenza virus vectors, such as recombinant replication-incompe- vaccine was developed that contains eight gene seg- tent adenoviral vector (89, 254), rALV, and rFPV ments of H5 HPAI virus (HA cleavage site altered (19) require injection to produce an effective immune from HP to LP) but includes the ectodomain of the response. One additional hurdle to the application of HN gene from NDV instead of the NA gene of the virus-vectored vaccines in the fi eld is the immune AI virus (168). The resulting virus was attenuated status of the birds against the vector. For example, as evident by inconsistent replication in 2-week-old most poultry raised in the developed world have chickens, but in ovo administered virus produced been immunized against NDV, which may limit the humoral antibody response and vaccinated chickens immunogenicity of the rNDV vectored vaccine, were protected from both H5 HPAI virus and viru- unless NDV immunity is serologically monitored lent NDV challenges (211). Truncation of NS1 gene and the vectored vaccine is applied only when anti- may also induce attenuation of AI viruses and may body levels are suffi ciently low to produce an anam- be another method of producing an attenuated live nestic response. Swayne et al. (237) described that AI virus vaccine (128). preexisting immunity, most likely cell-mediated immunity, against fowl poxvirus will interfere with Live Vectored Vaccines (Non–Infl uenza rFP-AIV-H5 vaccine in chickens (237), but fi eld A Viruses) data suggest maternal antibody against H5 AI virus Other types of live virus vaccines have been devel- or fowl poxvirus vector did not interfere with active oped for AI using alternative virus vectored con- immunity to rFP-AIV-H5 vaccine given at 1 day of structs and can provide some of the immunological age (40, 91). Finally, the host range is important in advantages of a live virus vaccine but without the knowing which bird species can be immunized with reassortment risk of using a live (replication-compe- each vectored vaccine. For example, rFPV and rILT tent) AI virus (Table 19.4) (233). These types of have produced protective immunity and are only vaccines use recombinant DNA technologies to used in chickens because of the host restriction of incorporate genetic material provided from the AI the viruses, but there is some evidence that rFP- genome into a viral backbone for gene expression in AIV-H5 can produce protective immune response in vivo. Many examples of these types of vaccines cats and geese (110, 114). In addition, rFPV with have been documented in the literature with varying NDV gene inserts are effi cacious in turkeys against levels of success, but the most frequently reported virulent NDV; thus, the rFPV with AI HA virus system has been the recombinant fowl poxvirus genes would potentially be effective in turkeys (rFPV) with H5 (rFP-AIV-H5) or H7 (rFP-AIV-H7) against AI virus challenge. The rFPV is best applied AI HA gene inserts (18, 29, 30, 36, 37, 58, 91, 111, in the hatchery at 1 day of age. So while it is clear 179–181, 240, 243, 244, 248, 255, 270). An rFP- that live vectored vaccines have advantages in terms 424 Avian Influenza of immunity, their use may be limited by other against clinical disease and death, but they do not factors. always provide absolute protection against mucosal infection or shedding of the virus from oropharynx DNA Vaccines and cloaca. The risk of infection of vaccinated birds Plasmid-based experimental DNA vaccines using and excretion of challenge or fi eld virus is greatly the HA gene have elicited protective immune reduced, but absolute prevention of infection is not response in chickens against a variety of H5 and H7 feasible under most fi eld conditions. The effective- HPAI viruses (86, 117, 118, 188, 225). Such vac- ness of reduction of virus excretion is linked to both cines produce antigen in situ, inducing both an adap- a reduction in titer of the virus excreted and the tive humoral and cellular immune responses, similar shortened duration of viral shedding. “Silent infec- to those produced by live virus infection or vaccinations” have been proposed based on experimental tion (128). Typically, DNA vaccines are naked studies where shedding is seen in chickens following 6 nucleic acids containing AI viral cDNA within high challenge doses (10 EID50). Such “silent infec- various plasmids under control of a mammalian pro- tions” have not been as convincingly demonstrated moter gene (86, 117, 225). Studies in mammals have in properly vaccinated poultry in the fi eld (207). shown DNA vaccines expressing HA genes pro- However, the risk of spreading virus from potential duced more effective protection against antigenic “silent” infections in vaccinated fl ocks is much variant infl uenza A viruses than inactivated vaccines lower than from infected nonvaccinated fl ocks as and inclusion of the NP gene augmented the protec- evident by nonvaccinated chickens excreting 100 to tion (128, 225). However, protection induced by 10,000 fold more virus when infected than vacci- DNA vaccine in an avian model (i.e., the chicken), nated chickens (98) and the nonvaccinated chickens has been less consistent than with inactivated whole are also 100 to greater than 100,000 times more AI virus vaccines or in vitro or in vivo expressed susceptible to infection than vaccinated chickens HA subunit vaccines (118, 128, 225). Currently, the (35, 48); that is, even a less than ideal vaccine can primary limitations for DNA vaccines in poultry be advantageous over no vaccine use. However, include that (1) requirement of large quantities of birds that receive vaccines of low quality (i.e., low expensive nucleic acid per dose to produce a protec- quantities of HA antigen or poor adjuvant systems) tive immune response in chickens, and (2) protec- may be protected from clinical signs and death, but tion is achievable only after three or more the challenge/fi eld virus may replicate with excre- vaccinations. DNA vaccines will be economically tion of signifi cant quantities of virus into the envi- prohibitive for use in the fi eld until promotors are ronment (90). In Italy, clinical infections in developed to reduce the number of immunizations vaccinated turkey fl ocks have been associated with and quantity of nucleic acids needed per bird. low antibody titers and multiple vaccinations have been needed to produce protective immune response FIELD USE OF VACCINE AND that last the entire production cycle (Giovanni Ortali, SPECIAL ISSUES personal communication, November 14, 2003). Only AI vaccines that are licensed by a country’s Avian Infl uenza Vaccination Issues national veterinary authority should be considered In terms of AI vaccination to protect poultry, there for use in the fi eld. In addition, such vaccines should are four ideal goals: (1) prevent clinical disease and meet the minimum requirements of World Organi- death (2) induce complete resistance to infection zation of Animal Health (Offi ce Internationale des after exposure (3) stop challenge or fi eld virus rep- Epizooties [OIE]) (164). International standards are lication and excretion from the vaccinated birds, and needed to assure uniform potency and effi cacy of AI (4) easily identify infected animals in a vaccinated vaccines (231). At a minimum, AI vaccines should population (i.e., differentiating infected from vac- meet the following criteria: (1) purity as determined cinated animals [DIVA] principle) (222). However, to not be adulterated but contain only the desired few, if any, commercially available or experimen- immunogens and other compounds, and must be tally tested vaccines consistently fulfi ll all of these consistent in composition; (2) safety as evident by requirements (128, 222). In experimental studies, no adverse effect on the vaccinated host or environ- most AI vaccines provide consistent protection ment; (3) effi cacy to protect in specifi c quantifi able 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 425 assays or tests against a specifi c challenge AI virus; dence for the lack of absolute broad protection and (4) potency indicating the vaccine has suffi cient within a subtype comes from H7 AI virus experi- HA antigen mass (inactivated vaccine) or dose (live mental studies using rFPV vectored vaccines (36). vectored vaccine) to ensure protection under a Chickens challenged with A/turkey/Italy/4580/1999 variety of conditions. (H7N1) HPAI virus were only protected if they had Protection of vaccinated birds against AI is received rFP-AIV-H7 vaccines with Eurasian H7 impacted by the following factors: (1) vaccine strain insert, and chickens vaccinated with rFPV contain- (2) vaccine dose (“vaccine quality”) (3) route of ing HA gene inserts from Austratia, A/chicken/ administration (4) management conditions, and (5) Victoria/1/1985 (H7N7) or North America, A/ fi eld application method and coverage. If ranked in turkey/Virginia/158512/2002 (H7N2) were not pro- order, vaccine potency and fi eld application would tective. Because H7 AI vaccines have had no or be one and two in importance (D. Halvorson, per- limited use in Australia and North America, the H7 sonal communication, May 31, 2007). AI virus drift is probably the result of geographic isolation of the H7 viruses in wild birds between Selection of Vaccine Strains different continents. Because the HA is the primary protective protein Historically, most inactivated AI vaccine strains and subtype specifi c, the vaccine strain must match have been selected from outbreak LPAI viruses of the HA subtype of the fi eld virus. However, unlike the same HA subtype. Many have been used as alum-adjuvanted human infl uenza A viruses where autogenous vaccines for control of the original LPAI antigenic drift of fi eld viruses requires changing the outbreak (85) or used at later dates in other LPAI vaccine strains every 3 to 4 years, the inactivated oil outbreaks of the same HA subtype (223). For HPAI, adjuvanted and rFP-AIV-H5 poultry vaccines have LPAI vaccine strains of the same HA subtype have been far less impacted by drift in fi eld viruses (224). been protective, and since 1995, some HPAI viruses For example, the H5 AI vaccine strains have pro- have been used as vaccine strains in some epidemics vided broad cross-protection from mortality against such as in Pakistan (H7N3) and Indonesia (H5N1) diverse H5 HPAI fi eld viruses, collected over 38 (60, 159). Contrary to rumor, HPAI strains do rep- years (1959–1997) and differing by as much as 12% licate to suffi cient titer in ECE to be used in inacti- in amino acid sequence at the HA1 (236, 239, 240) vated AI vaccines but to lower titer than many of compared to the challenge HPAI virus. In one study, the LPAI vaccine strains. However, the safe and the closer the HA gene sequence similarity between biosecure use of HPAI strains in vaccines requires vaccine and fi eld viruses, the greater was the reduc- specialized, high biocontainment manufacturing tion in challenge virus replication and shedding from facilities and/or special biosecurity and biosafety the respiratory tract (240). However, this subtype personnel procedures. The uses of LPAI virus strains specifi c broad protection is not absolute within all have fewer biosecurity and biosafety concerns subtypes and in all fi eld situations. The inactivated during manufacturing and are preferred over HPAI H5N2 AI vaccine used in Mexico from 1995 through viruses. When LPAI or HPAI outbreak viruses are 2007 has used a 1994 Mexican H5N2 LPAI strain used as vaccine strains, they should be a close and provided protection in chickens against the 1995 genetic match to the HA of the current outbreak HPAI fi eld virus and the early H5N2 LPAI fi eld virus and have high growth characteristics in ECE viruses (90, 125). However, the 1994 vaccine strain in order to produce suffi cient quantities of antigen was not protective against two later lineages of to be immunogenic and protective. H5N2 LPAI viruses isolated in 1998 from southern In the past 2 decades, biotechnology has advanced Mexico and 2003 from Guatemala as evident by to allow laboratory generation of infectious clone AI replication and shedding of same quantity of chal- virus strains for use in inactivated vaccines or HA lenge virus from the respiratory tract of vaccinated gene inserts for use in in vitro and in vivo vectors, and nonvaccinated chickens (125). It is unclear or in DNA vaccines. The infectious clone AI viruses whether the drift in fi eld viruses resulted from are produced by reverse genetics using the six inter- immunity following infections in nonvaccinated nal genes from an infl uenza A vaccine strain such poultry or improper vaccination and subsequent as PR8 and the HA and NA genes from the AI out- infections in vaccinated chickens. Additional evi- break or related viruses (126, 133, 273). The use of 426 Avian Influenza

PR8 internal genes imparts the ability to replicate to ulation is more uniform because of greater host high virus titers in ECE typically used as the subtrate genetic homogeneity than is present in the human in the manufacturing process. If the donor HA gene population; (4) the AI vaccine use in poultry is tar- is from an HPAI virus, the HA proteolytic cleavage geted to a relatively young, healthy population as site must be changed from a sequence of an HPAI compared to humans where the vaccine is optimized to LPAI virus, thus producing an LPAI vaccine for groups with the highest risk of severe illness and strain that can be manufactured at lower level of death; and (5) historically, less endemic infl uenza biosafety than an HPAI virus. The NA gene can be virus infection in poultry than in human populations. selected from among the existing nine NA subtypes For the latter in the developed countries, the uncom- to be different from the outbreak virus, thus creating mon AI virus exposure and infrequent AI vaccine a vaccine with a heterologous NA subtype that will use have exerted less selection pressure on AI allow a serological test to identify infected birds viruses to drift. However, the high prevalence of within the vaccinated populations (see Surveillance). H9N2 LPAI and H5N1 HPAI virus infections in The infectious clone vaccines are as effi cacious as poultry of some developing countries and the high the existing licensed vaccines based on outbreak usage and reliance on vaccine in control programs LPAI and HPAI viruses. Finally, the advent of bio- will contribute to increased immune pressure on AI technology has allowed rapid selection of HA gene fi eld viruses to drift away from current vaccine from outbreak viruses for insertion into vectored strains. As a result, vaccine strains may need to be vaccines such as adenovirus or rFPV (89), thus changed more frequently and may require increase allowing close genetic matching between vaccine usage of reverse genetics (126, 133) to generate and fi eld AI virus. In vectored vaccines, HA should replacement LPAI vaccine strains or replacement of be altered from HP to LPAI virus to maximize rep- AI HA genes in vectored vaccine products such as lication titers in the in vitro system without being new rFPV or rNDV vaccines to maintain effi cacious excessively cytolytic on cell cultures or ECE. vaccines for use in the fi eld. The initial virus strain selected should be very protective as demonstrated by effi cacy studies in Vaccine Quality avian species. However, over time, the fi eld viruses Quality control in vaccine manufacturing is critical will drift away from the vaccine strain to a point to ensure a safe and effi cacious product (250). The where protection is lacking as has been seen with quality of commercial vaccines greatly impacts the H5N2 LPAI vaccine in Central America in the past fi eld effi cacy in three ways: (1) antigen quantity, that decade. Therefore, vaccine strains should be reas- is, suffi cient HA antigen must be present (inacti- sessed in in vivo and in vitro protection assays on a vated vaccine) or high enough titer (live vaccine) to continual basis or at a minimum of every 2 years if produce a serologically measurable protective tested in vivo against current circulating fi eld viruses immune response and there should be minimal to assess protection (232). If the vaccine strain is no batch-to-batch variation in antigen content; (2) adju- longer protective, a new strain should be selected vant, i.e., for inactivated vaccine, high-quality adju- based on protection studies. vants are needed to enhance the bird’s immune response to the HA antigen and, in addition, should Broad Homosubtypic Protection be safe for administration, and (3) sterility; that is, This historically demonstrated broad and longer- for inactivated vaccines, the vaccine strain must not term homosubtypic protective effi cacy of poultry AI only be completely inactivated but also be pure for vaccines as compared to human infl uenza A (H1 and live vaccines—there must be absence of any bacte- H3) and B vaccine strains is potentially the result of rial or viral contaminates. In the manufacturing the following (232): (1) poultry inactivated AI vac- process, the production of inactivated vaccines uses cines use proprietary oil emulsion adjuvants, which a chemical process to inactivate the vaccine strain elicits more intense and longer-lived immune and the inactivation should be verifi ed by ECE or response in poultry than alum-adjuvant human infl u- tissue culture inoculation. In addition, the antigen enza A and B vaccines; (2) the AI vaccine immune should be sterility tested to ensure no contaminating response in poultry appears to be broader than in bacteria or other agents are present. For live vac- humans; (3) the immunity in domestic poultry pop- cines, the input products (ECE or tissue culture and 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 427 seed stock) should be tested to verify that no extra- quantity of antigen needed will vary depending on neous viral or bacterial pathogens are present. For vaccine strain, challenge virus strain and challenge inactivated vaccines, the use of oil adjuvants is nec- dose, and adjuvant used in the vaccine. essary to produce consistent, broad and long-lived protective antibody titers. Most veterinary biologics Route of Administration companies use proprietary adjuvant systems. The vaccine should be formulated with suffi cient Posthatch Parenteral Administration HA antigen to produce a consistent protective Parenteral administration to poultry involves the response in the target species. Garcia et al. (1998) catching, handling, and injecting individual birds examined six inactivated commercial H5N2 vac- with the vaccine to produce the protective immune cines from Mexico for protection in chickens against response. Parenteral administration is most com- an H5N2 HPAI virus challenge (90). All provided monly accomplished by subcutaneous (SQ) injec- similar good protection from mortality but the indi- tion in the nape of the neck, but some vaccines vidual vaccines varied in the ability to reduce shed- require intramuscular (IM) or wing web (WW) ding from the oropharynx and cloaca and to induce injection. Most licensed inactivated AI vaccines a serological response. Their results suggested a require SQ or IM injection while rFP-AIV-H5 vac- single immunization dose of 4 μg or greater of HA cines are administered via the SQ and WW routes. protein induced immune response that was the best Although wild-type fowl poxviruses can produce in SPF chickens for reducing replication of HPAI oral, pharyngeal, and tracheal infection and lesions, virus in the respiratory and alimentary tracts when the replication pattern of rFP-AIV-H5 is more challenged with intermediate dose of virus (100 restricted and the vaccine given intranasally (IN) or mean chicken lethal doses [CLD50]). In a study with in the drinking water did not protect against HPAI experimental vaccines, a dose of 3 to 8 μg HA virus challenge (19). In the United States, the costs protein from inactivated AI virus or 2 μg of baculo- of the vaccine plus administration are expensive, virus-vectored HA protein was protective in intrana- with costs for vaccines ranging between $0.05 and sally challenged chickens as compared to 0.1–0.3 μg $0.10 per dose and estimated administration costs of and 0.2 μg, respectively, which were not completely handling of the birds ranging at $0.05 to $0.07 per protective (239). In a contact transmission study, bird (232). However, the actual costs will vary 0.3 μg of HA in inactivated oil emulsifi ed vaccine, depending on the type and quality of the vaccine and but not 0.1 μg HA, prevented clinical disease in the costs for government control programs, surveil- chickens intranasally challenged with HPAI virus, lance, and labor for vaccination. while 0.9 μg of HA in the vaccine provided optimal protection without isolation of challenge virus from Mass Administration respiratory or alimentary swabs (277). In another Low-cost methods for mass immunization of poultry study, two immunizations with 0.5 μg HA was against AI would be advantageous, providing a minimal dose to give best protection (75). method to reduce the cost for administering AI vac- A more reproducible and accurate way to establish cines that would translate into an economic incen- the required HA antigen content is using a PD50 tive to vaccinate and would increase vaccine use in determination. In a chicken experimental study using regions or countries where vaccination is needed to inactivated experimental AI vaccines containing a control AI. Methods for mass immunization against standardized generic oil-emulsion adjuvant system AI could include in ovo administration or posthatch and experiencing a high challenge dose of H5N2 administration to the respiratory or alimentary

HPAI virus (1000 CLD50), the PD50 ranges based on tracts. mortality were 0.006 to 0.156 μg of HA and on morbidity were 0.008 to 0.156 μg of HA, which, when In ovo administration translated to a standard minimal dose range to achieve In the early 1980s, vaccination by injection into the

50 PD50, would require HA content per dose of 0.3 to developing chicken embryo (i.e., in ovo) was devel- 7.8 μg (236). As a summary of multiple experiments, oped to provide early posthatch protection against a minimum of 1 to 5 μg of HA/dose of poultry AI Marek’s disease herpesvirus (MDV) (206). The vaccines has been suggested (232). However, the methodology has been automated, allowing mass 428 Avian Influenza vaccination of chicken embryos at the 18th to 19th replication adenovirus vectored AI-H5 vaccine day of incubation. Currently, the majority of broiler failed to protect intranasally vaccinated chickens chickens produced in the United States are vacci- from intranasal challenge by homologous H5N1 nated against MDV by in ovo technology and this HPAI virus, indicating it is unlikely to be used as a methodology is being widely adopted by the inte- topical mass applied vaccine (89). grated commercial broiler industries around the world. Currently, there are no licensed AI vaccines Alimentary administration (drinking water for in ovo administration. However, multiple exper- and feed) imental studies have been conducted and have Currently, no licensed AI vaccines are available for shown proof-of-concept protection against AI and drinking water or feed administration. Inactivated have included an inactivated AI vaccine (213), defi - vaccines have been experimentally administered cient replicating adenovirus with AI H5 gene insert orally with protection from HPAI virus challenge, (254), and AI-NDV-HN chimera (211). In addition, but the immunization required very high vaccine use of a recombinant MDV vectored vaccine with doses and up to nine immunizations to produce pro- AI HA gene insert holds great potential for being tection in SPF chickens, making alimentary admin- effi cacious against AI and would be compatible with istration not feasible (67). Various methods for the existing in ovo MDV vaccines. Another theo- induction of protective antigens in plants for oral retical possibility for AI in ovo vaccine is an AI consumption have been proposed, but none have virus HA–antibody complex vaccine, similar to the been shown to protect poultry against HPAI virus infectious bursal disease virus (IBDV)-antibody challenge. Such nonlive HA AI vaccines may require vaccine, but this would require use of an inactivated high doses and special adjuvants to produce a pro- AI virus to use in the antigen–antibody complex tective immune response. (109). Recently, an attenuated ΔaroA Salmonella enteritidis (SE) vector with AI-M2e gene insert was Respiratory administration (spray and eyedrop) shown to produce neutralizing antibodies in alimen- Infectious bronchitis virus and NDV vaccines are tary-exposed chickens (63). However, in vivo chal- the most common mass applied live virus vaccines lenge studies are needed to determine if a for poultry, with billions of doses used each year bacteria-expressed AI M2e or SE-expressed AI HA around the world in all sectors of poultry production vaccine will protect poultry from LP or HPAI (230). Most often, these vaccines are mass applied viruses. in the hatchery by spray cabinet and in the fi eld by back-pack sprayers in the production house. Such Management and Environmental Conditions application results in conjunctival and upper respira- Protection induced by a vaccine applied under labo- tory tract exposure with vaccine virus replication ratory conditions in SPF poultry controls all con- and resulting production of mucosal and systemic founding variables and gives the best immune immune response. Because of the widespread use of response, but in the fi eld, vaccine-induced protec- live NDV vaccines, the concept of using an infection will be less than optimal unless all management tious clone system to produce vectored vaccines was variables that negatively impact immunity are con- developed (122, 160, 171). Such rNDV vectors with trolled. Some experimental AI vaccine studies have H5 (rND-AIV-H5) or H7 AI HA gene inserts have reported “sterilizing immunity” at mucosal sites, been used to immunize chickens and protection has but, in the fi eld, “sterilizing mucosal immunity” is been demonstrated against challenge by both NDV neither practical nor achievable. However, vaccines and HPAI viruses (92, 134, 168, 245, 264). Two can signifi cantly reduce AI virus replication and rND-AIV-H5 vaccines have been licensed in China shedding from the respiratory and alimentary tracts, and Mexico for fi eld use against NDV and H5 HPAI which translates into reduced environmental load of (134, 282). Recently, a spray application inactivated virus and reduction or elimination of virus transmis- H5N2 AI virus vaccine containing a special adjuvant sion (81). Management or environmental variables was proposed to prevent replication of an H5N2 that may inhibit or reduce protection in the fi eld LPAI virus when challenged by eyedrop exposure include the following: (1) failure to vaccinate suf- in the conjunctival sac (136). However, a defi cient- fi cient numbers of birds in the population to achieve 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 429 herd immunity; (2) administering less than the rec- (SIV) and SIV is endemic in pigs throughout the ommended vaccine dosage; (3) use of low cost, poor world (79). Therefore, in many developed countries, quality vaccines; (4) improper storage and transport turkey breeders and occasionally meat turkeys may of vaccines; (5) improper vaccination with numer- be strategically vaccinated against H1 and H3 SIV ous missed injections; and (6) immunosuppression in high-risk geographic areas where both swine and resulting from specifi c virus infections (e.g., infec- turkeys are produced. Vaccination is less commonly tious bursal disease, chicken anemia virus, adenovi- practiced against H5 and H7 LPAI. Vaccination of ruses, and others), exposure to naturally occurring poultry is very rarely utilized against HPAI in devel- compounds such as afl atoxins or exposure to stress- oped countries where early diagnosis and stamping- ful, or poor management conditions. Biosecurity, out without vaccination are most commonly education, diagnostics and surveillance, and elimi- practiced for immediate eradication. In developing nation of AI virus–infected poultry are essential countries, the use of vaccine against H5 and H7 because vaccines and their use are not perfect. Vac- LPAI and HPAI depends on the endemic status and cination should only be used as one tool in a com- the ability of the stamping-out programs to immedi- prehensive AI control program. ately eradicate the virus. With 22 of the 26 HPAI epidemics of HPAI of the past 50 years, stamping- Field Application Method and Coverage out for immediate eradication was the strategy and AI vaccination was not used. AI vaccination was Vaccination Programs implemented in 1995 for the H5N2 HPAI epidemic The development of a viable vaccination strategy in Mexico, in 1995 with the H7N3 HPAI epidemic requires examination of multiple facets including: in Pakistan, in 2002 with H5N1 HPAI epidemic in (1) the type of AI virus as either HPAI, H5 and H7 Asia, and in 2005 with H7N7 HPAI epidemic in LPAI (i.e., low pathogenicity notifi able avian infl u- North Korea. With H5 and H7 LPAI, the use of enza [LPNAI]) or other LPAI (H1-4, H6 and H8-16) vaccines may depend on the local situation and have viruses to be controlled; (2) the goal or desired been used as an adjunct to controlled marketing. outcome of the control program to either prevent, In the situations where vaccine is being consid- manage or eradicate AI; (3) the risk level for AI ered for use, the risk of AI infections should be introduction and role vaccination can play in the determined, and if the risk is high, proper vaccina- control strategy; (4) the species of birds to vaccinate; tion could be used as an effective tool when used as (5) the production sector or system involved; (6) the part of a multicomponent AI control strategy. For logistic support including regulatory oversight, example, in Hong Kong in 2002, compulsory vac- availability of trained vaccinators and surveillance cination was implemented because of the repeated capabilities; and (7) types and availability of introduction of H5N1 HPAI virus in imported vaccines. poultry from southern China and because of the The use of vaccine is universally accepted for presence of endemic infections within the region control of economically signifi cant and common (80). This strategy has been successful in preventing endemic poultry diseases such as infectious bronchi- further outbreaks in poultry of Hong Kong and pre- tis and infectious bursal disease and, within some venting exposure and infections in humans. However, countries, zones, and compartments (CZC) for LPAI blanket use of vaccination in all situations should control. For example, in much of Asia and the not be endorsed because vaccine use without proper Middle East, H9N2 LPAI viruses are endemic in biosecurity and surveillance may make early detec- poultry and vaccines are routinely used to manage tion of AI infection diffi cult and confound rapid the infection and disease in order to make poultry detection and stamping-out programs. Proper production economically viable, but eradication of vaccine programs should be designed to fi t a specifi c H9N2 LPAI has not been the goal. By contrast, in need and the epidemiology of the disease. most developed countries, AI is sporadic and local- Various strategies have been proposed for using ized in nature making widespread use of vaccines vaccines in both HPAI and LPAI control programs incompatible with general preventative or eradica- (42, 44, 46, 49–51, 96, 98, 128, 141, 223, 230, 231). tion strategies (16). However, domestic turkeys are Historically, vaccination against exotic diseases susceptible to H1 and H3 swine infl uenza viruses such as foot-and-mouth disease and rinderpest in 430 Avian Influenza

Ring Vaccination Surveillance or Buffer Zone

Quarantine or InfectedQ Zone Suppressor Vaccination

Figure 19.2. Concepts for application of AI vaccine in the ﬁ eld during an emergency vaccination program.

livestock has utilized a ring vaccination strategy— tion by placing vaccinated birds back into the envi- vaccination of a control or surveillance zone around ronment when there is still a risk for pockets of the outbreak, which is based on the epidemiological residual virus in environments that cannot be prop- concept of short distance movement of infected erly cleaned or disinfected or in areas where unde- large livestock (e.g., cattle and swine) and fomites tected, low infection rates may still be present in containing the virus (Fig. 19.2). Ring vaccination inaccessible village poultry. Repopulation with vac- has been proposed for HPAI control as an emer- cinated poultry should decrease the potential for AI gency vaccination program, but such a strategy may virus resurgence. Similar vaccination strategies can not always be effective because of easy and long be used with LPAI control. In the case of LPAI, distance movement of small poultry to and from live vaccination was practiced on several affected egg- market systems, especially in developing countries laying chicken farms containing multiple large where the small, compact birds are easily and com- houses that experienced H7N2 LPAI virus infec- monly moved in small crates on motor bikes over tions in Connecticut, United States, during 2003– great distances between farms and markets and 2005 (223). In this situation, the LPAI virus did not back. Another strategy would be as a “suppressor” produce consistent infection of all chickens within vaccination within the HPAI outbreak zone for non- the large houses and vaccination was implemented infected fl ocks, to increase resistance of fl ocks to AI to raise the immunity of all chickens to a consistent virus infection, and in infected fl ocks, to induce con- and protective level that allowed safe production and sistent high immune level with the goal of stopping marketing of table eggs from the fl ocks. After 18 AI virus shedding and transmission (Fig. 19.2). This months, all chickens on the affected farms had been latter use can also have the benefi t of stopping the replaced and no H7N2 LPAI virus was detected in AI virus spread within a premises or barn, even on surveillance (223). infected premises, as was demonstrated with fi eld Vaccine application within a geographic area vaccination experiments against H5N2 HPAI virus could be applied as a ring around the outbreak zone, in Pennsylvania during 1983 and H5N1 HPAI virus as blanket vaccination in a large endemic infected in Hong Kong during 2002 (80, 81). Alternatively, region, or in the case of a small high-risk area, tar- suppressor vaccination can be used during repopula- geted vaccination as defi ned by geography, compart- 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 431 ment, or poultry species. Recently, a detailed parent breeders in endemic areas; and, last (5) meat strategic plan for AI vaccination was proposed and production poultry. includes the following categories: (1) routine vaccination in endemic areas (2) emergency vaccination Biosecurity during Vaccination during an epidemic, and (3) preventative or prophy- Any people, equipment, and supply movement on or lactic vaccination when the risk of AI virus introduc- off a farm create a risk for introducing or spreading tion is high (141). In an emergency vaccination AI virus between premises. During vaccination cam- program, an exit strategy must be developed and paigns, the highest level of biosecurity must be prac- implemented as part of the eradication strategy; oth- ticed to reduce the risk of spreading fi eld virus by erwise, the vaccination program will quickly become vaccination crews as they move from premises to routine with acceptance of the AI infections and the premises. Such a danger was reported in the eradica- disease as endemic. tion efforts of the 1970s during the California New- In the laboratory setting, a single vaccination castle disease epidemic. The vaccination crews were typically produces protection from virulent chal- epidemiologically linked to prolonging the outbreak lenge. However, in the fi eld, a single vaccination is by spreading the fi eld virus from farm to farm (258). not suffi cient for all poultry species, all production However, any kind of traffi c on and off the farm systems, and all ages of birds. For example, immu- such as visitors (family, relative, friends, etc.), utility nity induced by a single vaccination in chickens with crews, catching crews, feed trucks, etc., carry a risk an inactivated AI vaccine lasted twice as long as the of spreading the AI virus (98). same vaccine given to turkeys (161). A single vaccination may provide protection for short-lived meat Avian Infl uenza Vaccine Use in the Field chickens under low dose challenge, but meat turkeys, long-lived chickens (layers and breeders), ducks, Licensed Avian Infl uenza Vaccines and geese may need two or more vaccinations to A variety of vaccine technologies and virus strains produce long-lived protective immunity. Little expe- have been licensed and used in the fi eld. In the rience is available on vaccines in chukar partridges, United States, only two AI vaccine technologies quail, pheasants, guinea fowl, ratites, and zoo birds. have been licensed: inactivated whole AI virus vac- Vaccination protocols must be developed that blend cines and an rFP-AIV-H5 vaccine with the H5 gene fi eld experience with laboratory data to produce a insert obtained from A/turkey/Ireland/83 (H5N8) viable program that will ensure adequate vaccina- (232). A variety of HA subtype vaccines have been tion of birds resulting in both individual and popula- licensed under autogenous, conditional, and full tion immunity. Such protocols could include licensure as inactivated AI vaccines. However, the inactivated as well as recombinant live vectored AI fi eld application of the licensed H5 and H7 vaccines vaccines. requires approval of both the state and federal governments, but other subtypes may only require Priorities for Vaccination approval of the state government when used in the In designing a vaccination program with limited species listed in the product license restrictions. His- resources, the sectors and species for AI vaccine use torically, vaccine has not been used in the United should be based on value and risk analysis. A simple States in HPAI epidemics but contingency plans for algorithm for AI vaccine use, in decreasing order of potential future outbreaks could allow use. Vaccines application, should be as follows: (1) poultry and against LPAI viruses have only been of limited use other birds in high risk compartments, sectors or in the United States. The rFP-AIV-H5 is also situations during an outbreak using a suppressor licensed and used in Mexico, El Salvador, and Gua- vaccine when immediate stamping-out is not feasi- temala. In Mexico, an rNDV-AIV-H5 vaccine is ble; (2) rare captive birds such as in zoological col- licensed (134) lections as a preventative when risk for introduction Globally, the majority of licensed AI vaccines are is high; (3) valuable genetic poultry stock such as inactivated whole AI virus vaccines, principally of pure lines or grandparent stocks whose individual the H5, H7, and H9 subtypes. For the current H5N1 value is high and there is a high risk for AI introduc- HPAI epidemic, licensed inactivated AI vaccines tion; (4) long-lived poultry, such as egg layers or used in Asia, Africa, and Europe have utilized the 432 Avian Influenza following seed strains from previous AI outbreaks: Historically, vaccines have been used most fre- A/turkey/England/N28/73 (H5N2) LPAI virus, A/ quently to control and eradicate LPAI viruses in chicken/Mexico/232/94 (H5N2) LPAI virus, A/ defi ned, small geographic areas of high risk such as chicken/Legok-Indonesia/03 (H5N1) HPAI virus, the emergency vaccination of Minnesota turkeys to A/duck/Potsdam/1402/86 (H5N2) LPAI virus, A/ control wild duck–origin LPAI viruses; between chicken/Italy/22A/98 (H5N9) LPAI virus, and A/ 1978 and 1996, only 22 million doses of inactivated turkey/Wisconsin/68 (H5N9) LPAI virus (34, 233, AI vaccines were used (96). As a response to the 242). In addition, three different reverse genetic pro- continuing risk, the Minnesota turkey industry elim- duced infectious clone seed strains have been devel- inated outdoor production in 1997, which reduced oped using the six internal genes from PR8 infl uenza the risk of infection with wild duck–origin infl uenza A vaccine strain and the following HA and NA viruses, and vaccine use against wild duck–origin genes: (1) H5 and N1 genes of A/goose/Guang- LPAI viruses has been eliminated (96, 99). In Utah, dong/96 (H5N1) (251), (2) H5 gene from A/chicken/ United States, during 1995, an outbreak of H7N3 Vietnam/c58/04 (H5N1) and N3 gene from A/Duck/ LPAI was eradicated by use of 2.03 million doses of Germany/1215/73 (H2N3) (273), and (3) an H5 and inactivated vaccine over 4 months along with other N1 genes from a recent H5N1 HPAI virus from components in the control program (85). In Califor- Shanxi, China (282). Two rFP-AIV-H5 vaccines nia turkey breeders, 2.3 million doses of inactivated have been licensed for use in Asia and contain AI AI vaccine was used between 1979 and 1985 (146). virus gene inserts as follows: (1) H5 gene from A/ Another example of emergency and preventative turkey/Ireland/83, and (2) H5 and N1 genes from vaccination has been in northern Italy, where A/goose/Guangdong/96. In both situations, the HA 202,140,000 doses of AI vaccine were used from gene was altered from an HPAI virus to an LPAI November 2002 to December 2006, mostly in laying virus. Finally, rNDV-AIV-H5 vaccine containing hens and meat turkeys but some in capons, guinea H5 gene insert from A/bar-headed goose/ fowls, and cockerels. Most of the AI vaccine was Qinghai/3/2005 (H5N1) has been licensed and used inactivated H7 used in an emergency vaccination in China (92). Requirements for licensing AI vac- campaign against LPAI, but some vaccine was biva- cines will vary with each country depending on spe- lent H5 and H7 used during 2005 and 2006 for a cifi c requirements of the national veterinary preventative LPAI campaign (S. Marangon, per- biologics authority in areas of safety, purity, potency, sonal communication, April 7, 2007). As an example and label approval for species, and age and route of of routine infl uenza vaccination, SIV causes infec- administration. tions and economic losses in turkey breeders located in specifi c states within the United States where pigs Field Usage of Avian Infl uenza Vaccine and turkeys are raised within the same geographic The quantity of AI vaccine manufactured and used areas. For example, 2.6 million doses of inactivated in poultry prior to the past two decades has been H1 infl uenza A vaccine was used in turkey breeders poorly documented, but global usage has been low in the United States during 2001 to protect against until the mid 1990s and manufacturing expanded SIV (229). However, the largest use of AI vaccine geometrically early within the fi rst decade of 21st against LPAI has been inactivated H9N2 vaccines century. For H5 and H7 vaccines, a survey in early in the Middle East and Asia, where, since the late 2002 identifi ed only two manufacturers in OIE 1990s, billions of doses have been used in layers, member countries that responded to the survey, but broilers, and other poultry. by 2006 and 2007, the OIE listed 38 manufacturers The use of AI vaccines for control of HPAI was of AI vaccines, and Food and Agriculture Organiza- fi rst reported in 1995 in Mexico during the H5N2 tion (FAO) listed 41 AI vaccines for use against H5 HPAI epidemic and in Pakistan during the H7N3 and H7 HPAI available from China, France, HPAI epidemic. The H5N2 HPAI virus was eradi- Germany, Italy, Mexico, Netherlands, Pakistan, and cated from Mexico during 1995, but the precursor United States (143, 165, 190). However, vaccines H5N2 LPAI virus continues to cause infections in for other HA subtypes are also produced globally poultry and has expanded into Guatemala and El such as H9N2 AI vaccines, but a compiled list for Salvador. From 1995 to 2006, 1.8 billion doses of such vaccines is not available. H5N2 inactivated vaccine and over 2 billion doses of 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 433 rFP-AIV-H5 vaccine have been used in chickens (36, and/or serological methods, depending upon the 233). In Pakistan, inactivated H7N3 vaccine was used goal and specifi c needs of the AI control program. in 1995 with expanded usage when H7N3 LPAI occurred in another region of Pakistan during 2001 Assessment of Immunity and H7N3 HPAI virus in Southern Pakistan during Humoral immunity produced against the HA protein, 2004 (158, 159). Vaccination against H9N2 was as measured in HI or virus neutralization tests, pro- implemented in 1998. In 2006 with outbreaks of vides a predictive measure of protection in the indi- H5N1 HPAI, Pakistan began using a trivalent inacti- vidual bird and, when evaluated collectively, the vated AI vaccine containing H5, H7, and H9 sub- immunity within the fl ock or population. When types. The emergence of H5N1 HPAI in Asia with using a standardized test method, serological results spread to Africa has led to emergency, routine, and can be compared with in vivo protection studies to preventative AI vaccination beginning with inacti- establish minimal protective serological titers and vated H5N2 AI vaccine usage in Hong Kong poultry determine the point at which booster vaccinations (2002), followed by vaccination implemented in should be administered to achieve optimal second- Indonesia (2003), China (2004), Vietnam (2005), ary or booster immune response. However, the Russia (2005), India (2006), Pakistan (2006), and establishment of a single minimal protective titer for Egypt (2006), which continues today (141, 233). The all bird species based on HI results is not possible greatest quantity of vaccine used has been in China, because HI titers vary depending on HI test proce- where from January 2004 through December 2006, dure, species and breed of bird, the length of time 22.7 billion doses of H5 AI vaccine were used, mostly measured post vaccination, and the type of vaccine inactivated H5 AI vaccine (19.5 billion doses), but and the adjuvants used. For example, one inactivated most recently, 608 million doses of rFP-AIV-H5N1 AI vaccine study in SPF laying chickens identifi ed vaccine, and 2.6 billion doses of rND-AIV-H5 HI geometric mean titers (GMT) of 10 to 40 were vaccine (282). In 2005, North Korea used inactivated associated with prevention of mortality, but some H7 AI virus vaccine against H7N7 HPAI virus. birds still had challenge HPAI virus replication and In addition to poultry, some inactivated vaccine shedding from oropharynx and cloaca, while titers has been used in European zoos as a means to protect of 80 or greater were associated with protection for valuable bird collections against H5 and H7 HPAI mortality and prevention of replication and shedding virus during high-risk periods of 2003 (H7N7 HPAI of challenge HPAI virus (123). In another study, a virus, the Netherlands) and 2006 (H5N1 HPAI virus, mean HI titer of 120 gave SPF chickens 90% to multiple European Union countries). These fi eld 100% protection from mortality and most did not trials used commercial poultry H5 and H7 AI vac- shed challenge HPAI virus or shed the HPAI virus cines administered in a two-dose regimen at zoos in in low quantities (242). However, with inactivated the Netherlands, Denmark, France, and Sweden (24, AI vaccines in SPF chickens, minimal GMT of 40 173, 174). No challenge studies were done, but pro- or greater are achievable, but most potent vaccines tective HI serological responses of 32 or greater achieve a minimal HI titer of greater than 120 fol- were reported for 76%, 80.5%, and 81.5% of the lowing a single vaccination, which provides protec- birds in the three studies. The serological response tion from mortality and greatly reduces challenge of birds from different orders varied between the virus shedding. Protective HI titers are usually lower individual reports. Total vaccine usage was low, in commercial broilers and breeders, turkeys, ducks, based on the approximately 7000 birds vaccinated and geese than in SPF laying chickens. In some (24, 68, 173, 174, 200). species of birds, a minimum of two vaccinations may be necessary to produce protective HI titers or SURVEILLANCE even for long-lived chickens such as breeders and Vaccinated fl ocks must be monitored to determine layers. if the vaccination has produced adequate fl ock immunity, to determine when immunity has declined Identifi cation of Infected Animals Within a suffi ciently to warrant a booster vaccination, and to Vaccinated Population determine if the fl ock has been infected by fi eld AI Vaccinated fl ocks should be monitored for AI virus virus. Such monitoring can be done with virological infection in order to assess the success of the 434 Avian Influenza

Vaccinated Flocks

Non-vaccinated Vaccinated Sentinels Birds

Virological Exam: Standard Serology: Dead or Ill Birds Special Serology: NP/M (AGID, ELISA), • Inactivated AI Vaccine HA (HI), NA (NI, (Heterologous NA, ELISA), NS1 NS1) VI, RT-PCR, • Vectored AI Vaccine RRT-PCR, (NP/M, NS1, NA) antigen capture

Figure 19.3. Flow diagram for virological and serological surveillance of vaccinated ﬂ ocks for detection of AI virus infection (i.e., DIVA strategies).

vaccination program and to identify infections in individuals were incorrectly vaccinated such as the vaccinated fl ocks that should be eliminated (i.e., vaccine was not deposited in the correct site, or for DIVA). Conceptually, the DIVA principle of moni- other reasons individual birds did not develop immu- toring can be accomplished by a variety of viro- nity. In HPAI-affected areas, mortality in highly logical and/or serological surveillance methods susceptible individual birds such as chickens or when using sensitive and appropriate tests based on other gallinaceous poultry serves a syndromic sur- adequate sample types and numbers of samples (Fig. veillance role as an early warning clinical system or 19.3) (223). Basic principles for such AI surveil- “biosensor” for quick identifi cation of potential lance are available from OIE (164). cases to be tested for AI virus. However, with LPAI virus, the infection will not consistently produce Virological Surveillance mortality and testing ill birds should also be included The most direct and accurate means to identify AI in syndromic surveillance. The use of mortality in virus infection is through the detection of the virus vaccinated fl ocks as biosensors has been most clearly within the vaccinated fl ock or population. The sim- demonstrated in experimental studies as follows: (1) plest and most sensitive surveillance system is mon- vaccinated birds that did not develop immunity (as itoring a highly susceptible subpopulation within the evident by lack of HI antibodies) were susceptible vaccinated population because the majority of vac- to infection and died following exposure to HPAI cinated birds will be immune and thus resistant to viruses (242), and (2) nonvaccinated birds placed in AI virus infection, thus making most vaccinated contact with intranasally challenged, vaccinated birds poor for attempting virus detection. Therefore, birds served as sentinels with detection of HPAI the virological surveillance program should empha- virus excretion (Table 19.3) (34). In the 2003–2005 size targeted, clinical or syndromic surveillance of Connecticut H7N2 LPAI vaccination program, suc- highly susceptible individuals within the vaccinated cessful surveillance for evidence of infection was population as follows: (1) permanently marked or conducted through combination of virological testing identifi ed, nonvaccinated sentinels that originated of daily mortality in vaccinated birds and serological from an external biosecure source or were obtained testing of sentinels (223). from the source fl ock, but intentionally left unvac- However, virological surveillance should only be cinated; or (2) a subpopulation of unmarked birds done if adequate and appropriate samples are col- that did not develop protective immunity because lected from the correct bird subpopulations and individuals were missed during the catching process, tested using the correct laboratory methodology. For 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 435

Table 19.5. Positive serological results against specifi c AI viral proteins following AI virus infection or vaccination with different type of AI vaccines. Serological test results against specifi c avian infl uenza viral proteins Homologous Heterologous Group NP/M HA fi eld virus NA vaccine NA NS1

Avian infl uenza fi eld virus ++ + Na + infection Inactivated avian infl uenza ++ + Na − vaccine: homologous NA Inactivated avian infl uenza ++ − + − vaccine: heterologous NA Vectored or DNA HA vaccine −+ − − − Nonvaccinated sentinels −− − − − a Not applicable. NP/M, nucleoprotein and matrix protein; HA, hemagglutinin; NA, neuraminidase; NS1, nonstructural 1 protein. example, sampling ill or dead birds, which typically sion risk. A variety of serological surveillance excrete high levels of AI virus from oropharyngeal systems have been described, each having specifi c and/or cloacal, have shown most consistent detec- advantages and disadvantages, but no one system tion of both HPAI and LPAI virus by using virus will work for all vaccines and all fi eld situations. All isolation, RRT-PCR, or antigen detection by various serological surveillance systems are based on dif- antigen capture ELISA test methods (69, 202). ferential production of antibodies against various AI However, sampling of preclinical infected birds, virus proteins following AI virus infection as com- which shed low levels of virus, will only detect virus pared to vaccination (Table 19.5). if using the most sensitive methods (i.e., virus isola- The primary challenge with serological surveil- tion and RRT-PCR). The use of antigen capture tests lance is identifi cation of antibodies in AI-vaccinated have shown a 79% to 80% sensitivity and 99% to birds that differ from those in infected birds. Anti- 100% specifi city in detecting virus in samples from bodies against NP/M proteins, detected by AGP or ill or dead LPAI virus–infected chickens (71, 72, ELISA tests, are present in all birds that are infected 202, 283). The RRT-PCR test had 95% sensitivity with any HA subtype of AI virus and cannot be used and 99% specifi city for the same data sets. There- as a differential test to distinguish infected from fore, to increase the sensitivity of detecting infected inactivated whole AI virus–vaccinated birds. birds, collection and testing of the daily mortality or However, several systems can allow identifi cation visibly sick birds should be used for virological of AI virus infections within a vaccinated population monitoring of vaccinated populations for possible of birds. AI virus infections and the most sensitive virological tests should be used. Sentinel birds Since the late 1970s in Minnesota, sentinel birds Serological Surveillance have been used successfully for serological detec- Serological monitoring provides a historical view of tion of LPAI virus infections within inactivated AI infection within the fl ock, which can primarily be virus–vaccinated turkey fl ocks (97). This sentinel used to assess the success of the vaccination program, system has been expanded for use in other outbreaks and cannot be used to identify actively infected of LPAI in turkeys and chickens in the United States fl ocks. Virological surveillance is needed to deter- and elsewhere in the world (85, 142, 202). The mine if the fl ock is currently infected and what risk, detection of antibodies against any of the 10 AI viral if any, the fl ock may have as a reservoir or transmis- proteins is indicative of infection and sentinels can 436 Avian Influenza be used as a mitigation step to detect “silent infec- subsequent years against other H5 and H7 LPAI tions” within AI-vaccinated fl ocks (194) (Table viruses (41, 43–45, 47, 53, 54). Basically, the AI 19.5). However, the sentinels must be permanently vaccine has the same HA subtype but a different NA marked or identifi ed in order to properly manage the than the fi eld AI virus, thus allowing development surveillance system for the following reasons: (1) to of protective HA antibodies, but the vaccinated birds allow easy observation of susceptible birds within would not have antibodies against the fi eld NA the fl ock for syndrome surveillance; i.e., observation unless they were also infected (Table 19.5). However, of clinical signs or death that would trigger an AI to use this strategy as a preventative vaccination investigation (2) to allow easy access to birds for program, a preexisting vaccine bank is needed with quick collection of appropriate samples to use in a minimum of two different NA subtypes for each serological or virological testing, and (3) to prevent HA subtype vaccine, or the vaccine should have a unethical and unlawful tampering with sentinels by rare NA subtype such as N5 (20). Vaccine strains unscrupulous individuals with intent to alter test can be LPAI outbreak viruses, can be generated by results through replacing offi cial sentinel birds with classic reassortment techniques (20, 227), or can be other birds. Typically, the state/provincial or federal produced by an infectious clone system (126). Mul- government should manage the sentinel bird tiple inactivated AI vaccines for poultry and humans program. During the 2000–2002 H7N1 LPAI out- have been developed using the H5 HA from current breaks in Italy, 1% of birds in vaccinated fl ocks were fi eld isolates but engineered to contain various NA maintained as nonvaccinated sentinels with a (e.g., N9) subtypes (74, 126, 129, 133, 145, 226, minimum of 10 birds per barn were bled every 45 251, 273). In the United States, U.S. Department of days for serological determination of infection (45). Agriculture–Animal and Plant Health Inspection The birds should be identifi ed by a tamper-proof, Service (USDA/APHIS) AI vaccine banks for visual or electronic tag system which uniquely iden- poultry typically contain vaccines against only H5 tifi es each sentinel. Management of sentinels is and H7 subtypes. The heterologous NA system easiest for cage-housed birds such as laying chick- should not be viewed as the only DIVA system. ens because the birds can be individually identifi ed Virological and other serological surveillance and they can be placed within tagged cages that can systems can and have been used successfully as be physically mapped for quick, repeated observa- DIVA strategies. tion. Floor-reared birds are more diffi cult to manage Many inactivated AI vaccines use strains that and may require development and use of some cre- contain an NA subtype homologous to the fi eld ative visual identifi cation in addition to the perma- virus; thus, antibodies against the homologous NA nent number tagged system such as confi ning the cannot be used to identify infection. However, anti- sentinels in several fl oor cages spread throughout the bodies against the NS1 protein have been proposed house or, with free-roaming birds, quick visual iden- as a differentiation test because NS1 protein is only tifi cation by using large, bright-colored wing tags, produced in the infected cell and is not packaged in painting the feathers of white birds, or using breeds the AI virus. In two separate studies, antibodies as sentinels that differ in color from the vaccinated against NS1 were not seen in chickens or turkeys birds. immunized with experimental or commercial inactivated AI vaccines unless they had also been infected Vaccinated birds with an AI virus (257, 281). The majority of AI vaccines used in poultry are A simpler surveillance strategy for detecting inactivated AI vaccines. Because protection is HA infection in AI-vaccinated birds is through the use subtype specifi c, the inactivated AI vaccine does not of AI vaccines generated by biotechnology that require a NA subtype match between vaccine and contain only the HA protein such as in vitro or in fi eld virus to be protective (15). In the mid-1980s vivo expressed HA protein, or an HA DNA vaccine, and 1990s, serological detection of heterologous NA accompanied by use of standard serological tests. was proposed as a method to identify infected among These AI-vaccinated birds have protective antibod- vaccinated birds (15, 77, 214). This concept was ies only against the specifi c HA protein in the developed and fi rst used successfully during an out- vaccine, but lack antibodies against the other AI break of H7N1 LPAI in Italy during 2000 and in viral proteins. Thus, the AI-vaccinated birds will be 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 437 positive for antibodies on the specifi c HI test, but Several issues are important for fi eld use of AI negative for antibodies on the standard AGP or com- vaccines: (1) AI vaccine use should be part of a mercial ELISA tests used around the world unless comprehensive total AI control program; (2) the the birds have been infected. For example, chickens vaccine strain must be of the same HA subtype and vaccinated with rFP-AIV-H5 vaccine at 1 day of age should be protective in the target species based on had antibodies at 3 weeks of age against the homo- in vivo studies against a recent circulating fi eld logous HA but were negative for antibodies against virus; (3) the vaccine should have suffi cient HA NP/M as measured by AGP test (235). Following AI content (inactivated or in vitro expressed HA system virus infection, some chickens developed antibodies vaccine with minimum titer of 1 to 5 μg HA/dose) against NP/M as measured by AGP tests. All sero- or adequate live virus titer (vectored vaccines) suf- logical DIVA strategies in vaccinated birds should fi cient to produce a protective immune response, or be interpreted as a fl ock and not individual bird tests suffi cient HI serological titers are demonstrated to because proper vaccination will increase resistance indicate protection; (4) inactivated or in vitro to infection in the majority of birds. expressed HA system vaccines should be emulsifi ed within a good oil adjuvant system; (5) manufactur- CONCLUSIONS ing of AI vaccines must be standardized in order to AI vaccines provide protection to birds, principally produce consistent and effi cacious vaccine batches; through mucosal and systemic humoral immunity (6) procedures must be established for proper against the HA protein, and such protection is HA storage, distribution, and administration of the subtype specifi c. Protection can be directly assessed vaccine; (7) establishment of biosecurity practices by prevention of clinical signs and death, a decrease to prevent vaccination crews or other service person- in number of birds infected, reduction in the quantity nel from accidental spreading of fi eld virus; (8) of challenge virus shed from respiratory and alimen- proper serological or virological surveillance tary tracts, and prevention of contact transmission in systems must be in place to determine if vaccination in vivo experimental studies. Protection can also be has produced protective immunity and to monitor assessed indirectly through measurement of protec- vaccinated populations for possible fi eld virus circu- tive antibody levels in vaccinated birds or assaying lation (i.e., DIVA); and (9) an exit strategy from the quantity of HA protein in the vaccine. AI vac- emergency vaccine use should be developed to cines should be suffi ciently potent to protect birds prevent vaccination from becoming a routine under a variety of fi eld conditions. program with associated AI virus endemicity. AI AI vaccines are based on four technology plat- vaccines must be periodically reevaluated to deter- forms: (1) inactivated whole AI virus vaccines; (2) mine if they are still effective against circulating in vitro expressed HA protein; (3) in vivo expressed fi eld virus strains and if no longer protective, vaccine HA protein in vectored systems; and (4) HA-based strains should be replaced. Requirements for licens- DNA vaccine. However, only inactivated AI virus, ing AI vaccines will vary with each country depend- rFP-AIV-H5 and rND-AIV-H5 vaccines are curing on specifi c requirements of the national veterinary rently licensed and used in the fi eld in various coun- biologics authority in areas of safety, purity, potency, tries around the world. Historically, inactivated AI and label approval for species, and age and route of virus vaccines have used seed virus strains predom- administration. inantly based on LPAI outbreak viruses and, occasionally, HPAI outbreak viruses. Such seed strains REFERENCES have been broadly protective within an HA subtype 1. Abraham, A., V. Sivanandan, D. Karunakaran, and requiring less frequent change of seed strains D.A. Halvorson, and J.A. Newman. 1988. Comparative serological evaluation of avian than has been necessary for human infl uenza A vac- infl uenza vaccine in turkeys. Avian Diseases cines. In the past 5 years, some seed strains have 32:659–662. been produced by reverse genetics utilizing the HA 2. Ada, G.L., and P.D. Jones. 1986. The immune and NA genes from an outbreak virus and the six response to infl uenza infection. Current Topics in internal genes of a high-growth infl uenza A vaccine Microbiology and Immunology 128:1–54. strain. AI vaccines have been used in emergency, 3. Albini, B., G. Wick, E. Rose, and E. Orlans. 1974. routine, or preventative vaccination programs. Immunoglobulin production in chicken Harderian 438 Avian Influenza

glands. International Archives of Allergy and 14. Beard, C.W. 1981. Turkey infl uenza vaccination Immunology 47(1):23–34. [letter]. Veterinary Record 108:545. 4. Alexander, D.J., and G. Parsons. 1980. Protection 15. Beard, C.W. 1987. To vaccinate or not to vacci- of chickens against challenge with virulent infl u- nate. In: B.C. Easterday and C.W. Beard (eds.). enza A viruses of Hav5 subtype conferred by Proceedings of the Second International Sympo- prior infection with infl uenza A viruses of Hsw1 sium on Avian Infl uenza, Georgia Center for subtype. Archives of Virology 66:265–269. Continuing Education, the University of Georgia, 5. Allan, W.H., C.R. Madeley, and A.P. Kendal. Athens, Georgia, USA, September 3–5, 1986. 1971. Studies with avian infl uenza A viruses: Symposium on Avian Infl uenza, US Animal cross protection experiments in chickens. Journal Health Association: Richmond, VA, pp. 258– of General Virology 12:79–84. 263. 6. Allison, A.C., and N.E. Byars. 1992. Syntex adju- 16. Beard, C.W. 1992. The role of vaccines and vac- vant formulation. Research in Immunology cination. In: B.C. Easterday (ed.). Proceedings of 143(5):519–525. the Third International Symposium on Avian 7. Altstein, A.D., A.K. Gitelman, Y.A. Smirnov, Infl uenza, May 27–29, 1992, Madison, Wiscon- L.M. Piskareva, L.G. Zakharova, G.V. Pashvykina, sin. Symposium on Avian Infl uenza, US Animal M.M. Shmarov, O.P. Zhirnov, N.P. Varich, P.O. Health Association: Richmond, VA, pp. 409– Ilyinskii, and A.M. Shneider. 2006. Immuniza- 420. tion with infl uenza A NP-expressing vaccinia 17. Beard, C.W., and B.C. Easterday. 1973. A- virus recombinant protects mice against experi- Turkey-Oregon-71, an avirulent infl uenza isolate mental infection with human and avian infl u- with the hemagglutinin of fowl plague virus. enza viruses. Archives of Virology 151(5):921– Avian Diseases 17:173–181. 931. 18. Beard, C.W., W.M. Schnitzlein, and D.N. Tripa- 8. Anderson, R.W., J.R. Bennink, J.W. Yewdell, thy. 1991. Protection of chickens against highly W.L. Maloy, and J.E. Coligan. 1992. Infl uenza pathogenic avian infl uenza virus (H5N2) by basic polymerase 2 peptides are recognized by recombinant fowlpox viruses. Avian Diseases infl uenza nucleoprotein-specifi c cytotoxic T lym- 35:356–359. phocytes. Molecular Immunology 29(9):1089– 19. Beard, C.W., W.M. Schnitzlein, and D.N. Tripa- 1096. thy. 1992. Effect of route of administration on the 9. Andrew, M.E., B.E. Coupar, G.L. Ada, and D.B. effi cacy of a recombinant fowlpox virus against Boyle. 1986. Cell-mediated immune responses to H5N2 avian infl uenza. Avian Diseases 36:1052– infl uenza virus antigens expressed by vaccinia 1055. virus recombinants. Microbial Pathogenesis 20. Beato, M.S., M. Rigoni, A. Milani, and I. Capua. 1(5):443–452. 2007. Generation of avian infl uenza reassortant 10. Aucouturier, J., L. Dupuis, and V. Ganne. 2001. viruses of the H7N5 subtype as potential vaccine Adjuvants designed for veterinary and human candidates to be used in the framework of a vaccines. Vaccine 19(17–19):2666–2672. “DIVA” vaccination strategy. Avian Dis 51(1 11. Bang, B.G., and F.B. Bang. 1968. Localized lym- Suppl.):479–480. phoid tissues and plasma cells in paraocular and 21. Beato, M.S., A. Toffan, R. De Nardi, A. Cristalli, paranasal organ systems in chickens. American C. Terregino, G. Cattoli, and I. Capua. 2007. A Journal of Pathology 53(5):735–751. conventional, inactivated oil emulsion vaccine 12. Barun, K., M.W. Shaw, P.A. Rota, M.W. Harmon, suppresses shedding and prevents viral meat col- J.J. Esposito, R. Rott, N.J. Cox, A.P. Kendal, and onisation in commercial (Pekin) ducks challenged K. De-Barun. 1988. Protection against virulent with HPAI H5N1. Vaccine 25(20):4064–4072. H5 avian infl uenza virus infection in chickens by 22. Beaudette, F.R., C.B. Hudson, and A.H. Saxe. an inactivated vaccine produced with recombi- 1932. An outbreak of fowl plague in New Jersey nant vaccinia virus. Vaccine 6:257–261. in 1929. Journal of Agriculture Research 49:83– 13. Beard, C.W. 1981. Immunization approaches to 92. avian infl uenza. In: R.W. Bankowski (ed.). Pro- 23. Beck, I., H. Gerlach, E. Burkhardt, and E.F. ceedings of the First International Symposium Kaleta. 2003. Investigation of several selected on Avian Infl uenza, April 1981, Beltsville, adjuvants regarding their effi cacy and side effects Maryland. Symposium on Avian Infl uenza, US for the production of a vaccine for parakeets to Animal Health Association: Richmond, VA, pp. prevent a disease caused by a paramyxovirus type 172–177. 3. Vaccine 21(9–10):1006–1022. 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 439

24. Bertelsen, M.F., J. Klausen, E. Holm, C. Grondahl, USA, September 3–5, 1986. Symposium on and P.H. Jorgensen. 2007. Serological response Avian Infl uenza, US Animal Health Association: to vaccination against avian infl uenza in zoo- Richmond, VA, pp. 283–292. birds using an inactivated H5N9 vaccine. Vaccine 34. Bublot, M., F.X. Le Gros, D. Nieddu, N. Pritchard, 25(22):4345–4349. T.R. Mickle, and D.E. Swayne. 2007. Effi cacy of 25. Bienenstock, J., A.D. Befus, M. McDermott, S. two H5N9-inactivated vaccines against challenge Mirski, K. Rosenthal, and A. Tagliabue. 1983. with a recent H5N1 highly pathogenic avian The mucosal immunological network: compart- infl uenza isolate from a chicken in Thailand. mentalization of lymphocytes, natural killer cells, Avian Diseases 51:332–337. and mast cells. Annals of the New York Academy 35. Bublot, M., N. Pritchard, J.S. Cruz, T.R. Mickle, of Sciences 409:164–170. P. Selleck, and D.E. Swayne. 2007. Effi cacy of a 26. Bouma, A., T. Tiensin, I. Claassen, M. Nielen, M. fowlpox-vectored avian infl uenza H5 vaccine van Boven, and J.A. Stegeman. 2007. Estimation against Asian H5N1 highly pathogenic avian of the critical proportion of chickens in a fl ock to infl uenza virus challenge. Avian Diseases 51: be immunized to prevent major outbreaks of 498–500. HPAI H5N1. Abstracts of OIE/FAO conference 36. Bublot, M., N. Pritchard, D.E. Swayne, P. Selleck, on vaccination: a tool for the control of avian K. Karaca, D.L. Suarez, J.C. Audonnet, and T.R. infl uenza 20–22 March 2007, Verona, Italy, Mickle. 2006. Development and use of fowlpox p. 60. vectored vaccines for avian infl uenza. Annals of 27. Bourlet, Y., G. Behar, F. Guillemot, N. Frechin, the New York Academy of Sciences 1081:193– A. Billault, A.M. Chausse, R. Zoorob, and C. 201. Auffray. 1988. Isolation of chicken major histo- 37. Bublot, M., D.E. Swayne, P. Selleck, E. Montiel, compatibility complex class II (B-L) b chain N. Pritchard, M. Lee, and T.R. Mickle. 2006. sequences: comparison with mammalian b chains TROVAC AI H5, an avian infl uenza fowlpox and expression in lymphoid organs. EMBO vector vaccine, as an alternative vaccine for Journal 7(4):1031–1039. hatcheries. Developments in Biologicals 124:248– 28. Bowie, A.G., and I.R. Haga. 2005. The role of 249. Toll-like receptors in the host response to viruses. 38. Burns, R.B. 1982. Histology and immunology of Molecular Immunology 42(8):859–867. Peyer’s patches in the domestic fowl (Gallus 29. Boyle, D.B., and B.E.H. Coupar. 1988. Construc- domesticus). Research in Veterinary Science tion of recombinant fowlpox viruses as vectors 32(3):359–367. for poultry vaccines. Virus Research 10:343–356. 39. Butterfi eld, W.K., and C.H. Campbell. 1978. 30. Boyle, D.B., P. Selleck, and H.G. Heine. 2000. Vaccination for fowl plague. American Journal of Vaccinating chickens against avian infl uenza Veterinary Research 39(4):671–674. with fowlpox recombinants expressing the H7 40. Calderon, H.M.L., V.H. Rodriguez, L.D. Garcia, haemagglutinin. Australian Veterinary Journal M.R. Palacios, R.T. Nickle, N.E.H. Montiel, G. 78(1):44–48. Tinoco, and G.J. Garcia. 2007. Interference of 31. Brown, D.W., Y. Kawaoka, R.G. Webster, and passive immunity with the effi ciency of a recom- H.L. Robinson. 1992. Assessment of retrovirus- bining vaccine on the fi rst day of life to prevent expressed nucleoprotein as a vaccine against bird fl u. In: Proceedings of the XXII ANECA lethal infl uenza virus infections of chickens. Annual Congress, pp. 245–252. Avian Diseases 36:515–520. 41. Capua, I., S. Marangon, P. Dalla, and U. Santucci. 32. Brugh, M., C.W. Beard, and H.D. Stone. 1979. 2000. Vaccination for avian infl uenza in Italy. Immunization of chickens and turkeys against Veterinary Record 147(26):751. avian infl uenza with monovalent and polyvalent 42. Capua, I., and D.J. Alexander. 2004. Avian infl u- oil emulsion vaccines. American Journal of enza: recent developments. Avian Pathology Veterinary Research 40:165–169. 33:393–404. 33. Brugh, M., and H.D. Stone. 1987. Immunization 43. Capua, I., G. Cattoli, and S. Marangon. 2004. of chickens against infl uenza with hemagglutinin- DIVA—a vaccination strategy enabling the detec- specifi c (H5) oil emulsion vaccine. In: B.C. tion of fi eld exposure to avian infl uenza. Devel- Easterday and C.W. Beard (eds.). Proceedings of opments in Biologicals 119:229–233. the Second International Symposium on Avian 44. Capua, I., and S. Marangon. 2003. The use of Infl uenza, Georgia Center for Continuing Educa- vaccination as an option for the control of avian tion, the University of Georgia, Athens, Georgia, infl uenza. Avian Pathology 32(4):335–343. 440 Avian Influenza

45. Capua, I., and S. Marangon. 2003. Vaccination Virology 167:414–421. policy applied for the control of avian infl uenza 57. Chen, H., Y. Matsuoka, D. Swayne, Q. Chen, N.J. in Italy. Developments in Biologicals 114:213– Cox, B.R. Murphy, and K. Subbarao. 2003. Gen- 219. eration and characterization of a cold-adapted 46. Capua, I., and S. Marangon. 2007. Control and infl uenza A H9N2 reassortant as a live pandemic prevention of avian infl uenza in an evolving infl uenza virus vaccine candidate. Vaccine scenario. Vaccine 25(30):5645–5652. 21:4430–4436. 47. Capua, I., C. Terregino, G. Cattoli, F. Mutinelli, 58. Chen, S., L. Sun, W. Liu, X. Sun, and X. Liu. and J.F. Rodriguez. 2003. Development of a 2006. Recombinant fowlpox virus coexpressing DIVA (differentiating infected from vaccinated HA and NA gene from subtype H5N1 of avian animals) strategy using a vaccine containing a infl uenza virus and its protective effi cacy. Weish- heterologous neuraminidase for the control of engwu Xuebao 46(1):111–114. avian infl uenza. Avian Pathology 32(1):47–55. 59. Cherbonnel, M., J. Rousset, and V. Jestin. 2003. 48. Capua, I., C. Terregino, G. Cattoli, and A. Toffan. Strategies to improve protection against low- 2004. Increased resistance of vaccinated turkeys pathogenicity H7 avian infl uenza virus infection to experimental infection with an H7N3 low- using DNA vaccines. Avian Diseases 47:1181– pathogenicity avian infl uenza virus. Avian Pathol- 1186. ogy 33(2):158–163. 60. Claassen, I.J.T.M., E.J. Jahja, A. Sahesti, A. 49. Capua, I., and D.J. Alexander. 2006. The chal- Farida, F. Balk, and A. Bouma. 2007. PD50 lenge of avian infl uenza to the veterinary com- experiments demonstrate that avian infl uenza munity. Avian Pathology 35(3):189–205. vaccines used in Indonesia differ greatly in pro- 50. Capua, I., and S. Marangon. 2004. Vaccination tective capacity. Abstracts of OIE/FAO Confer- for avian infl uenza in Asia. Vaccine 22(31– ence on Vaccination: A Tool for the Control of 32):4137–4138. Avian Infl uenza OIE/FAO, Verona, Italy, p. 73. 51. Capua, I., and S. Marangon. 2006. Control of 61. Clancy, R., and J. Bienenstock. 1974. The prolif- avian infl uenza in poultry. Emerging Infectious erative response of bronchus-associated lymphoid Diseases 12(9):1319–1324. tissue after local and systemic immunization. 52. Cardona, C.J., B.R. Charlton, and P.R. Wool- Journal of Immunology 112(6):1997–2001. cock. 2006. Persistence of immunity in commer- 62. Clancy, R., A. Cripps, K. Murree-Allen, S. cial egg-laying hens following vaccination with a Yeung, and M. Engel. 1985. Oral immunisation killed H6N2 avian infl uenza vaccine. Avian with killed Haemophilus infl uenzae for protection Diseases 50(3):374–379. against acute bronchitis in chronic obstructive 53. Cattoli, G., A. Milani, F. Bettini, B.M. Serena, M. lung disease. Lancet 2(8469–8470):1395–1397. Mancin, C. Terregino, and I. Capua. 2006. Devel- 63. Cole, K., S.L. Layton, M.M. Cox, Y.M. Kwon, opment and validation of an anti-N3 indirect W.J. Berghman, W.J. Bottje, and B.M. Hargis. immunofl uorescent antibody test to be used as a 2007. Development and primary evaluation of a companion diagnostic test in the framework of a recombinant salmonella expressing M2e and “DIVA” vaccination strategy for avian infl uenza CD154 as a vaccine vector for avian infl uenza. infections in poultry. Avian Pathology 35(2):154– In: Abstracts of the FAO/OIE Conference on 159. Vaccination: A Tool for the Control of Avian 54. Cattoli, G., C. Terregino, V. Brasola, J.F. Infl uenza, Verona, Italy, p. 54. Rodriguez, and I. Capua. 2003. Development and 64. Couch, R.B., and J.A. Kasel. 1983. Immunity to preliminary validation of an ad hoc N1-N3 dis- infl uenza in man. Annual Review of Microbiol- criminatory test for the control of avian infl uenza ogy 37:529–549. in Italy. Avian Diseases 47:1060–1062. 65. Cox, J.C., and A.R. Coulter. 1997. Adjuvants—a 55. Cauthen, A.N., D.E. Swayne, M.J. Sekellick, P.I. classifi cation and review of their modes of action. Marcus, and D.L. Suarez. 2007. Amelioration of Vaccine 15(3):248–256. infl uenza virus pathogenesis in chickens attrib- 66. Crawford, J., B. Wilkinson, A. Vosnesensky, G. uted to the enhanced interferon-inducing capacity Smith, M. Garcia, H. Stone, and M.L. Perdue. of a virus with a truncated NS1 gene. Journal of 1999. Baculovirus-derived hemagglutinin vac- Virology 81(4):1838–1847. cines protect against lethal infl uenza infections by 56. Chambers, T.M., Y. Kawaoka, and R.G. Webster. avian H5 and H7 subtypes. Vaccine 17:2265– 1988. Protection of chickens from lethal infl uenza 2274. infection by vaccine-expressed hemagglutinin. 67. Crawford, J.M., M. Garcia, H. Stone, D. Swayne, 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 441

R. Slemons, and M.L. Perdue. 1998. Molecular national Symposium on Avian Infl uenza, May characterization of the hemagglutinin gene and 29–31, 1997, Athens, Georgia. Symposium on oral immunization with a waterfowl-origin avian Avian Infl uenza, US Animal Health Association: infl uenza virus. Avian Diseases 42(3):486–496. Richmond, VA, pp. 228–236. 68. Czifra, G., and U. Carlsson. 2007. Vaccination of 78. Donovan, B.W., J.D. Reuter, Z. Cao, A. Myc, zoo birds in Sweden 2006. In: Abstracts of the K.J. Johnson, and J.R. Baker, Jr. 2000. Prevention FAO/OIE Conference on Vaccination: A Tool for of murine infl uenza A virus pneumonitis by sur- the Control of Avian Infl uenza, Verona, Italy, factant nano-emulsions. Antiviral Chemistry & p. 67. Chemotherapy 11(1):41–49. 69. Das, A.M., E. Spackman, C. Thomas, D.E. 79. Easterday, B.C., and K. Van Reeth. 1999. Swine Swayne, and D.L. Suarez. 2007. Detection of infl uenza. In: B.E. Straw, S. D’Allaire, W.L. H5N1 high pathogenicity avian infl uenza virus in Mengeling, and D.J. Taylor (eds.). Diseases of meat and tracheal samples from experimentally Swine. Iowa State University Press: Ames, IA, infected chickens. Avian Diseases, in press. pp. 277–290. 70. Davies, I.A. 1986. Immunological adjuvants of 80. Ellis, T.M., C.W. Leung, M.K. Chow, L.A. Bissett, natural origin and their adverse effects. Adverse W. Wong, Y. Guan, and J.S.M. Peiris. 2004. Vac- Drug Reactions and Acute Poisoning Reviews cination of chickens against H5N1 avian infl uenza 5(1):1–21. in the face of an outbreak interrupts virus trans- 71. Davison, S., R.J. Eckroade, and A.F. Ziegler. mission. Avian Pathology 33(4):405–412. 2003. A review of the 1996–1998 nonpathogenic 81. Eskelund, K.H. 1984. Use of inactivated vaccine H7N2 avian infl uenza outbreak in Pennsylvania. to control avian infl uenza outbreaks. Proceedings Avian Diseases 47(Suppl.):823–827. of the Western Poultry Disease Conference 33:8– 72. Davison, S., A.F. Ziegler, and R.J. Eckroade. 10. 1998. Comparison of an antigen-capture enzyme 82. Fatunmbi, O.O., J.A. Newman, V. Sivanandan, immunoassay with virus isolation for avian infl u- and D.A. Halvorson. 1992. Enhancement of anti- enza from fi eld samples. Avian Diseases 42(4): body response of turkeys to trivalent avian infl u- 791–795. enza vaccine by positively charged liposomal 73. De, B.K., M.W. Shaw, P.A. Rota, M.W. Harmon, avridine adjuvant. Vaccine 10:623–626. J.J. Esposito, R. Rott, N.J. Cox, and A.P. Kendal. 83. Fearon, D.T., and R.M. Locksley. 1996. The 1988. Protection against virulent H5 avian infl u- instructive role of innate immunity in the acquired enza virus infection in chickens by an inactivated immune response. Science 272(5258):50–53. vaccine produced with recombinant vaccinia 84. Folitse, R., D.A. Halvorson, and V. Sivanandan. virus. Vaccine 6:257–261. 1998. Effi cacy of combined killed-in-oil emul- 74. Desheva, J.A., X.H. Lu, A.R. Rekstin, L.G. sion and live Newcastle disease vaccines in Rudenko, D.E. Swayne, N.J. Cox, J.M. Katz, and chickens. Avian Diseases 42(1):173–178. A.I. Klimov. 2006. Characterization of an infl u- 85. Frame, D.D., B.J. McCluskey, R.E. Buckner, and enza A H5N2 reassortant as a candidate for live- F.D. Halls. 1996. Results of an H7N3 avian infl u- attenuated and inactivated vaccines against highly enza vaccination program in commercial meat pathogenic H5N1 viruses with pandemic poten- turkeys. Proceedings of the Western Poultry tial. Vaccine 24(47–48):6859–6866. Disease Conference 45:32. 75. Di Trani, L., P. Cordioli, E. Falcone, G. 86. Fynan, E.F., H.L. Robinson, and R.G. Webster. Lombardi, A. Moreno, G. Sala, and M. Tollis. 1993. Use of DNA encoding infl uenza hemag- 2003. Standardization of an inactivated H7N1 glutinin as an avian infl uenza vaccine. DNA and avian infl uenza vaccine and effi cacy against A/ Cell Biology 12:785–789. chicken/Italy/13474/99 high-pathogenicity virus 87. Fynan, E.F., R.G. Webster, D.H. Fuller, J.R. infection. Avian Diseases 47:1042–1046. Haynes, J.C. Santoro, and H.L. Robinson. 1993. 76. Diebold, S.S., T. Kaisho, H. Hemmi, S. Akira, DNA vaccines: protective immunizations by par- and C.R.E. Sousa. 2004. Innate antiviral responses enteral, mucosal, and gene-gun inoculations. Pro- by means of TLR7-mediated recognition of ceedings of the National Academy of Sciences single-stranded RNA. Science 303(5663):1529– U S A 90:11478–11482. 1531. 88. Gall-Recule, G., M. Cherbonnel, N. Pelotte, P. 77. Donahoe, J.P. 1998. Inactivated avian infl uenza Blanchard, Y. Morin, and V. Jestin. 2007. Impor- whole virus vaccines. In: D.E. Swayne and R.D. tance of a prime-boost DNA/protein vaccination Slemons (eds.). Proceedings of the Fourth Inter- to protect chickens against low-pathogenic H7 442 Avian Influenza

avian infl uenza infection. Avian Diseases 51(1 International Symposium on Avian Infl uenza, Suppl.):490–494. May 29–31, 1997, Athens, Georgia. Symposium 89. Gao, W., A.C. Soloff, X. Lu, A. Montecalvo, on Avian Infl uenza, US Animal Health Associa- D.C. Nguyen, Y. Matsuoka, P.D. Robbins, D.E. tion: Richmond, VA, pp. 223–227. Swayne, R.O. Donis, J.M. Katz, S.M. Barratt- 98. Halvorson, D.A. 2002. The control of H5 or H7 Boyes, and A. Gambotto. 2006. Protection of mildly pathogenic avian infl uenza: a role for inac- mice and poultry from lethal H5N1 avian infl u- tivated vaccine. Avian Pathology 31(1):5–12. enza virus through adenovirus-based immuniza- 99. Halvorson, D.A. 2002. Twenty-fi ve years of tion. Journal of Virology 80(4):1959–1964. avian infl uenza in Minnesota. In: Proceedings of 90. Garcia, A., H. Johnson, D.K. Srivastava, D.A. the 53rd North Central Avian Disease Confer- Jayawardene, D.R. Wehr, and R.G. Webster. ence, NCADC: Minneapolis, 65–69. 1998. Effi cacy of inactivated H5N2 infl uenza 100. He, H., K.J. Genovese, D.J. Nisbet, and M.H. vaccines against lethal A/chicken/Queretaro/19/95 Kogut. 2006. Profi le of Toll-like receptor expres- infection. Avian Diseases 42:248–256. sions and induction of nitric oxide synthesis by 91. Garcia-Garcia, J., H. Rodriguez, and H. Hernan- Toll-like receptor agonists in chicken monocytes. dez. 1998. Experimental studies and fi eld trials Molecular Immunology 43(7):783–789. with recombinant fowlpox vaccine in broilers in 101. He, Q., A. Mitchell, T. Morcol, and S.J. Bell. Mexico. In: D.E. Swayne and R.D. Slemons 2002. Calcium phosphate nanoparticles induce (eds.). Proceedings of the Fourth International mucosal immunity and protection against herpes Symposium on Avian Infl uenza, May 29–31, simplex virus type 2. Clinical and Diagnostic 1997, Athens, Georgia. Symposium on Avian Laboratory Immunology 9(5):1021–1024. Infl uenza, US Animal Health Association: 102. Hilton, L.S., A.G.D. Bean, and J.W. Lowenthal. Richmond, VA, pp. 245–252. 2002. The emerging role of avian cytokines as 92. Ge, J., G. Deng, Z. Wen, G. Tian, Y. Wang, J. immunotherapeutics and vaccine adjuvants. Vet- Shi, X. Wang, Y. Li, S. Hu, Y. Jiang, C. Yang, erinary Immunology and Immunopathology K. Yu, Z. Bu, and H. Chen. 2007. Newcastle 85(3–4):119–128. disease virus-based live attenuated vaccine com- 103. Holmgren, J. 1991. Mucosal immunity and vac- pletely protects chickens and mice from lethal cination. FEMS Microbiology and Immunology challenge of homologous and heterologous H5N1 4(1):1–9. avian infl uenza viruses. Journal of Virology 104. Holt, P.S., H.D. Stone, R.W. Moore, and R.K. 81(1):150–158. Gast. 2005. Development of a lavage procedure 93. Glenn, G.M., T. Scharton-Kersten, R. Vassell, to collect lung secretions from chickens for eval- G.R. Matyas, and C.R. Alving. 1999. Trans- uating respiratory humoral immunity. Avian cutaneous immunization with bacterial ADP- Pathology 34(5):396–398. ribosylating exotoxins as antigens and adjuvants. 105. Hu, K.F., K. Lovgren-Bengtsson, and B. Morein. Infection and Immunity 67(3):1100–1106. 2001. Immunostimulating complexes (ISCOMs) 94. Gough, R.E., W.H. Allan, D.J. Knight, and J.W. for nasal vaccination. Advanced Drug Delivery Lieper. 1975. Further studies on the adjuvant Reviews 51(1–3):149–159. effect of an interferon inducer (BRL 5907) on 106. Hunt, L.A., D.W. Brown, H.L. Robinson, C.W. Newcastle disease and avian infl uenza inactivated Naeve, and R.G. Webster. 1988. Retrovirus- vaccines. Research in Veterinary Science 19:185– expressed hemagglutinin protects against lethal 188. infl uenza virus infections. Journal of Virology 95. Gradon, J.D., and L.I. Lutwick. 1999. Maintain- 62:3014–3019. ing and enhancing vaccine immunogenicity. 107. Illum, L., I. Jabbal-Gill, M. Hinchcliffe, A.N. Infectious Disease Clinics of North America Fisher, and S.S. Davis. 2001. Chitosan as a novel 13(1):39–60, vi. nasal delivery system for vaccines. Advanced 96. Halvorson, D.A. 1998. Epidemiology and control Drug Delivery Reviews 51(1–3):81–96. of avian infl uenza in Minnesota. In: Proceedings 108. Janeway, C.A., Jr., B. Jones, and A. Hayday. of the 47th New England Poultry Health Con- 1988. Specifi city and function of T cells bearing ference, New England Poultry Association: gamma delta receptors. Immunology Today Portsmouth, New Hampshire, 5–11. 9(3):73–76. 97. Halvorson, D.A. 1998. Strengths and weaknesses 109. Jeurissen, S.H., E.M. Janse, P.R. Lehrbach, E.E. of vaccines as a control tool. In: D.E. Swayne and Haddad, A. Avakian, and C.E. Whitfi ll. 1998. R.D. Slemons (eds.). Proceedings of the Fourth The working mechanism of an immune complex 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 443

vaccine that protects chickens against infectious Shaw, and D.A. Halvorson. 1994. A type-specifi c bursal disease. Immunology 95(3):494–500. avian infl uenza virus subunit vaccine for turkeys: 110. Jia, L., X. Liu, Y. Zhang, D. Peng, and R. Zhang. induction of protective immunity to challenge 2004. Protective effi cacy of recombinant fowl infection. Vaccine 12:1467–1472. pox virus-HA vaccine against H5 subtype of 121. Kogut, M.H., M. Iqbal, H. He, V. Philbin, P. avian infl uenzavirus in domestic geese. Chinese Kaiser, and A. Smith. 2005. Expression and func- Journal of Veterinary Science 24:417–419. tion of Toll-like receptors in chicken heterophils. 111. Jia, L., D. Peng, Y. Zhang, H. Liu, and X. Liu. Developmental and Comparative Immunology 2003. Construction, genetic stability and protec- 29(9):791–807. tive effi cacy of recombinant fowlpox virus 122. Krishnamurthy, S., Z.H. Huang, and S.K. Samal. expressing hemagglutinin gene of H5N1 subtype 2000. Recovery of a virulent strain of Newcastle avian infl uenza virus. Weishengwu Xuebao 43(6): disease virus from cloned cDNA: expression of a 722–727. foreign gene results in growth retardation and 112. Kaiser, P., T.Y. Poh, L. Rothwell, S. Avery, S. attenuation. Virology 278(1):168–182. Balu, U.S. Pathania, S. Hughes, M. Goodchild, S. 123. Kumar, M., H.J. Chu, J. Rodenberg, S. Krauss, Morrell, M. Watson, N. Bumstead, J. Kaufman, and R.G. Webster. 2007. Association of serologic and J.R. Young. 2005. A genomic analysis of and protective responses of avian infuenza chicken cytokines and chemokines. Journal of vaccines in chickens. Avian Diseases 51:481– Interferon and Cytokine Research 25(8):467– 483. 484. 124. Lamb, R.A., and R.M. Krug. 1996. Orthomyxo- 113. Kapczynski, D.R. 2006. Sequence analysis of the viridae: the viruses and their replication. In: B.N. toll-like receptor 7 from gallinaceous and non- Field, D.M. Knipe, and P.M. Howley (eds.). gallinaceous species. In: G.C. Perry (ed.). Poultry Fields Virology. Lippincott-Raven: New York, Science Symposium Series, 28th ed. Carfax Pub- pp. 1353–1395. lishing Company: Oxfordshire, p. 405. 125. Lee, C.W., D.A. Senne, and D.L. Suarez. 2004. 114. Karaca, K., D.E. Swayne, D. Grosenbaugh, M. Effect of vaccine use in the evolution of Mexican Bublot, A. Robles, E. Spackman, and R. Nor- lineage H5N2 avian infl uenza virus. Journal of dgren. 2005. Immunogenicity of fowlpox virus Virology 78(15):8372–8381. expressing the avian infl uenza virus H5 gene 126. Lee, C.W., D.A. Senne, and D.L. Suarez. 2004. (TROVAC AIV-H5) in cats. Clinical and Diag- Generation of reassortant infl uenza vaccines by nostic Laboratory Immunology 12(11):1340–1342. reverse genetics that allows utilization of a DIVA. 115. Karunakaran, D., J.A. Newman, D.A. Halvorson, Vaccine 22(23–24):3175–3181. and A. Abraham. 1987. Evaluation of inactivated 127. Lee, C.W., and D.L. Suarez. 2004. Application of infl uenza vaccines in market turkeys. Avian Dis- real-time RT-PCR for the quantitation and com- eases 31:498–503. petitive replication study of H5 and H7 subtype 116. King, D.J. 1991. Evaluation of different methods avian infl uenza virus. Journal of Virological of inactivation of Newcastle disease virus and Methods 119(2):151–158. avian infl uenza virus in egg fl uids and serum. 128. Lee, C.W., and D.L. Suarez. 2005. Avian infl u- Avian Diseases 35:505–514. enza virus: prospects for prevention and control 117. Kodihalli, S., J.R. Haynes, H.L. Robinson, and by vaccination. Animal Health Research Reviews R.G. Webster. 1997. Cross-protection among 6(1):1–15. lethal H5N2 infl uenza viruses induced by DNA 129. Lee, C., D.A. Senne, and D.L. Suarez. 2004. Gen- vaccine to the hemagglutinin. Journal of Virology eration of reassortant infl uenza vaccines by 71(5):3391–3396. reverse genetics that allows utilization of a DIVA 118. Kodihalli, S., D.L. Kobasa, and R.G. Webster. (Differentiating Infected from Vaccinated 2000. Strategies for inducing protection against Animals) strategy for the control of avian infl u- avian infl uenza A virus subtypes with DNA vac- enza. Vaccine 22(23–24):3175–3181. cines. Vaccine 18(23):2592–2599. 130. Lee, Y.J., H.W. Sung, J.G. Choi, E.K. Lee, O.M. 119. Kodihalli, S., V. Sivanandan, K.V. Nagaraja, Jeong, Y.K. Kwon, J.H. Kwon, C.S. Song, and S.M. Goyal, and D.A. Halvorson. 1993. Antigen- J.H. Kim. 2007. Effects of homologous and het- capture enzyme-immunoassay for detection of erologous neuraminidase vaccines in chickens avian infl uenza-virus in turkeys. American against H5N1 highly pathogenic avian infl uenza. Journal of Veterinary Research 54:1385–1390. Avian Diseases 51:476–478. 120. Kodihalli, S., V. Sivanandan, K.V. Nagaraja, D. 131. Lillehoj, H.S., X. Ding, R.A. Dalloul, T. Sato, A. 444 Avian Influenza

Yasuda, and E.P. Lillehoj. 2005. Embryo vacci- ogy Research Group Meeting, Paris, France, nation against Eimeria tenella and E. acervulina p. 19. infections using recombinant proteins and cyto- 141. Marangon, S., L. Busani, and I. Capua. 2007. kine adjuvants. Journal of Parasitology 91(3):666– Practicalities of the implementation of a vaccina- 673. tion campaign for avian infl uenza. Avian Dis- 132. Lim, O.J., and Maheswaran. 1977. Purifi cation eases 51:297–303. and identifi cation of turkey immunoglobulin-A. 142. Marangon, S., I. Capua, G. Pozza, and U. Avian Diseases 21:675–696. Santucci. 2004. Field experiences in the control 133. Liu, M., J.M. Wood, T. Ellis, S. Krauss, P. Seiler, of avian infl uenza outbreaks in densely populated C. Johnson, E. Hoffmann, J. Humberd, D. Hulse, poultry areas. Developments in Biologicals Y. Zhang, R.G. Webster, and D.R. Perez. 2003. 119:155–164. Preparation of a standardized, effi cacious agricul- 143. Martin, V., A. Forman, and J. Lubroth. 2006. tural H5N3 vaccine by reverse genetics. Virology Preparing for Highly Pathogenic Avian Infl uenza. 314(2):580–590. FAO: Rome, pp. 1–62. 134. Lozano, B., E. Soto, D. Sarfati, F. Castro, M. 144. Martinet, W., X. Saelens, T. Deroo, S. Neirynck, Gay, and A. Antillon. 2007. A novel engineered R. Contreras, J.W. Min, and W. Fiers. 1997. Pro- live viral vaccine against Newcastle disease and tection of mice against a lethal infl uenza chal- avian infl uenza subtype H5. In: Abstracts of the lenge by immunization with yeast-derived FAO/OIE Conference on Vaccination: A Tool for recombinant infl uenza neuraminidase. European the Control of Avian Infl uenza, Verona, Italy, p. Journal of Biochemistry 247(1):332–338. 50. 145. Matsuoka, Y., H. Chen, N. Cox, K. Subbarao, J. 135. Lu, X., L.E. Edwards, J.A. Desheva, D.C. Nguyen, Beck, and D. E. Swayne. 2003. Safety evaluation A. Rekstin, I. Stephenson, K. Szretter, N.J. Cox, in chickens of candidate human vaccines against L.G. Rudenko, A. Klimov, and J.M. Katz. 2006. potential pandemic strains of infl uenza. Avian Cross-protective immunity in mice induced by Diseases 47:926–930. live-attenuated or inactivated vaccines against 146. McCapes, R.H., and R.A. Bankowski. 1987. Use highly pathogenic infl uenza A (H5N1) viruses. of avian infl uenza vaccines in California turkey Vaccine 24(44–46):6588–6593. breeders. In: B.C. Easterday and C.W. Beard 136. Lucio, E., D. Rodriguez, A. Morales, J. Chapa, (eds.). Proceedings of the Second International and P. Munoz. 2007. Mucosal protection afforded Symposium on Avian Infl uenza, Georgia Center by a killed spray avian infl uenza vaccine. In: for Continuing Education, the University of Abstracts of the FAO/OIE Conference on Vac- Georgia, Athens, Georgia, USA, September 3–5, cination: A Tool for the Control of Avian Infl u- 1986. Symposium on Avian Infl uenza, US Animal enza, Verona, Italy, p. 66. Health Association: Richmond, VA, pp. 271– 137. Lund, J.M., L. Alexopoulou, A. Sato, M. Karow, 278. N.C. Adams, N.W. Gale, A. Iwasaki, and R.A. 147. McDermott, M.R., and J. Bienenstock. 1979. Flavell. 2004. Recognition of single-stranded Evidence for a common mucosal immunologic RNA viruses by Toll-like receptor 7. Proceedings system. I. Migration of B immunoblasts into of the National Academy of Sciences U S A intestinal, respiratory, and genital tissues. Journal 101(15):5598–5603. of Immunology 122(5):1892–1898. 138. Luschow, D., O. Werner, T.C. Mettenleiter, and 148. Mcghee, J.R., J. Mestecky, M.T. Dertzbaugh, W. Fuchs. 2001. Protection of chickens from lethal J.H. Eldridge, M. Hirasawa, and H. Kiyono. avian infl uenza A virus infection by live-virus 1992. The mucosal immune system: from funda- vaccination with infectious laryngotracheitis mental concepts to vaccine development. Vaccine virus recombinants expressing the hemagglutinin 10(2):75–88. (H5) gene. Vaccine 19(30):4249–4259. 149. McNulty, M.S., G.M. Allan, and B.M. Adair. 139. Maas, R.A., M.P.M. de Winter, S. Venema, H.L. 1986. Effi cacy of avian infl uenza neuraminidase- Oei, and I.J.T.M. Claassen. 2000. Antigen quan- specifi c vaccines in chickens. Avian Pathology tifi cation as in vitro alternative for potency testing 15:107–115. of inactivated viral poultry vaccines. Veterinary 150. Medina, E., and C.A. Guzman. 2000. Modulation Quarterly 22(4):223–227. of immune responses following antigen adminis- 140. MacDonald, M.R.W., A.L. Smith, and K.E. tration by mucosal route. FEMS Immunology and Magor. 2006. The toll-like receptor 7 (TLR7) Medical Microbiology 27(4):305–311. genomic locus in ducks. In: 9th Avian Immunol- 151. Medzhitov, R., and C.A. Janeway, Jr. 1997. 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 445

Innate immunity: impact on the adaptive immune Sciences 34:72–79. response. Current Opinions in Immunology 162. Nicholson, K.G., A.E. Colegate, A. Podda, I. 9(1):4–9. Stephenson, J. Wood, E. Ypma, and M.C. 152. Medzhitov, R., and C.A. Janeway, Jr. 1998. An Zambon. 2001. Safety and antigenicity of non- ancient system of host defense. Current Opinions adjuvanted and MF59-adjuvanted infl uenza in Immunology 10(1):12–15. A/Duck/Singapore/97 (H5N3) vaccine: a ran- 153. Middleton, D., J. Bingham, P. Selleck, S. Lowther, domised trial of two potential vaccines against L. Gleeson, P. Lehrbach, S. Robinson, J. Roden- H5N1 infl uenza. Lancet 357(9272):1937–1943. berg, M. Kumar, and M. Andrew. 2007. Effi cacy 163. O’Hagan, D.T., G.S. Ott, and G. Van Nest. 1997. of inactivated vaccines against H5N1 avian Recent advances in vaccine adjuvants: the devel- infl uenza infection in ducks. Virology 359(1):66– opment of MF59 emulsion and polymeric mic- 71. roparticles. Molecular Medicine Today 3(2): 154. Mingxiao, M., J. Ningyi, W. Zhenguo, W. Ruilin, 69–75. F. Dongliang, Z. Min, Y. Gefen, L. Chang, J. 164. Offi ce Internationale des Epizooties. 2007. Avian Leili, J. Kuoshi, and Z. Yingjiu. 2006. Construc- infl uenza. Terrestrial Animal Health Code. Article tion and immunogenicity of recombinant fowlpox 2.7.12.1. Available at http://www.oie.int/eng/ vaccines coexpressing HA of AIV H5N1 and normes/mcode/en_chapitre_2.7.12.htm. Offi ce chicken IL18. Vaccine 24(20):4304–4311. Internationale des Epizooties: Paris, France. 155. Moriya, O. 1983. Ontogeny of lymphocytes Accessed April 24, 2007. expressing J chain in chickens. Cellular Immu- 165. Offi ce Internationale des Epizooties. 2007. Avian nology 80(1):78–83. infl uenza vaccine manufacturers. http://www.oie. 156. Muir, W.I., W.L. Bryden, and A.J. Husband. int/eng/AVIAN_INFLUENZA/AI_vaccines.pdf. 2000. Immunity, vaccination and the avian intes- Offi ce Internationale des Epizooties: Paris, tinal tract. Developmental and Comparative France. Accessed May 25, 2007. Immunology 24(2–3):325–342. 166. Olah, I., and B. Glick. 1979. Structure of the 157. Myers, T.J., and A.P. Morgan. 1998. Policy and germinal centers in the chicken caecal tonsil: light guidance for licensure of avian infl uenza vaccines and electron microscopic and autoradiographic in the United States. In: D.E. Swayne and R.D. studies. Poultry Science 58(1):195–210. Slemons (eds.). Proceedings of the Fourth Inter- 167. Olah, I., B. Glick, and R.L. Taylor, Jr. 1984. national Symposium on Avian Infl uenza, May Meckel’s diverticulum. II. A novel lymphoepi- 29–31, 1997, Athens, Georgia. Symposium on thelial organ in the chicken. Anatomical Record Avian Infl uenza, US Animal Health Association: 208(2):253–263. Richmond, VA, pp. 373–378. 168. Park, M., J. Steel, A. Garcia-Sastre, D. Swayne, 158. Naeem, K. 1998. The avian infl uenza H7N3 out- and P. Palese. 2006. Engineered viral vaccine break in South Central Asia. In: D.E. Swayne and constructs with dual specifi city: avian infl uenza R.D. Slemons (eds.). Proceedings of the Fourth and Newcastle disease. Proceedings of the International Symposium on Avian Infl uenza, National Academy of Sciences U S A 103(21): May 29–31, 1997, Athens, Georgia. Symposium 8203–8208. on Avian Infl uenza, US Animal Health Associa- 169. Pasare, C., and R. Medzhitov. 2004. Toll- tion: Richmond, VA, pp. 31–35. dependent control mechanisms of CD4 T cell 159. Naeem, K., and N. Siddique. Use of strategic activation. Immunity 21(5):733–741. vaccination for the control of avian infl uenza in 170. Payne, L.M., and P.C. Powell. 1984. The lym- Pakistan. Developments in biologicals 124:145– phoid system. In: B.M. Freeman (ed.). Physiol- 150. ogy and Biochemistry of the Domestic Fowl. 160. Nakaya, T., J. Cros, M.S. Park, Y. Nakaya, H.Y. Academic Press: Orlando, FL, p. 278. Zheng, A. Sagrera, E. Villar, A. Garcia-Sastre, 171. Peeters, B.P., O.S. de Leeuw, G. Koch, and A.L. and P. Palese. 2001. Recombinant Newcastle Gielkens. 1999. Rescue of Newcastle disease disease virus as a vaccine vector. Journal of virus from cloned cDNA: evidence that cleav- Virology 75(23):11868–11873. ability of the fusion protein is a major determi- 161. Narayan, O., B.T. Rouse, and G. Lang. 1970. A nant for virulence. Journal of Virology new infl uenza A virus infection in turkeys. VI. 73(6):5001–5009. Artifi cial immunization against the malignant 172. Philbin, V.J., M. Iqbal, Y. Boyd, M.J. Goodchild, virus strain turkey-Ontario 7732–66. Canadian R.K. Beal, N. Bumstead, J. Young, and A.L. Journal of Comparative Medicine and Veterinary Smith. 2005. Identifi cation and characterization 446 Avian Influenza

of a functional, alternatively spliced Toll-like infl uenza virus in SPF chickens. Scientia Agricul- receptor 7 (TLR7) and genomic disruption of tura Sinica 37:605–608. TLR8 in chickens. Immunology 114(4):507– 182. Quere, P., and F. Girard. 1999. Systemic adjuvant 521. effect of cholera toxin in the chicken. Veterinary 173. Philippa, J., C. Baas, W. Beyer, T. Bestebroer, R. Immunology and Immunopathology 70(1– Fouchier, D. Smith, W. Schaftenaar, and A. 2):135–141. Osterhaus. 2007. Vaccination against highly 183. Ratcliffe, M.J., O. Lassila, J. Reynolds, J.R. Pink, pathogenic avian infl uenza H5N1 virus in zoos and O. Vainio. 1987. A re-evaluation of the func- using an adjuvanted inactivated H5N2 vaccine. tion of the bursa of Fabricius. Progress in Clinical Vaccine 25(19):3800–3808. and Biological Research 238:3–14. 174. Philippa, J.D., V.J. Munster, H. Bolhuis, T.M. 184. Reis e Sousa, C. 2004. Toll-like receptors and Bestebroer, W. Schaftenaar, W.E. Beyer, R.A. dendritic cells: for whom the bug tolls. Seminars Fouchier, T. Kuiken, and A.D. Osterhaus. 2005. in Immunology 16(1):27–34. Highly pathogenic avian infl uenza (H7N7): vac- 185. Renegar, K.B., and P.A.J. Small. 1991. Immuno- cination of zoo birds and transmission to non- globulin A mediation of murine nasal anti- poultry species. Vaccine 23(50):5743–5750. infl uenza virus immunity. Journal of Virology 175. Porter, R.E., Jr., and P.S. Holt. 1992. Use of a 65:2146–2148. pilocarpine-based lavage procedure to study 186. Renegar, K.B., and P.A.J. Small. 1991. Passive secretory immunoglobulin concentration in the transfer of local immunity to infl uenza virus alimentary tract of White Leghorn chickens. infection by IgA antibody. Journal of Immunol- Avian Diseases 36(3):529–536. ogy 146:1972–1978. 176. Prel, A., G. Gall-Recule, M. Cherbonnel, B. 187. Rimmelzwaan, G.F., E.C. Claas, G. van Ameron- Grasland, M. Amelot, and V. Jestin. 2007. Assess- gen, J.C. de Jong, and A.D. Osterhaus. 1999. ment of the protection afforded by triple baculo- ISCOM vaccine induced protection against a virus recombinant coexpressing H5, N3, M1 lethal challenge with a human H5N1 infl uenza proteins against a homologous H5N3 low- virus. Vaccine 17(11–12):1355–1358. pathogenicity avian infl uenza virus challenge in 188. Robinson, H.L., L.A. Hunt, and R.G. Webster. Muscovy ducks. Avian Diseases 51(1 Suppl.):484– 1993. Protection against a lethal infl uenza virus 489. challenge by immunization with a haemaggluti- 177. Price, R.J. 1981. Commercial avian infl uenza nin-expressing plasmid DNA. Vaccine 11:957– vaccines. In: R.W. Bankowski (ed.). Proceedings 960. of the First International Symposium on Avian 189. Roth, J.A. 1999. Mechanistic bases for adverse Infl uenza, April 1981, Beltsville, Maryland. Sym- vaccine reactions and vaccine failures. Advances posium on Avian Infl uenza, US Animal Health in Veterinary Medicine 41:681–700. Association: Richmond, VA, pp. 178–179. 190. Roth, J.A., and A.R. Spickler. 2003. A survey of 178. Purchase, H.S. 1930. Active immunisation of vaccines produced for OIE list A diseases in OIE fowls against fowl plague. Journal of Compari- member countries. Development in Biologicals tive Pathology and Therapeutics 43:151–157. 114:5–25. 179. Qiao, C., K. Yu, Y. Jiang, C. Li, G. Tian, X. 191. Rott, R., H. Becht, and M. Orlich. 1974. The Wang, and H. Chen. Development of a recombi- signifi cance of infl uenza virus neuraminidase in nant fowlpox virus vector-based vaccine of H5N1 immunity. Journal of General Virology 22:35– subtype avian infl uenza. Developments in Bio- 41. logicals 124:127–132. 192. Saelens, X., P. Vanlandschoot, W. Martinet, M. 180. Qiao, C.L., K.Z. Yu, Y.P. Jiang, Y.Q. Jia, G.B. Maras, S. Neirynck, R. Contreras, W. Fiers, and Tian, M. Liu, G.H. Deng, X.R. Wang, Q.W. W.M. Jou. 1999. Protection of mice against a Meng, and X.Y. Tang. 2003. Protection of chick- lethal infl uenza virus challenge after immuniza- ens against highly lethal H5N1 and H7N1 avian tion with yeast-derived secreted infl uenza virus infl uenza viruses with a recombinant fowlpox hemagglutinin. European Journal of Biochemis- virus co-expressing H5 haemagglutinin and N1 try 260(1):166–175. neuraminidase genes. Avian Pathology 32(1): 193. Sambhara, S., A. Kurichh, R. Miranda, T. 25–31. Tumpey, T. Rowe, M. Renshaw, R. Arpino, A. 181. Qiao, C., Y. Jiang, K. Yu, G. Tian, and H. Chen. Tamane, A. Kandil, O. James, B. Underdown, M. 2004. Immune effi cacy of a recombinant fowlpox Klein, J. Katz, and D. Burt. 2001. Heterosubtypic virus co-expressing HA and NA genes of avian immunity against human infl uenza A viruses, 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 447

including recently emerged avian H5 and H9 of Virology 75(6):2516–2525. viruses, induced by FLU-ISCOM vaccine in mice 205. Sharma, J.M. 1999. Introduction to poultry vac- requires both cytotoxic T-lymphocyte and macro- cines and immunity. Advances in Virus Medicine phage function. Cellular Immunology 211(2):143– 41:481–494. 153. 206. Sharma, J.M., and R.L. Witter. 1983. Embryo 194. Savill, N.J., S.G. St Rose, M.J. Keeling, and E.J. vaccination against Marek’s disease with sero- Woolhouse Mark. 2007. Silent spread of H5N1 types 1, 2 and 3 vaccines administered singly or in vaccinated poultry. Nature 442(7104):757. in combination. Avian Diseases 27(2):453–463. 195. Schat, K.A., and T.J. Meyers. 1991. Avian intes- 207. Sims, L.D. 2007. Lessons learned from Asian tinal immunity. Critical Reviews in Poultry H5N1 outbreak control. Avian Diseases 51(1 Biology 3:19–34. Suppl.):174–181. 196. Schijns, V.E. 2000. Immunological concepts of 208. Smith, D.J., S. Bot, L. Dellamary, and A. Bot. vaccine adjuvant activity. Current Opinions in 2003. Evaluation of novel aerosol formulations Immunology 12(4):456–463. designed for mucosal vaccination against infl u- 197. Schijns, V.E. 2001. Induction and direction of enza virus. Vaccine 21(21–22):2805–2812. immune responses by vaccine adjuvants. Critical 209. Smith, V.W., W. Coakley, D. Maker, J.S. Reviews in Immunology 21(1–3):75–85. MacKenzie, and P.A. Lalor. 1980. The serologi- 198. Schijns, V.E. 2003. Mechanisms of vaccine adju- cal response of chickens to an avian infl uenza A vant activity: initiation and regulation of immune virus administered by various routes. Research in responses by vaccine adjuvants. Vaccine 21(9– Veterinary Science 29:248–250. 10):829–831. 210. Staats, H.F., R.J. Jackson, M. Marinaro, I. Taka- 199. Schijns, V.E., and A. Tangeras. 2005. Vaccine hashi, H. Kiyono, and J.R. Mcghee. 1994. adjuvant technology: from theoretical mecha- Mucosal immunity to infection with implications nisms to practical approaches. Developments in for vaccine development. Current Opinions in Biologicals 121:127–134. Immunology 6(4):572–583. 200. Schmitz, A., C. Guillemoto, I. Pierre, J. Lamande, 211. Steel, J., S.V. Burmakina, C. Thomas, E. Spack- C. Allee, D. Gomis, A. Lecu, Y. Roman, E. man, A. Garcia-Sastre, D.E. Swayne, and P. Bureau, T. Petit, J. Francart, and V. Jestin. 2007. Palese. 2007. A combination in-ovo vaccine for Avian infl uenza preventive vaccination of zoo avian infl uenza virus and Newcastle disease virus. birds in France. In: Abstracts of the FAO/OIE Vaccine. Conference on Vaccination: A Tool for the 212. Steensels, M., S. Van Borm, B. Lambrecht, J. De Control of Avian Infl uenza, Verona, Italy, p. 53. Vriese, F.X. Le Gros, M. Bublot, and T. van den 201. Schultz-Cherry, S., J.K. Dybing, N.L. Davis, C. Berg. 2007. Effi cacy of an inactivated and a Williamson, D.L. Suarez, R. Johnston, and M.L. fowlpox-vectored vaccine in Muscovy ducks Perdue. 2000. Infl uenza virus (A/HK/156/97) against an Asian H5N1 highly pathogenic avian hemagglutinin expressed by an alphavirus repli- infl uenza viral challenge. Avian Diseases 20:325– con system protects chickens against lethal infec- 331. tion with Hong Kong-origin H5N1 viruses. 213. Stone, H., B. Mitchell, and M. Brugh. 1997. In Virology 278:55–59. ovo vaccination of chicken embryos with experi- 202. Senne, D.A., T.J. Holt, and B.L. Akey. 2005. An mental Newcastle disease and avian infl uenza overview of the 2002 outbreak of low-pathogenic oil-emulsion vaccines. Avian Diseases 41(4): H7N2 avian infl uenza in Virginia, West Virginia 856–863. and North Carolina. In: R.S. Schrijver and G. 214. Stone, H.D. 1987. Effi cacy of avian infl uenza oil- Koch (eds.). Avian Infl uenza. Prevention and emulsion vaccines in chickens of various ages. Control, Springer: Dordrecht, 41–47. Avian Diseases 31:483–490. 203. Seo, K.H., P.S. Holt, L.E. Vaughn, R.K. Gast, and 215. Stone, H.D. 1988. Optimization of hydrophile- H.D. Stone. 2003. Detection of Salmonella enter- lipophile balance for improved effi cacy of New- itidis-specifi c immunoglobulin A antibodies in castle disease and avian infl uenza oil-emulsion crop samples from chickens infected with Salmo- vaccines. Avian Diseases 32:68–73. nella enteritidis. Poultry Science 82(1):67–70. 216. Stone, H.D. 1989. Effi cacy of oil-emulsion vac- 204. Seo, S.H., and R.G. Webster. 2001. Cross- cines prepared with pigeon paramyxovirus-1, reactive, cell-mediated immunity and protection Ulster, and La Sota Newcastle disease viruses. of chickens from lethal H5N1 infl uenza virus Avian Diseases 33(1):157–162. infection in Hong Kong poultry markets. Journal 217. Stone, H.D. 1991. The preparation and effi cacy 448 Avian Influenza

of manually emulsifi ed Newcastle disease oil- 104:469–471. emulsion vaccines. Avian Diseases 35(1):8–16. 230. Swayne, D.E. 2003. Vaccines for list A poultry 218. Stone, H.D. 1993. Effi cacy of experimental diseases: emphasis on avian infl uenza. Develop- animal and vegetable oil-emulsion vaccines for ments in Biologicals 114:201–212. Newcastle disease and avian infl uenza. Avian 231. Swayne, D.E. 2004. Application of new vaccine Diseases 37:399–405. technologies for the control of transboundary dis- 219. Stone, H.D. 1997. Newcastle disease oil emulsion eases. Developments in Biologicals 119:219– vaccines prepared with animal, vegetable, and 228. synthetic oils. Avian Diseases 41(3):591–597. 232. Swayne, D.E. 2006. Principles for vaccine pro- 220. Stone, H.D., M. Brugh, S.R. Hopkins, H.W. tection in chickens and domestic waterfowl Yoder, and C.W. Beard. 1978. Preparation of against avian infl uenza: emphasis on Asian H5N1 inactivated oil-emulsion vaccines with avian viral high pathogenicity avian infl uenza. Annals of the or Mycoplasma antigens. Avian Diseases New York Academy of Sciences 1081:174–181. 22(4):666–674. 233. Swayne, D.E. 2008. Current developments in 221. Stone, H.D., and Z.X. Xie. 1990. Effi cacy of avian infl uenza vaccines including food safety experimental Newcastle disease water-in-oil oil- aspects in vaccinated birds. Development in emulsion vaccines formulated from squalane and Biologicals. squalene. Avian Diseases 34(4):979–983. 234. Swayne, D.E., and B. Akey. 2005. Avian infl u- 222. Suarez, D.L. 2005. Overview of avian infl uenza enza control strategies in the United States of DIVA test strategies. Biologicals 33(4):221–226. America. In: R.S. Schrijver and G. Koch (eds.). 223. Suarez, D.L., C.W. Lee, and D.E. Swayne. 2006. Avian Infl uenza. Prevention and Control. Avian infl uenza vaccination in North America: Springer: Dordrecht, pp. 113–130. strategies and diffi culties. Developments in Bio- 235. Swayne, D.E., G.E. Avellaneda, T.R. Mickle, N. logicals 124:117–124. Pritchard, J. Cruz, and M. Bublot. 2007. Improve- 224. Suarez, D.L., and C.S. Schultz. 2000. Immunol- ments to the hemagglutination inhibition (HI) test ogy of avian infl uenza virus: a review. Develop- for serological assessment of recombinant mental and Comparative Immunology fowlpox-H5-avian-infl uenza vaccination in chick- 24(2–3):269–283. ens and its use along with agar gel immunodiffu- 225. Suarez, D.L., and S. Schultz-Cherry. 2000. The sion test in a differentiating infected from effect of eukaryotic expression vectors and adju- vaccinated animals (DIVA) strategy. Avian Dis- vants on DNA vaccines in chickens using an eases 51(3):697–704. avian infl uenza model. Avian Diseases 44(4):861– 236. Swayne, D.E., J.R. Beck, M. Garcia, and H.D. 868. Stone. 1999. Infl uence of virus strain and antigen 226. Subbarao, K., H.L. Chen, D. Swayne, L. Mingay, mass on effi cacy of H5 avian infl uenza inacti- E. Fodor, G. Brownlee, X.Y. Xu, X.H. Lu, J. vated vaccines. Avian Pathology 28:245–255. Katz, N. Cox, and Y. Matsuoka. 2003. Evaluation 237. Swayne, D.E., J.R. Beck, and N. Kinney. 2000. of a genetically modifi ed reassortant H5N1 infl u- Failure of a recombinant fowl poxvirus vaccine enza A virus vaccine candidate generated by containing an avian infl uenza hemagglutinin gene plasmid-based reverse genetics. Virology to provide consistent protection against infl uenza 305(1):192–200. in chickens preimmunized with a fowl pox 227. Subbarao, K., R.G. Webster, Y. Kawaoka, and vaccine. Avian Diseases 44(1):132–137. B.R. Murphy. 1995. Are there alternative avian 238. Swayne, D.E., J.R. Beck, and T.R. Mickle. 1997. infl uenza viruses for generation of stable attenu- Effi cacy of recombinant fowl poxvirus vaccine in ated avian-human infl uenza A reassortant viruses? protecting chickens against a highly pathogenic Virus Research 39:105–118. Mexican-origin H5N2 avian infl uenza virus. 228. Suguitan, A.L., Jr., J. McAuliffe, K.L. Mills, H. Avian Diseases 41(4):910–922. Jin, G. Duke, B. Lu, C.J. Luke, B. Murphy, D.E. 239. Swayne, D.E., J.R. Beck, M.L. Perdue, and C.W. Swayne, G. Kemble, and K. Subbarao. 2006. Beard. 2001. Effi cacy of vaccines in chickens Live, attenuated infl uenza A H5N1 candidate against highly pathogenic Hong Kong H5N1 vaccines provide broad cross-protection in mice avian infl uenza. Avian Diseases 45(2):355–365. and ferrets. PLoS Medicine 3(9):e360. 240. Swayne, D.E., M. Garcia, J.R. Beck, N. Kinney, 229. Swayne, D.E. 2001. Avian infl uenza vaccine use and D.L. Suarez. 2000. Protection against diverse during 2001. Proceedings of the Annual Meeting highly pathogenic avian infl uenza viruses in of the United States Animal Health Association chickens immunized with a recombinant fowl 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 449

pox vaccine containing an H5 avian infl uenza demic Publishers: Dordrecht, the Netherlands, hemagglutinin gene insert. Vaccine 18(11– pp. 347–365. 12):1088–1095. 251. Tian, G., S. Zhang, Y. Li, Z. Bu, P. Liu, J. Zhou, 241. Swayne, D.E., and D.A. Halvorson. 2003. Infl u- C. Li, J. Shi, K. Yu, and H. Chen. 2005. Protec- enza. In: Y.M. Saif, H.J. Barnes, A.M. Fadly, J. tive effi cacy in chickens, geese and ducks of an R. Glisson, L.R. McDougald, and D.E. Swayne H5N1-inactivated vaccine developed by reverse (eds.). Diseases of Poultry, 11th ed. Iowa State genetics. Virology 34(1):153–162. University Press: Ames, IA, pp. 135–160. 252. Todd, C. 1928. Experiments on the Virus of Fowl 242. Swayne, D.E., C.W. Lee, and E. Spackman. 2006. Plague (II). British Journal of Experimental Inactivated North American and European H5N2 Pathology 9:101–106. avian infl uenza virus vaccines protect chickens 253. Toivanen, A., P. Toivanen, J. Eskola, and O. from Asian H5N1 high pathogenicity avian infl u- Lassila. 1981. Ontogeny of the chicken lymphoid enza virus. Avian Pathology 35(2):141–146. system. In: M.E. Rose, L.N. Payne, and B.M. 243. Swayne, D.E., and T.R. Mickle. 1996. Protection Freeman (eds.). Avian Immunology. British of chickens against highly pathogenic Mexican- Poultry Science, Ltd: Edinburgh, p. 45. origin H5N2 avian infl uenza virus by a recombi- 254. Toro, H., D.C. Tang, D.L. Suarez, M.J. Sylte, J. nant fowlpox vaccine. Proceedings of the Annual Pfeiffer, and K.R. Van Kampen. 2006. Protective Meeting of the United States Animal Health avian infl uenza in ovo vaccination with non- Association 100:557–563. replicating human adenovirus vector. Vaccine 244. Swayne, D.E., and H.D. Stone. 1996. Evaluation 25(15):2886–2891. of inactivated H5 avian infl uenza virus and 255. Tripathy, D.N., and W.M. Schnitzlein. 1991. fowlpox recombinant vaccines and diagnostic Expression of avian infl uenza virus hemaggluti- test reagents: implications in avian infl uenza nin by recombinant fowlpox virus. Avian Dis- control and prevention stratagies. Proceedings of eases 35:186–191. the Western Poultry Disease Conference 45:35– 256. Tumpey, T.M., D.R. Kapczynski, and D.E. 38. Swayne. 2004. Comparative susceptibility of 245. Swayne, D.E., D.L. Suarez, S. Schultz-Cherry, chickens and turkeys to avian infl uenza A H7N2 T.M. Tumpey, D.J. King, T. Nakaya, P. Palese, virus infection and protective effi cacy of a com- and A. Garcia-Sastra. 2003. Recombinant Para- mercial avian infl uenza H7N2 virus vaccine. myxovirus type 1-avian infl uenza-H7 virus as a Avian Diseases 48(1):167–176. vaccine for protection of chickens against infl u- 257. Tumpey, T.M., R. Alvarez, D.E. Swayne, and enza and Newcastle disease. Avian Diseases D.L. Suarez. 2005. Diagnostic approach for dif- 47:1047–1050. ferentiating infected from vaccinated poultry on 246. Sylte, M.J., B. Hubby, and D.L. Suarez. 2007. the basis of antibodies to NS1, the nonstructural Infl uenza neuraminidase antibodies provide protein of infl uenza A virus. Journal of Clinical partial protection for chickens against high patho- Microbiology 43(2):676–683. genic avian infl uenza infection. Vaccine 258. Utterback, W.W., and J.H. Schwartz. 1973. Epi- 25(19):3763–3772. zootiology of velogenic viscerotropic Newcastle 247. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll- disease in Southern California, 1971–1973. like receptors. Annual Review of Immunology Journal of the American Veterinary Medical 21:335–376. Association 163(9):1080–1088. 248. Taylor, J., R. Weinberg, Y. Kawaoka, R.G. 259. van Boven, M., J.A. van der Goot, A.R.W. Elbers, Webster, and E. Paoletti. 1988. Protective immu- G. Koch, G. Nodelijk, M.C.M. de Jong, T.S. de nity against avian infl uenza induced by a fowlpox Vries, A. Bouma, and J.A. Stegeman. 2005. Vac- virus recombinant. Vaccine 6:504–508. cination of poultry against avian infl uenza: epide- 249. Terregino, C., A. Toffan, M.S. Beato, R. De miological rules of thumb and experimental Nardi, A. Drago, and I. Capua. 2007. Conven- quantifi cation of the effectiveness of vaccination. tional H5N9 vaccine suppresses shedding in spe- In: R.S. Schrijver and G. Koch (eds.). Avian cifi c-pathogen-free birds challenged with HPAI Infl uenza. Prevention and Control. Springer: H5N1 A/chicken/Yamaguchi/7/2004. Avian Dis- Dordrecht, pp. 85–92. eases 51:495–497. 260. van der Goot, J.A., G. Koch, M.C.M. de Jong, 250. Thornton, D.H. 1988. Quality control of vaccines. and M. van Boven. 2005. Quantifi cation of the In: D.J. Alexander (ed.). Developments in Veter- effect of vaccination on transmission of avian inary Virology: Newcastle Disease. Kluwer Aca- infl uenza (H7N7) in chickens. Proceedings of the 450 Avian Influenza

National Academy of Sciences U S A 102(50): 272. Webster, R.G., J. Taylor, J. Pearson, E. Rivera, 18141–18146. and E. Paoletti. 1996. Immunity to Mexican 261. van der Goot, J.A., M. van Boven, M.C. de Jong, H5N2 avian infl uenza viruses induced by a fowl and G. Koch. 2007. Effect of vaccination on pox-H5 recombinant. Avian Diseases 40(2):461– transmission of HPAI H5N1: the effect of a single 465. vaccination dose on transmission of highly patho- 273. Webster, R.G., R.J. Webby, E. Hoffmann, J. genic avian infl uenza H5N1 in Peking ducks. Rodenberg, M. Kumar, H. Chu, P. Seiler, S. Avian Diseases 51(1 Suppl.):323–324. Krauss, and T. Songserm. 2006. The immunoge- 262. van der Lubben, I.M., J.C. Verhoef, G. Borchard, nicity and effi cacy against H5N1 challenge of and H.E. Junginger. 2001. Chitosan for mucosal reverse genetics-derived H5N3 infl uenza vaccine vaccination. Advanced Drug Delivery Reviews in ducks and chickens. Virology 351(2):303– 52(2):139–144. 311. 263. Veits, J., D. Luschow, K. Kindermann, O. Werner, 274. Wernery, U., S. Joseph, W. Tarello, and M. J.P. Teifke, T.C. Mettenleiter, and W. Fuchs. Theneyan. 2006. Serological response of houbara 2003. Deletion of the non-essential UL0 gene of bustards to an H5N1 vaccine. Veterinary Record infectious laryngotracheitis (ILT) virus leads to 158(24):840. attenuation in chickens, and UL0 mutants express- 275. White, R.G. 1981. The structural organization of ing infl uenza virus haemagglutinin (H7) protect avian lymphoid tissues. In: M.E. Rose, L.N. against ILT and fowl plague. Journal of General Payne, and B.M. Freeman (eds.). Avian Immu- Virology 84:3343–3352. nology. British Poultry Science, Ltd: Edinburgh, 264. Veits, J., D. Wiesner, W. Fuchs, B. Hoffmann, H. p. 21. Granzow, E. Starick, E. Mundt, H. Schirrmeier, 276. Wight, P.A., R.B. Burns, B. Rothwell, and T. Mebatsion, T.C. Mettenleiter, and A. Romer- G.M. Mackenzie. 1971. The Harderian gland Oberdorfer. 2006. Newcastle disease virus of the domestic fowl. I. Histology, with refer- expressing H5 hemagglutinin gene protects ence to the genesis of plasma cells and Russell chickens against Newcastle disease and avian bodies. Journal of Anatomy 110(Pt 2):307– infl uenza. Proceedings of the National Academy 315. of Sciences U S A 103(21):8197–8202. 277. Wood, J.M., Y. Kawaoka, L.A. Newberry, E. 265. Villareal, C.L. 2006. Control and eradication Bordwell, and R.G. Webster. 1985. Standardiza- strategies of avian infl uenza in Mexico. Develop- tion of inactivated H5N2 infl uenza vaccine and ments in Biologicals 124:125–126. effi cacy against lethal A/chicken/Pennsylva- 266. Villarreal-Chavez, C. 2007. AI control experi- nia/1370/83 infection. Avian Diseases 29:867– ences in the Americas: a regional summary. Pre- 872. sented March 22, 2007, FAO/OIE Conference on 278. Xie, Z.X., and H.D. Stone. 1990. Immune Vaccination: A Tool for the Control of Avian response to oil-emulsion vaccines with single or Infl uenza, Verona, Italy. mixed antigens of Newcastle disease, avian infl u- 267. Vogel, F.R. 2000. Improving vaccine perfor- enza, and infectious bronchitis. Avian Diseases mance with adjuvants. Clinical Infectious Dis- 34:154–162. eases 30(Suppl. 3):S266-S270. 279. Zanella, A., G. Poli, and M. Bignami. 1981. 268. Waldman, R.H., and C.S. Henney. 1971. Cell- Avian infl uenza: approaches in the control mediated immunity and antibody responses in the of disease with inactivated vaccines in oil respiratory tract after local and systemic immuni- emulsion. In: R.W. Bankowski (ed.). Proceedings zation. Journal of Experimental Medicine of the First International Symposium on Avian 134(2):482–494. Infl uenza, April 1981, Beltsville, Maryland. 269. Wareing, M.D., and G.A. Tannock. 2001. Live Symposium on Avian Infl uenza, US Animal attenuated vaccines against infl uenza; an histori- Health Association: Richmond, VA, pp. 180– cal review. Vaccine 19(25–26):3320–3330. 183. 270. Webster, R.G., Y. Kawaoka, J. Taylor, R. 280. Zarzaur, B.L., and K.A. Kudsk. 2001. The Weinberg, and E. Paoletti. 1991. Effi cacy of mucosa-associated lymphoid tissue structure, nucleoprotein and haemagglutinin antigens ex- function, and derangements. Shock 15(6):411– pressed in fowlpox virus as vaccine for infl uenza 420. in chickens. Vaccine 9:303–308. 281. Zhao, S., M. Jin, H. Li, Y. Tan, G. Wang, R. 271. Webster, R.G., P.A. Reay, and W.G. Laver. 1988. Zhang, and H. Chen. 2005. Detection of antibod- Protection against lethal infl uenza with neuramin- ies to the nonstructural protein (NS1) of avian idase. Virology 164:230–237. infl uenza viruses allows distinction between vac- 19 / Vaccines, Vaccination, and Immunology for Avian Influenza Viruses in Poultry 451

cinated and infected chickens. Avian Diseases 283. Ziegler, A.F., S. Davison, H. Acland, and 49(4):488–493. R.J. Eckroade. 1999. Characteristics of H7N2 282. Zhigao, B. 2007. Field trials with chimera (nonpathogenic) avian influenza virus infec- vaccines. Presented March 22, 2007, FAO/OIE tions in commercial layers, in Pennsyl- Conference on Vaccination: A Tool for the vania, 1997–98. Avian Diseases 43(1):142– Control of Avian Inﬂ uenza, Verona, Italy. 149. 20 Public Health Implications of Avian Influenza Viruses

Nancy J. Cox and Timothy M. Uyeki

INTRODUCTION Human infl uenza A viruses are enveloped single- All human infl uenza pandemics have been caused stranded negative sense RNA viruses of the Ortho- by viruses that contain viral genetic components myxoviridae family (180). The genome of infl uenza from avian infl uenza (AI) viruses. At the present viruses contains eight gene segments that code for time, highly pathogenic avian infl uenza (HPAI) at least 11 proteins: HA, NA, matrix proteins M1 H5N1 viruses are panzootic in poultry and pose an and M2, nonstructural proteins NS1 and NS2, the ominous threat to global public health due to their nucleocapsid protein (NP), polymerase basic protein ability to infect humans, often with fatal conse- 1 (PB1), polymerase basic protein 2 (PB2), poly- quences. While these viruses are now ineffi ciently merase acidic protein (PA), and a relatively recently transmitted from infected birds to humans there is discovered protein encoded by the PB1, PB1-F2 a danger that H5N1 HPAI viruses might gain the protein (21). Human infl uenza viruses are classifi ed ability to transmit among humans in a sustained and as type A, B, or C on the basis of antigenic differ- effi cient manner and cause the next infl uenza pan- ences in the nucleoprotein and matrix proteins (59). demic. There is no certainty that these H5N1 HPAI The functions of these proteins are described in viruses will cause the next infl uenza pandemic, detail elsewhere (25, 76). but there is a particular concern regarding H5N1 Human infl uenza is a highly contagious acute HPAI infl uenza viruses because of the unusually respiratory disease caused by infection with human high case-fatality ratio among infected humans and infl uenza viruses. Although usually a self-limited the devastating consequences that might occur if disease, complications of infl uenza result in sub- these viruses gain the ability to transmit effi ciently stantial morbidity and mortality worldwide. Human among humans. However, predicting precisely when infl uenza viruses cause an annual toll of approxi- the next infl uenza pandemic will occur and the mately 300,000 to 500,000 deaths worldwide, and particular virus hemagglutinin (HA) and neuramini- in pandemic years this number increased to approx- dase (NA) subtypes that will cause it is impossible. imately 1 million in the 1957–1958 pandemic and In this context, it must be emphasized that HPAI an estimated 40 to 50 million during the devastating viruses along with low pathogenic avian infl uenza 1918–1919 pandemic (7, 99, 164). An annual (LPAI) viruses circulating in poultry have both dem- average of more than 200,000 hospitalizations and onstrated the ability to infect humans. Here we 36,000 deaths attributable to complications from provide a brief background describing the impact, infl uenza occur in the United States (142, 143). virology, epidemiology, and control of seasonal The public health impact of infl uenza is primarily human infl uenza and discuss the public health due to infl uenza A and B virus infections. Type A implications of both H5N1 HPAI viruses and those viruses are further divided into subtypes by anti- of other AI viruses that have infected humans genic differences in the two main surface glycopro- (Table 20.1). teins, HA and NA. While 16 HA and 9 NA subtypes

Avian Influenza Edited by David E. Swayne 453 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 Table 20.1. Human illness from reported infections with avian inﬂ uenza viruses, 1996—July 16, 2007.a No. of cases Virus Location(s) Year(s) [ages affected] Clinical ﬁ ndings

H7N2 LPAI United States 2002, 2003 1, 1 [adults] Infl uenza-like illness H7N2 LPAI United Kingdom 2007 4 [adults] Infl uenza-like illness, lower respiratory tract disease, conjunctivitis H7N3 LPAI United Kingdom 2006 1 [adult] Conjunctivitis H7N3 LPAI Canadab 2004 1 [adult] Conjunctivitis H7N7 LPAI United Kingdom 1996 1 [adult] Conjunctivitis H9N2 LPAI Hong Kong SAR of 1999, 2003, 2007 2, 1, 1 [children] Infl uenza-like illness China H9N2 LPAI China 1998, 1999 4, 1 [adults, child] Infl uenza-like illness H7N3 HPAI Canada 2004 1 [adult] Conjunctivitis H7N7 HPAI Netherlands 2003 89 (1 death) Infl uenza-like illness, [primarily adults] conjunctivitis, severe pneumonia with ARDS H5N1 HPAI Hong Kong SAR of 1997 18 (6 deaths) Infl uenza-like illness, China [adults, children] pneumonia, multiorgan failure, Reye syndrome, reactive hemophagocytosis H5N1 HPAI Hong Kong SAR of 2003 2 (1 death) Infl uenza-like illness, China; travelers [adult, child] pneumonia, ARDS visited Fujian Province, China H5N1 HPAI China 2003 1 (1 death) Pneumonia, ARDS, 2005 8 (5 deaths) multiorgan failure 2006 13 (8 deaths) 2007 3 (2 deaths) [adults, children] H5N1 HPAI Vietnam 2003 3 (3 deaths) Pneumonia, ARDS, 2004 29 (20 deaths) multiorgan failure, 2005 61 (19 deaths) encephalitis 2007 2 (0 deaths) [adults, children] H5N1 HPAI Thailand 2004 17 (12 deaths) Pneumonia, ARDS, 2005 5 (2 deaths) multiorgan failure 2006 3 (3 deaths) [adults, children] H5N1 HPAI Indonesia 2005 20 (13 deaths) Infl uenza-like illness, 2006 55 (45 deaths) pneumonia, ARDS, 2007 27 (23 deaths) multiorgan failure [adults, children] H5N1 HPAI Cambodia 2005 4 (4 deaths) Pneumonia, ARDS, 2006 2 (2 deaths) multiorgan failure 2007 1 (1 death) [adults, children]

454 20 / Public Health Implications of Avian Influenza Viruses 455

Table 20.1. Continued No. of cases Virus Location(s) Year(s) [ages affected] Clinical ﬁ ndings

H5N1 HPAI Azerbaijan 2006 8 (5 deaths) Pneumonia, ARDS, [children] multiorgan failure H5N1 HPAI Djibouti 2006 1 [child] Infl uenza-like illness H5N1 HPAI Egypt 2006 18 (10 deaths) Infl uenza-like illness, 2007 19 (5 deaths) pneumonia, ARDS [adults, children] H5N1 HPAI Iraq 2006 3 (2 deaths) Pneumonia, ARDS [adult, children] H5N1 HPAI Turkey 2006 12 (4 deaths) Infl uenza-like illness, [children] pneumonia, ARDS, multiorgan failure H5N1 HPAI Laos 2007 2 (2 deaths) Pneumonia, ARDS [adult, child] H5N1 HPAI Nigeria 2007 1 (1 death) Pneumonia, ARDS [adult] a Published data, selected conference presentations, or reported to WHO as of July 16, 2007. b Isolated during an H7N3 HPAI poultry outbreak; characterized as an LPAI virus infection. have been identifi ed in birds (163), currently only ate climates. Antigenic drift leads to seasonal infl u- infl uenza A subtypes H1N1, H1N2, and H3N2 enza epidemics of unpredictable and variable viruses have circulated widely among humans in severity that occur during winter months in temper- recent years. Seasons during which A (H3N2) ate climates of the northern (October to April) and viruses predominate generally have a greater impact southern (May to September) hemispheres. In tem- in terms of hospitalization and mortality than do perate climates, peak infl uenza activity may last for seasons when A (H1) or B viruses are circulating 6 to 8 weeks in communities, although infl uenza (131). Infl uenza viruses circulating in humans are virus infections may occur for several weeks longer. evolving continuously and unpredictably through a In tropical and subtropical countries, infl uenza activ- process called “antigenic drift” in which random ity generally occurs year-round with increases point mutations in the HA gene during replication during cooler months and rainy periods, although result in amino acid changes in the HA surface the epidemiology of infl uenza is less well described protein. These amino acid changes confer antigenic in many developing countries. differences in the evolving viruses. Relatively few Infl uenza attack rates are usually highest in pre- amino acid changes in the HA proteins of infl uenza school and younger school-aged children and can viruses can create a new variant that is antigenically lead to high school absenteeism, and high rates of distinguishable from previously circulating viruses. outpatient visits (41, 42, 47, 48, 89, 103). Infl uenza Antigenic variation and the consequent epidemio- patients can manifest different signs and symptoms logical behavior of infl uenza A viruses follow a depending on age, underlying chronic disease, and fairly uniform pattern, with each successive variant complications associated with infl uenza (16, 26, replacing the previous one such that co-circulation 112). While abrupt onset of febrile upper respiratory of distinct antigenic variants of a given subtype gen- disease is common, severe nonpulmonary manifes- erally occurs only for short periods. Intensive sur- tations (e.g., myocarditis, rhabdomyolysis, encepha- veillance in communities has shown that infl uenza litis), invasive bacterial co-infection, exacerbation activity can often be detected, albeit at low levels, of chronic disease, asymptomatic infection and mild during the summer months in countries with temper- illness all occur. Persons aged 65 years and older, 456 Avian Influenza individuals with certain chronic conditions (e.g., although some laboratories use a rapid culture cardiopulmonary disease), and young infants have method that allows virus to be detected within 18 to the highest hospitalization rates from complications 24 hours (187). Infl uenza viruses can be detected of infl uenza, and elderly persons have the highest directly in clinical samples using a number of sensi- mortality rates attributable to infl uenza, but deaths tive and specifi c fl uoroimmunoassays, radioimmu- have occurred in previously healthy children (8, 16, noassays, and enzyme immunoassays. These assays 62, 96, 112). are often less sensitive than virus isolation and Most infl uenza transmission is believed to be require specialized laboratory equipment and human-to-human via viral particles expelled in large reagents but can produce a result within a few hours droplets through coughing by a contagious person (67). to a susceptible person within very close proximity Numerous commercially available “point of care” (9). The role of small particle droplet nuclei and tests for rapid diagnosis of infl uenza are now avail- contact transmission of infl uenza viruses is unknown. able. Such tests use immunoassays that detect infl u- Human infl uenza viruses primarily infect noncili- enza viral proteins or detect viral NA activity in ated epithelial cells of the upper respiratory tract specimens. According to their design, “point of (but can also infect and replicate in lower respiratory care” tests either detect infl uenza A and B viruses tract tissue) (87). Following infection, the incuba- but do not distinguish between them, or they may tion period is generally 2 days, with a range of 1 to detect only infl uenza A viruses, or detect and distin- 4 days (39). Most infected children and adults shed guish between infl uenza A and B. In general, these infl uenza viruses beginning the day prior to illness diagnostic tests are useful for determining rapidly if onset for approximately 4 to 5 days after symptoms infl uenza is the cause of outbreaks in institutional or occur. Young infants can shed infl uenza viruses for other outbreaks and for documenting circulation of 1 to 3 weeks and immunosuppressed or immuno- infl uenza viruses in populations of patients. These compromised persons can shed viruses for longer tests have been reported to have a wide range of periods (43, 120). sensitivities (40% to 100%) and specifi cities (52% to 100%) (153) and are even less sensitive in detect- DIAGNOSIS ing H5N1 virus infections in humans (6, 38, 65, Infl uenza infections are diffi cult to identify reliably 104). by clinical examination and routine laboratory fi nd- Molecular methods now are applied widely to ings alone; use of diagnostic tests is essential for diagnose infl uenza virus infections and are likely to determining when infl uenza is circulating in the become the “gold standard” for virus detection due community and to guide patient management. Such to increased sensitivity compared to virus culture. tests include virus isolation (culture) and identifi ca- Reverse transcription of viral RNA followed by tion, direct detection of infl uenza virus in clinical amplifi cation with polymerase chain reaction (RT- specimens, rapid point of care tests, molecular PCR) is more rapid and sensitive than virus culture methods, and serological tests. and has been in wide use for several years (67). The Isolation of infl uenza viruses in cell culture or time required for RT-PCR assays is 6 to 8 hours eggs followed by hemagglutination-inhibition (HI) inclusive of the time required for RNA extractions testing to identify the type and subtype of virus has and analysis of the amplifi cation products. More been considered the “gold standard” for infl uenza recently, PCR methods using fl uorescent probes for diagnosis in humans. Viral isolates can then be detection and/or quantifi cation of amplifi ed DNA in typed, subtyped, and further characterized antigeni- real time have been adopted (37, 84). The use of cally and genetically. However, the sensitivity of real-time PCR shortens the time to results to approx- this technique depends on the timing and quality of imately 4 hours, increases sensitivity and specifi city specimen collections. The specimen can be inocu- of diagnosis, allows quantifi cation of the gene target, lated into a variety of cell culture systems or embry- and decreases the risk of PCR cross-contamination onated hens’ eggs (67) and virus growth in tissue through the use of a closed system (67). By using culture or allantoic fl uid is then detected by hemag- primers and probes that are specifi c for conserved glutination, hemadsorption, or cytopathic effect. genes such at the infl uenza M gene along those tar- Results usually are not available for 3 days or longer, geted to a specifi c set of infl uenza A HA and NA 20 / Public Health Implications of Avian Influenza Viruses 457 subtypes, it is possible to determine the type and to the need for paired sera, serodiagnosis of infec- subtype of an infl uenza infection within hours of tion is necessarily retrospective and is not useful for arrival of clinical specimens in the laboratory. Nev- patient management. Techniques for measuring ertheless, due to the rapid evolution of the HA and antibody against infl uenza in sera include HI using NA genes, it is necessary to constantly evaluate the a variety of types of red blood cells, virus neutraliza- need to modify primers or probes over time based tion, and enzyme immunoassays. In general, these on available sequence data for these genes. Alterna- tests are considered sensitive and may provide the tive molecular detection methods such as loop- only means for documenting infl uenza infection in mediated isothermal amplifi cation tests (61, 113) situations where appropriate respiratory specimens and DNA microarrays containing nucleic acid probes are not available. The microneutralization (MN) test to type and subtype infl uenza viruses (27, 80, 145) is generally more sensitive and specifi c than the HI are being developed to detect both human and avian test for detecting antibodies to human infl uenza infl uenza A viruses with pandemic potential, such as viruses and has become the “gold standard” for H5N1 and H9N2; however, these methods are not detection of antibodies to AI viruses in human sera. in routine use. This assay is sensitive and specifi c, can yield results In addition, it must be emphasized that despite the in 2 days, and can detect H5-specifi c antibody at high sensitivity and specifi city of the real-time RT- titers that could not be detected by the HI assay PCR assay and the ease and rapidity of the point-of- (122). care assays, it is crucial to use clinical specimens for viral isolation in order to have infl uenza viruses VIRUS ENTRY AND RELEASE FOR available to test for changes in antigenicity, genetic HUMAN AND AVIAN INFLUENZA VIRUSES sequences, and antiviral susceptibility; this informa- Current evidence suggests that during infection with tion is essential for public health purposes and the human infl uenza A viruses the HA protein attaches virus isolates are essential for production of licensed to sialic acid receptors present on glycoproteins and infl uenza vaccines. It is important to note that viral glycolipids located on nonciliated columnar epithe- isolation should be carried out in laboratory facili- lial cells of the respiratory tract. The HAs of human ties with Biosafety Level 3 enhancements for clini- infl uenza A viruses bind preferentially to sialic acid cal specimens obtained from persons suspected of (SA) residues linked to galactose by α-2,6 linkages having HPAI infections. World Health Organization present on these cells. In contrast, avian viruses pref- (WHO) guidelines for the safe handling of speci- erentially bind to sialic acid linked to galactose by mens from suspected H5N1 cases are posted at α-2, 3 linkages (121). These differences in the spec- http://www.who.int/csr/disease/avian_influenza/ ifi city of receptor binding for human and AI viruses guidelines/handlingspecimens/en/index.html. Infl u- were previously thought to provide a host species enza H5N1-specifi c RT-PCR testing conducted barrier that generally prevented avian viruses from under Biosafety Level 2 conditions is the preferred infecting humans. However, infections of humans method for diagnosis of human infection with H5N1. by HPAI viruses in 1997 during the Hong Kong The WHO has established criteria for accepting H5 H5N1 HPAI outbreak led to studies that have testing results from national reference laboratories increased our understanding of the presence of dif- (166). Ideally, clinical specimens should be made ferent terminal sialic acid receptors in various tissues available for confi rmation, viral culture, and detailed of the human respiratory tract. It has been shown analyses at WHO H5 Reference Laboratories (176). recently that epithelial cells of the terminal bronchi- In the United States, all state public health laborato- oles and alveoli have SA with both α-2,6 and α-2,3 ries, several local public health laboratories, and linkages and H5N1 HPAI viruses have been shown the Centers for Disease Control and Prevention to infect and replicate in ex vivo cultures of human (CDC) are able to perform infl uenza H5N1 RT-PCR lung fragments (101, 127, 155). Furthermore, it has testing and are the recommended sites for initial been shown that human airway epithelium harbors diagnosis. α-2,3 linked SA on ciliated cells, a fi nding that helps Infl uenza virus infections can also be detected by explain the ability of H5N1 and other avian viruses measuring increases in infl uenza-specifi c antibody to replicate in humans despite their avian virus–like between acute and convalescent serum samples. Due receptor specifi city (86, 87). Glycan arrays have 458 Avian Influenza revealed that the situation is more complex than diabetes mellitus, respiratory diseases, renal dis- initially thought; different viruses bind to receptor eases, and heart diseases. Understanding infl uenza structures such as sulfated and sialylated glycans pathogenesis is thus greatly complicated by the in addition to those structures described earlier. many different pathways that lead to severe disease Elucidation of the full range of host cell receptors and death. to which infl uenza viruses bind and the differences There is growing evidence that early cytokines in the ability of different infl uenza A viruses to produced at the site of infl uenza virus infection are bind these receptors prior to entering host cells involved in mediation of the clinical and pathologi- should provide a better understanding of interspe- cal manifestations of the disease. Infl uenza infected cies transmission. patients were shown to have their highest concentra- Mucins in the respiratory tract that are rich in tions of cytokines such as interleukin-6 (IL-6) and sialic acid provide another barrier to infection by interferon α (IFN-α) in nasal fl uids within 48 hours infl uenza viruses. Mucins are able to bind infl uenza of infection, correlating with the onset of fever (53). viruses entering the respiratory tract and thus trap A variety of other studies have shown that cytokines and prevent viruses from reaching cell surface recep- and chemokines, including IL-1α/β, tumor necrosis tors. By cleaving sialic acids in mucins, the NA factor α/β (TNF α/β), IL-6, INF α/γ, IL-8, and mac- allows the viruses to escape entrapment and allows rophage infl ammatory protein (MIP)-1 α can be them to infect host cells. Once a cell is infected and detected in nasopharyngeal washes of humans viral replication occurs, release of viral particles infected with human infl uenza viruses (13). Cyto- during budding from the host cell surface is medi- pathic effects occur after infection in the epithelial ated by the enzymatic activity of the viral NA, which cells of the respiratory tract and acute disease of the disrupts viral aggregates and facilitates release of airways and lungs takes place. Obstruction of small infectious virus particles (1). Thus the NA is critical airways and impaired diffusion capacity is common both for entry into host cells and release from them. in infl uenza infection (52, 60). Replication of infl u- The NA of human infl uenza viruses has a preference enza viruses in the respiratory tract also leads to cell for the hydrolyzation of human SA α-2,6 galactose damage through downregulation of host protein syn- linkage, while the opposite is true for the NAs of AI thesis, cytolytic activity, and apoptosis. Primary viruses. It appears that for optimal virus replication viral pneumonia, although rare, is often fatal and is a balance between the ability of the HA to bind and accompanied by infl ammation of the upper and attach to SA on the host cell surface of a particular lower respiratory tracts where hyaline membrane species and for the NA to allow virus entry and then coverage of alveolar walls together with extensive to facilitate virus release after replication has to be intra-alveolar edema and hemorrhage have been balanced, and therefore co-evolution of HA and NA observed (56, 85, 181). Development of secondary has occurred to match receptor specifi city in human bacterial pneumonia following infl uenza infection is and avian infl uenza A viruses (158). more common and is usually associated with Strep- tococcus pneumoniae, Staphylococcus aureus, or PATHOGENESIS Haemoplilus infl uenzae infectons as described else- Much remains to be learned about the pathogenesis where (83). Complete recovery from the symptoms of human infl uenza virus infections and the relation- of uncomplicated infl uenza often requires 1 to 2 ship of pathogenesis to the clinical manifestations weeks and can take longer. The high pathogenicity and complications associated with this disease. In of H5N1 infections in humans has been attributed to humans, the replication of seasonal infl uenza viruses higher levels of viral replication in the lower respira- is generally restricted to the epithelial cells of the tory tract, wider dissemination in a variety of organs upper and lower respiratory tract but can cause beyond the respiratory tract, and induction of hyper- severe constitutional symptoms. Infl uenza also cytokinemia. These features are described in the causes complications and death by a variety of H5N1 HPAI virus section below and in more detail means, including inducing primary viral pneumonia, elsewhere (109). secondary bacterial pneumonia in virus-damaged lungs, and an acute respiratory distress syndrome IMMUNITY TO INFLUENZA VIRUSES possibly associated with immune responses, and Both strain-specifi c humoral immunity and cellular by exacerbating serious chronic diseases such as immunity contribute to recovery from acute infl u- 20 / Public Health Implications of Avian Influenza Viruses 459 enza virus infection. While strain-specifi c virus-neu- First, antigenic characteristics of infl uenza viruses tralizing antibody directed against the HA is the isolated through WHO’s Global Infl uenza Surveil- primary immune mediator of protection against lance Network (GISN) are analyzed to detect new infection, antibody to the NA reduces the severity antigenic variants that have recently emerged. of disease through enhancing viral clearance (44). Second, epidemiological and virological data are Humoral immunity to HA proteins is acquired via combined to determine if the new variant is spread- natural infection or vaccination and is specifi c to the ing geographically in association with respiratory particular variant that previously infected an indi- disease outbreaks. Third, the ability of the existing vidual or that was received in the infl uenza vaccine. vaccine strains to induce an antibody response in CD4+ and CD8+ T cells also play an important role humans to the newly detected variants is examined in immunity to infl uenza and, in contrast to the in order to determine if immunization with the exist- strain-specifi c antibody response of humoral immu- ing vaccine could protect against disease caused by nity, tend to be more cross-reactive among subtypes the new variant. Vaccine formulations are updated by recognizing common epitopes on the surface of accordingly. internal viral proteins. CD4+ T cells provide help for Infl uenza virus strains are grown in embryonating the antibody response and the induction of CD8+ T chicken eggs for vaccine production. Most infl uenza cells, while CD8+ T cells are associated with the vaccines used are embryonating chicken egg-grown accelerated clearance of virus and recovery from and formaldehyde-inactivated, split virus pre- infection (141). The ability to recognize a given T- parations for intramuscular injection (108). Live- cell epitope is restricted by MHC molecules and attenuated cold-adapted infl uenza virus vaccine for depends on the HLA phenotype of an individual. intranasal administration is approved in the United Serum and mucosal anti-HA immunoglobulin M States for certain healthy persons (168). When cir- (IgM), IgG, and IgA antibodies can be detected after culating infl uenza virus strains are well matched by primary infl uenza infection. While IgM and IgA the vaccine strains, infl uenza vaccine is approxi- serum antibodies peak approximately 2 weeks after mately 70% to 90% effective in preventing infl uenza onset of symptoms and then decline, the IgG response illness in nonelderly persons and can prevent hospi- to HA peaks a few weeks after infection and may talizations and death in the elderly and persons with persist for years and are thought to be responsible chronic health conditions (39). When signifi cant for the protection of individuals over 20 years of age antigenic drift in circulating strains distinct from from infection during the reemergence of H1N1 vaccine strains occurs, infl uenza vaccine effective- viruses in 1977 after a 20-year absence from the ness is reduced. In the United States, annual infl u- human population. In primed individuals, infection enza vaccination is recommended for persons at with related infl uenza viruses results in a rise in high risk for complications from infl uenza, their serum IgG and IgA and mucosal IgA in many cases. household contacts, and for all health care providers A more complete description of acquired immunity (39). as well as the innate early host immune response is reviewed elsewhere (25). New Vaccine Strategies Research into new vaccines includes continuing PREVENTION development of tissue cell culture-grown vaccines, Annual infl uenza vaccination is the best public antigen sparing technologies, new adjuvants, recom- health intervention to prevent human infl uenza. binant protein vaccines, vector-based vaccines, Infl uenza vaccine is available in two trivalent for- DNA vaccines, and vaccines targeted at conserved mulations—inactivated and live-attenuated—that epitopes. The use of mammalian cells to replicate contain an A (H1N1), an A (H3N2), and a B virus infl uenza viruses for use in inactivated vaccine has strain. A semiannual strain selection process is coor- received a great deal of attention in recent years and dinated by the WHO to determine the composition is anticipated to provide several advantages, includ- of the northern and southern hemisphere infl uenza ing the potential availability of a product for use by vaccines (165). Updating of the three infl uenza virus those who are allergic to eggs or egg proteins. Anti- vaccine strains is based on global strain surveillance genic changes in viruses often occur when human data. Three types of data are used to make the WHO infl uenza viruses are grown in eggs. Similar changes infl uenza vaccine strain recommendations each year. are rarely seen in infl uenza viruses grown in 460 Avian Influenza mammalian tissue culture. Therefore, human infl u- surge in global vaccine demand will accompany the enza viruses grown in mammalian tissue cultures are emergence of a new pandemic virus, and therefore more likely to produce vaccines that are antigeni- new approaches for immunization strategies are cally more representative of viruses circulating in greatly needed to optimize protection of unprimed humans and therefore provide somewhat increased individuals when early production capacity for vaccine effectiveness (68, 97). vaccine antigen is expected to be limited. To over- Similarly, the diversity of infl uenza virus sub- come the inherent safety concerns surrounding pro- types and the time pressures for production of vac- duction of vaccines with HPAI viruses, alternative cines based on HA content have prompted a renewed strategies have been explored including use of re- effort to develop vaccines with broad protective verse genetic systems to generate modifi ed recom- effects by way of innate and adaptive immune binant strains, the use of baculovirus-expressed HA responses. Vaccines in development include those or related LPAI strains, and the use of adjuvants to targeting the ectodomain of M2 (M2e), which is enhance immunogenicity. Following the emergence very highly conserved among infl uenza A strains. of H5N1 HPAI viruses in humans in 1997, two Other conserved targets are also being examined. vaccine strategies were developed to overcome these Alternative strategies using vectors containing limitations. One approach was to use an antigeni- infl uenza HA or NA genes have been used to infect cally similar H5 LP vaccine strain, overcoming the tissue cultures in which the viral protein of interest need to grow and purify vaccine under high bio- (HA or NA) is produced in large quantities and can containment conditions. A surface-antigen vaccine be purifi ed for use as a vaccine. Clinical trials of based on the LPAI A/duck/Singapore/97 (H5N3) purifi ed HA or NA vaccines produced by using a virus that was antigenically similar to A/Hong baculovirus vector have demonstrated that these Kong/156/97 (H5N1) was administered with or vaccines are immunogenic and have reactogenicity without MF59 adjuvant in two doses of 7.5, 15, or profi les similar to those of vaccines produced by 30 μg of H5 HA given 3 weeks apart (102). Although viral replication (63, 75, 114). DNA vaccines, which both vaccines were well tolerated, the nonadjuvanted are based on plasmids containing the relevant viral vaccine was poorly immunogenic, with only a 36% gene(s), also have been investigated. In animal response rate after two 30-μg doses of vaccine. Indi- models, DNA vaccines can stimulate both humoral viduals who received the H5N3 vaccine formulated and cellular immune mechanisms (151); however, with MF59 achieved signifi cantly higher antibody when tested in humans the immune response was responses with a majority of individuals showing relatively poor. It has been shown that specifi c CpG seroconversion to the vaccine strain. Revaccination motifs in bacterial DNA have differential effects on with the same vaccine formulation 16 months later the immune response, and work is proceeding to substantially boosted antibody titers and increased identify optimal motifs for enhancing immunogenic cross-reactive antibodies against 2004 H5N1 viruses and protective effects of vaccines in humans (51, 88, in those receiving the adjuvanted, but not the non- 156). adjuvanted vaccine (135, 136). These results suggest that the use of adjuvants may be dose-sparing and Prepandemic Vaccines enhancing the cross-reactivity of the antibody Development of prepandemic H5N1 vaccines response. Another approach relied on production of against the HPAI viruses currently widely circulat- a baculovirus-expressed purifi ed H5 HA protein ing in birds have the highest priority for vaccine based on the HA gene cloned from the prototype development at present. However, H9 and H7 AI H5N1 strain. Unfortunately, even the highest dose subtypes, which have also transmitted from avian (two doses of 90 μg) of the baculovirus-expressed species to humans, are also considered a pandemic recombinant H5 vaccine elicited seroconversion in threat for which vaccines are needed. Because H2N2 only 52% subjects as measured by an MN assay, viruses continue to circulate in birds and previously unlike the results for the seasonal vaccine, where, in demonstrated their ability to cause the 1957 pan- a primed population, lower doses were immuno- demic, these viruses are also important candidates genic (146, 148). for vaccine development because individuals born Because LP antigenically similar viruses of the after 1968 lack immunity to this virus subtype. A H5 subtype have not been identifi ed, experimental 20 / Public Health Implications of Avian Influenza Viruses 461

H5N1 vaccines have been generated from human individuals were low and modest, respectively, in H5N1 isolates using reverse genetics technology these two studies. (95). This process had fi rst been used to generate In earlier studies, two traditional inactivated vac- reassortant vaccine candidates based on the HA and cines against avian H9N2 or an early human H2N2 NA genes from H5N1 strains isolated from humans were evaluated in human studies (54, 137). Stephen- in 1997 and either the internal protein genes of the son et al. (136) compared subunit versus whole virus A/Ann Arbor/6/60 ca vaccine donor virus (81) or A/Hong Kong/1073/99 (H9N2) vaccines in subjects PR8, the high growth donor for inactivated vaccines aged 18 to 60 years. Among individuals older than (138). Using reverse genetics, the H5 HA gene is 32 years a level of anti-H9 HA antibody response genetically modifi ed to remove the multibasic amino that was associated with protection followed a single acid motif associated with HP for chickens and the dose of the H9N2 vaccine. However, in unprimed resulting reassortant vaccine strain is avirulent for individuals younger than 32 years, even two doses experimentally infected chickens, and is attenuated of vaccine failed to induce antibody titers associated in mammalian species (160). A plasmid rescue- with protection in many recipients. In this naïve derived subvirion vaccine based on A/ population, a whole virus vaccine was more immu- Vietnam/1203/2004 (H5N1) virus was evaluated in nogenic than the subunit vaccine. In another study, 450 healthy adults receiving two doses of 7.5, 15, alum adjuvant was shown to enhance responses to 45, or 90 μg of antigen. The frequency of serum whole virus H9N2 and H2N2 vaccines and unprimed antibody response was highest among subjects individuals required two doses of vaccine to achieve receiving two doses of 90 μg; 54% of individuals maximal mean antibody titers (54). Vaccine doses receiving this vaccine dose achieved neutralizing of less than 15 μg formulated with adjuvant-induced antibody titers of 1 : 40 or greater and was well toler- antibody titers similar to those induced by nonadju- ated (147). In another study, an inactivated split vanted full-dose vaccine. Taken together, these vaccine based on A/Vietnam/1194/2004 (H5N1) studies suggest that in unprimed populations, two and administered in two doses of 7.5, 15, and 30 μg, doses of inactivated AI vaccines are necessary to with or without aluminum hydroxide adjuvant, was elicit a protective antibody response to AI viruses. evaluated in 300 healthy adults. The adjuvanted 30- μg formulation was safe and induced the highest rate Antivirals of response (67%) after two vaccinations (10). Antiviral medications are available for chemopro- Taken together, these early studies confi rmed previ- phylaxis and treatment of infl uenza. Two classes, ous fi ndings, suggesting that very high doses of the adamantanes (amantadine and rimantadine) and unadjuvanted H5 vaccine are needed to elicit anti- the NA inhibitors (oseltamivir and zanamivir), are body responses in a majority of subjects, and that approved in many countries. The adamantane drugs addition of an adjuvant may allow for some level of have activity against infl uenza A viruses only, high dose-sparing. levels of adamantane resistance have been detected More recent studies generally have shown that in recently circulating infl uenza A viruses, and use aluminum-containing adjuvants are inconsistent in of amantadine or rimantadine is not recommended their ability to enhance the immune response, in the United States (39, 108). In the United States, depending on the vaccine formulation and type and early treatment of infl uenza with oseltamivir is rec- the amount of adjuvant used per dose, and therefore ommended (39). Studies have shown that early anti- dosage-dependence and formulation difference viral treatment can decrease the signs and symptoms require additional study. In contrast, whole virus of infl uenza by approximately 1 day compared to H5N1 vaccines (70), a novel proprietary adjuvanted placebo (39). (AS) vaccine (55), and MF-59 adjuvanted vaccines (5) show considerable promise for improved im - INFLUENZA PANDEMICS munogenicity. Live-attenuated vaccines developed Rarely, the emergence of a novel infl uenza A subtype using a reverse genetics modifi ed 2004 H5N1 HPAI virus in humans can lead to a global infl uenza pan- vaccine (66) or using traditional reassortment with demic with widespread global morbidity and mortal- a H5N1 LPAI virus (123) have shown promise but ity over a short period of time. “Antigenic shift” antibody responses and virus shedding in unprimed refers to the emergence of a novel infl uenza A 462 Avian Influenza subtype virus in humans with a new HA, or a new the world is the Pandemic Alert Period, Phase 3, in HA and new NA. Antigenic shift can occur through which human infections with a new subtype (primar- direct transmission of a novel infl uenza A virus from ily H5N1 HPAI), but no human-to-human spread, or an animal host or through genetic reassortment at most rare instances of spread to a close contact. between human infl uenza A and animal infl uenza A The determination of a change in phases is made by viruses. Antigenic shift does not necessarily result the WHO in consultation with infl uenza and public in an infl uenza pandemic. In order for a novel infl u- health experts. enza A virus that infects humans to cause a pandemic, it must cause disease and, more important, HUMAN INFECTIONS WITH LOW acquire the ability for sustained human-to-human PATHOGENIC AVIAN INFLUENZA transmission. Sequencing of the eight genes of the A VIRUSES 1918 H1N1 pandemic virus revealed that this pan- A relatively small number of human infections with demic virus likely emerged by mutation and adapta- LPAI viruses of different subtypes have been tion to mammals of a wholly AI virus without reported to cause human illness (see Table 20.1). genetic reassortment (116, 117, 139, 140). In con- The majority of human infections with LPAI viruses trast, the H2N2 virus that caused the “Asian infl u- have been linked to direct contact with poultry. enza” pandemic in 1957–1958 and the H3N2 virus While some human infections were linked to expo- that caused the 1968–1969 “Hong Kong infl uenza” sures during poultry outbreaks of LPAI, other cases pandemic both originated through genetic resassort- have occurred from poultry contact without identi- ment between previously circulating human infl u- fi ed outbreaks of LPAI, for some cases of LPAI enza A and LPAI virus gene segments. The H2N2 human infections the source of exposure preceding virus contained HA, NA, and PB1 genes from an infection remains unknown. avian virus, whereas the H3N2 virus contained HA Illness from infection with LPAI viruses gener- and PB1 genes from an avian virus (143). The dev- ally has been mild clinically without serious com- astating 1918 H1N1 pandemic resulted in an esti- plications or fatal cases reported, and has ranged mated 50 to 100 million deaths worldwide during from focal mild signs and symptoms (e.g., conjunc- three observed “waves” (90). The 1957 H2N2 and tivitis) to more acute systemic illness (fever and 1968 H3N2 pandemics were relatively milder, but upper respiratory tract disease) with full recovery. still resulted in an estimated 70,000 and 34,000 The most common clinical fi nding with LPAI virus excess deaths, respectively, in the United States infection is conjunctivitis, which has been observed (58). Following the emergence of a pandemic virus, in adults with H7 subtype virus infections, including the virus may continue to circulate among humans a 43-year-old woman with unilateral conjunctivitis through antigenic drift with substantial public health due to H7N7 virus in the United Kingdom, 1996 impact. H3N2 viruses continue to evolve and circu- (74), a man with bilateral conjunctivitis due to H7N3 late among humans worldwide. virus in the United Kingdom, 2006 (94, 100), and The WHO issued a Global Infl uenza Preparedness adults with conjunctivitis due to H7N2 virus in the Plan that designates three periods (Interpandemic, United Kingdom, 2007 (98). In addition, severe uni- Pandemic Alert, and Pandemic) and six phases lateral conjunctivitis associated with H7N7 LPAI according to the risk of a pandemic. Each ascending virus of avian origin was reported in the United phase describes increasing human infections with States in 1976 when an experimentally infected seal novel infl uenza A viruses with pandemic potential sneezed in the face of an adult animal handler who and increasing risk of a pandemic described in phase became ill within 40 hours of this exposure (161). 6; this plan also outlines public health objectives at Infl uenza-like illness (ILI) with fever and upper the global and national levels and recommended respiratory tract signs and symptoms was reported actions (167). Public health goals for each phase are in two adults with H7N2 LPAI virus infection in the divided into the following fi ve categories: (1) plan- United States during 2002 and 2003, respectively ning and coordination; (2) situation monitoring and (17, 18, 36), and in three adults in the United assessment; (3) prevention and containment; (4) Kingdom during 2007 (98). ILI with fever and upper health system response; and (5) communications. As respiratory tract signs and symptoms were reported of mid-2007, WHO has continued to indicate that with H9N2 LPAI virus infection in one pediatric 20 / Public Health Implications of Avian Influenza Viruses 463 patient in China in 1999, four hospitalized children Likewise, the source of H9N2 virus infection aged 9 months to 10 years old in Hong Kong during could not be determined for a 1-year-old or a 4-year- 1999 through 2007, and four adult outpatients in old girl who were hospitalized in Hong Kong during China during 1998 (15, 32, 49, 50, 111, 154, 184). 1999 with fever and upper respiratory tract symp- There are no controlled clinical data about antiviral toms and in whom H9N2 virus was isolated from treatment of cases of LPAI virus infection. While nasopharyngeal specimens. Seroprevalence studies two H7N2 LPAI cases with respiratory disease were among family members and health care workers treated with oseltamivir in the United Kingdom in who had close contacts with two H9N2 cases did not 2007 (98), most cases of LPAI virus infection have detect any serologic evidence of human-to-human been confi rmed after resolution of illness without transmission of H9N2 viruses (154). A 5-year-old antiviral treatment. boy did not have contact with poultry and the source The relatively small number of detected human of his H9N2 virus infection in Hong Kong in 2003 infections with LPAI viruses has precluded analy- is unknown, and no information was available about tical risk factor studies. Case investigations have poultry exposures for other human H9N2 cases that observed that transmission of LPAI viruses to have been reported to date (32, 49, 184). humans has been associated with direct contact with Diagnosis of acute human infection with LPAI well-appearing poultry, with sick poultry, or with viruses has been predominantly through detection of material likely contaminated with poultry feces. In viral RNA by RT-PCR or viral isolation from con- 1996, a 43-year-old woman developed unilateral junctival, nasopharyngeal, nasal, and throat swabs. conjunctivitis 1 day after a piece of straw had con- Serological diagnosis of acute LPAI virus infection tacted her eye while she was cleaning a duck house has been reported for H7N2 LPAI in an adult who that contained 26 ducks of multiple breeds, none of had fever and respiratory illness without conjuncti- which were sick. H7N7 LPAI virus was isolated vitis in the United States. In this case, a serum spec- from a conjunctival swab specimen from the woman imen collected 10 days after symptom onset had an (74). A worker at a farm that experienced an out- H7N2 virus–neutralizing antibody titer of 1 : 80 or break of H7N3 LPAI among poultry in the United greater by MN assay and confi rmatory Western blot Kingdom during 2006 was diagnosed with conjunc- using the H7N2 LPAI virus strain that caused the tivitis, and H7N3 virus was isolated from conjunc- poultry outbreak. In addition, ELISA detected H7- tival, nasopharyngeal, and throat swabs (72, 100). A specifi c IgM antibodies (36). H7N2 IgG–neutraliz- culler who was involved with the disposal of poultry ing antibodies were detected, but there was no developed ILI with fever and upper respiratory tract evidence of H7-specifi c IgM antibodies in a serum symptoms and was confi rmed serologically with specimen obtained 5 months after illness onset. In a H7N2 virus infection during a widespread multistate human case of H7N3, serum obtained 28 days after outbreak of H7N2 LPAI among turkeys and chick- illness onset had low levels of H7N3 antibodies by ens at commercial poultry farms in the United States MN, HI, and Western blot assay (72). In two chil- during 2002 (17, 18). Four human infections with dren in whom H9N2 virus was isolated from naso- H7N2 LPAI virus were detected through RT-PCR pharyngeal aspirate specimens, serum obtained 35 by testing of conjunctival and respiratory specimens and 39 days after illness onset had detectable neu- from adults who had contact with poultry linked to tralizing, H9-specifi c IgM, and IgG titers (154). H7N2 outbreaks in the United Kingdom during Limited data are available about the risk of human 2007 (100). Three of these cases were hospitalized infection with LPAI viruses from exposure to poultry with lower respiratory disease and one had conjunc- or wild birds. No routine human surveillance for tivitis (100). However, the source of H7N2 LPAI LPAI virus infection is conducted, and because most virus infection could not be determined for an immu- human infections with LPAI viruses have been clin- nocompromised adult male who was hospitalized ically mild, it likely that such infections have been with febrile upper and lower respiratory tract illness underdetected. Small serosurveys have reported and in whom H7N2 virus was isolated from a respi- detection of antibodies to LPAI subtype viruses ratory specimen (CDC, unpublished data) (18). He using HI, single radial hemolysis (SRH), and MN denied any contact with well-appearing or sick assays. Sero-surveys conducted among participants poultry (CDC, unpublished data). in China, Taiwan, and Hong Kong using single 464 Avian Influenza radial hemolysis assay reported detection of anti- including live poultry market workers, poultry farm bodies to numerous infl uenza A virus subtypes, but workers, persons raising backyard poultry, people the titers were not provided (128). In a study of 149 visiting live poultry markets, poultry veterinarians, workers at a poultry farm in Peru, no evidence of wild bird handlers, zoo workers, subsistence hunters, neutralizing antibodies to any AI virus subtypes was and bird hunters, appears very rare, but additional found (105). Another study of 798 workers at farms studies are required to better quantify this risk. in Italy that experienced poultry outbreaks of H7N1 LPAI found no evidence of antibodies to H7N1 by HUMAN INFECTIONS WITH HIGHLY HI, MN, or SRH assays (115). Among 185 workers PATHOGENIC AVIAN INFLUENZA at Italian farms that experienced poultry outbreaks VIRUSES of H7N3 LPAI, 7 participants were found to be Human infections with HPAI viruses of different seropositive to H7N3 by MN and confi rmatory subtypes have caused human illness and have Western blot assays (115). In a study of 39 duck attracted much public health, government, and media hunters and 68 workers with wild duck exposures in attention in recent years. Most human cases of infec- the United States, 1 hunter was found to have an tion with HPAI viruses have been associated with H11N9-neutralizing antibody titer of 1 : 40, suggest- poultry exposure. The widespread ongoing epizootic ing possible H11N9 LPAI virus infection in the past of (H5N1) HPAI viruses has resulted in more than (45). Another study among 42 veterinarians and 66 300 human H5N1 cases with high mortality in 12 adult controls in the United States reported a sig- countries since 1997 (Fig. 20.1). As H5N1 HPAI nifi cant association between geometric mean neu- virus strains continue to circulate and evolve among tralizing antibody titers for H5, H6, and H7 LPAI poultry in many countries, further human H5N1 viruses in veterinarians compared to controls (93). virus infections are expected. In addition, HPAI Because LPAI viruses have infected nonavian infections with H7N3 and H7N7 viruses have been species such as pigs and seals, human infection with well documented. LPAI viruses from contact with other animals is possible (79, 161). H7 Highly Pathogenic Avian Infl uenza Viruses It is diffi cult to interpret the available published Illness from infection with H7 HPAI viruses has results from limited LPAI virus antibody cross- ranged from relatively mild illness (conjunctivitis) sectional seroprevalence studies for several reasons. with H7N3 and H7N7 to severe and fatal disease Some studies failed to specify criteria for a positive with H7N7 virus infection. Very mild illness was antibody titer, did not specify what viruses were reported for human infection with H7N3 viruses in used as antigens in the assays, and did not specify two persons involved in culling activities during a whether adsorption was performed using human large poultry outbreak of H7N3 HPAI in British infl uenza A viruses so that cross-reactive antibodies Columbia, Canada, during 2004. The fi rst case was to human infl uenza viruses were removed. Some a 40-year-old male poultry worker who was not studies considered very low antibody titers to be wearing protective goggles, and developed unilat- evidence for past LPAI virus infection. Internation- eral conjunctivitis and coryza three days after contact ally accepted standardized methods are not estab- with dead poultry (149). H7N3 virus was isolated lished for serological testing methods or for what from a nasal specimen from this individual. Although antibody titers constitute a positive result for detec- it was presumed that this H7N3 virus was HP, tion of LPAI virus antibodies, so it is not possible detailed analyses indicated that the poultry worker to compare fi ndings across different studies. Fur- was infected with an H7N3 LPAI virus strain, sug- thermore, the possible underdetection of antibodies gesting that a back mutation had occurred or that to LPAI viruses due to lack of a detectable humoral both H7N3 LPAI and H7N3 HPAI viruses were co- immune response with local infection (e.g., conjunc- circulating during this outbreak (132). The second tivitis), and the expected decline in detectable anti- case was a 45-year-old poultry worker, who was body levels with time after initial infection must wearing glasses that did not prevent direct eye be taken into consideration. Therefore, the risk of contact with a feather, and who developed unilateral LPAI virus transmission to humans among persons conjunctivitis and headache 1 day after exposure. in contact with domestic poultry and wild birds, H7N3 HPAI virus was isolated from a conjunctival 20 / Public Health Implications of Avian Influenza Viruses 465

Figure 20.1. Geographic distribution of human H5N1 cases reported to World Health Organization, November 2003 to July 11, 2007. Source: World Health Organization. swab from this individual (149). Both H7N3 cases prophylaxis with oseltamivir, and only three cases were treated with oseltamivir and recovered fully. received oseltamivir treatment (71). All cases recov- In 1959, a 46-year-old man who had traveled in ered fully except for a 57-year-old Dutch male vet- several countries in Asia, the Middle East, and erinarian, who was previously healthy, developed Europe was diagnosed with hepatitis and H7N7 high fever and severe headache without respiratory HPAI virus was reportedly isolated from a blood symptoms 2 days after visiting a farm with H7N7 specimen collected more than one month after he HPAI virus–infected chickens. Nine days after returned to the United States (31). He did not have exposure and 7 days after illness onset, he was hos- any respiratory symptoms and serological testing of pitalized with pneumonia and treated with antibiot- convalescent sera did not detect H7N7-neutralizing ics but not antiviral medication because initial antibodies; thus, the relationship of this virus to the workup at admission was negative for H7N7 virus disease is unknown (31). During a widespread out- by PCR. His condition deteriorated, he developed break of H7N7 HPAI among poultry at commercial bilateral pneumonia, multiorgan failure, respiratory farms in the Netherlands, 89 cases of human infec- failure, and the acute respiratory distress syndrome tion with H7N7 HPAI virus were identifi ed, with a (ARDS) and died 13 days after illness onset (40, 71). mean age of 30.4 years (range, 13 to 59 years) (71). H7N7 HPAI virus was detected by real-time RT- The majority of H7N7 cases (88%) had conjunctivi- PCR testing of bronchoalveolar lavage fl uid and tis only, while fi ve had ILI and conjunctivitis, two H7N7 virus was isolated from postmortem lung had ILI only, and four had other symptoms. Cullers tissue specimens (40). The H7N7 virus strain that and veterinarians had the highest estimated attack was isolated from the fatal case was shown to be rates for H7N7 virus infection. Only one of the con- distinct from the H7N7 strains isolated from another fi rmed H7N7 cases had been taking antiviral chemo- patient with conjunctivitis but similar to viruses 466 Avian Influenza isolated from birds in the same area (92) (Ron eight reported conjunctivitis or ILI, and 4 of 5 had Fouchier, personal communication, 2007). There are detectable HI antibodies to H7 virus. However, this no controlled clinical data about antiviral treatment study considered a positive H7 HI antibody titer to of human cases of H7 HPAI virus infection. be 1 : 10 or greater and overall, 33 of 56 participants Investigations of the H7N3 cases in Canada had detectable H7 antibodies although most had no suggest that direct contact with contaminated mate- health complaints, and none of the participants had rial was the likely route of transmission that resulted evidence of neutralizing antibodies to H7N7 virus in conjunctivitis in two poultry workers (149). In the when tested by using the MN assay (35). Because 2003 H7N7 HPAI outbreak in the Netherlands, 86 most H7 HPAI virus infections have been clinically cases of primary H7N7 virus infection were identi- mild such as conjunctivitis or ILI, it is likely that fi ed of an estimated 4500 people who were exposed additional H7 HPAI virus infections have been to H7N7 virus–infected poultry (71). Presumably, underdetected, even during recognized H7 HPAI these individuals were infected with H7N7 virus poultry outbreaks, and that serosurveys may not through direct or close contact with infected poultry detect all H7 HPAI infections. The limitations noted or contaminated material. In addition, three second- for serological detection of LPAI virus infections ary H7N7 cases were identifi ed in family members above also apply to serology for H7 HPAI virus who did not have exposure to poultry, but were in infections. contact with primary H7N7 cases, suggesting possible human-to-human transmission of H7N7 viruses H5N1 HPAI Virus (71). Two family members of a male poultry worker Although H5N1 HPAI virus was fi rst isolated from with H7N7 were also confi rmed to be infected with poultry in 1959, human infection with H5N1 virus H7N7 viruses, including his 13-year-old daughter was not recognized until 1997 when human H5N1 with conjunctivitis and ILI, and his 37-year-old wife HPAI cases were associated with poultry die offs in with conjunctivitis (71). Both received oseltamivir live poultry markets in Hong Kong (20). During the treatment and recovered. A 44-year-old father of a 1997 Hong Kong outbreak, 18 human cases with 6 poultry worker with H7N7 conjunctivitis developed deaths were identifi ed. No additional human H5N1 conjunctivitis 1 day after onset of H7N7-associated cases were reported until in early 2003 when two conjunctivitis in his son. None of the contacts of the H5N1 HPAI cases were identifi ed in Hong Kong veterinarian who died of H7N7 HPAI had evidence residents who had traveled to Fujian Province, of H7N7 virus infection (71). southern China, in early 2003 (110). By mid to late Diagnosis of acute human infection with H7 2003, it appears that H5N1 HPAI viruses had spread HPAI viruses has been predominantly through from southern China to Southeast Asia and caused detection of viral RNA by RT-PCR or viral isolation widespread poultry outbreaks in several Asian coun- from conjunctival swabs, respiratory specimens, or tries, with associated human H5N1 cases (162). lung tissue specimens. Serological diagnosis of Since late 2005, H5N1 HPAI viruses have spread acute H7 HPAI virus infection may be limited by among poultry to Europe, the Middle East, and the lack of detectable antibody response with local Africa. Die-offs of multiple wild bird species and infection (conjunctivitis) from H7N3 virus. Both poultry have been detected in more than 60 countries confi rmed H7N3 cases in Canada had no detectable as of mid-2007. From 1997 to mid-2007, more than H7N3 virus antibodies by serological testing of 300 human H5N1 cases were reported in the follow- serum collected greater than 21 days after illness ing 12 countries and regions (in chronological onset by HI or MN assays (149). There are no pub- order): Hong Kong SAR, China; Vietnam; Thailand; lished data available from studies that assessed the Cambodia; Indonesia; People’s Republic of China; neutralizing antibody response to H7N7 HPAI virus Turkey; Iraq; Azerbaijan; Egypt; Djibouti; Nigeria; infection in confi rmed H7N7 cases. and Lao PDR. Most human H5N1 cases have had Limited data are available about the risk of human severe disease with a case-fatality proportion of infection with H7 HPAI viruses from exposure to approximately 60%. poultry. One household cohort study of 62 family In the 1997 Hong Kong outbreak, while the members of 25 poultry worker index cases with median age was 9.5 years, human H5N1 cases H7N7 who did not have poultry exposure found that occurred in a wide age range (1 to 60 years), and 20 / Public Health Implications of Avian Influenza Viruses 467 nearly all cases were previously healthy. Among the with H5N1 virus infection and reported in June 2006 18 cases, 6 fatal cases occurred, including 2 children (186). The “second wave” of human H5N1 cases and 4 adults. The most signifi cant risk factor for in Vietnam (4 cases, 4 deaths) and Thailand (5 H5N1 cases was visiting a live poultry market in the cases, 4 deaths) occurred during August through week prior to illness onset (91). No further human October 2004 and was associated with increases in H5N1 cases were identifi ed after the Hong Kong poultry outbreaks of H5N1 HPAI. The “third wave” government implemented a widespread cull of of human H5N1 cases began in December 2004 approximately 1.4 million poultry, temporarily through mid-2005 with most cases reported in stopped importation of poultry from China, and Vietnam and Cambodia. It appears that a “fourth enacted measures to improve biosecurity in the wave” of H5N1 cases began in June/July 2005 with live poultry markets (20). An epidemiological study the fi rst H5N1 cases identifi ed in Indonesia, and conducted among health care workers that cared H5N1 cases identifi ed in new countries associated for H5N1 patients identifi ed two individuals who with the spread of clade 2 H5N1 viruses among had a four-fold rise in H5N1-neutralizing antibodies poultry from Asia to eastern Europe (Azerbaijan, in paired sera, suggesting that nosocomial transmis- Turkey) and the Middle East (Iraq, Egypt) and sion of H5N1 virus had occurred (11). A seroepide- Africa during the second half of 2005 through 2006. miological study of poultry workers and cullers In 2007, the fi rst human H5N1 cases in Nigeria and reported an estimated seroprevalence of H5N1- Laos were reported, and cases continued to occur in neutralizing antibodies of 10% among 1525 par- Egypt and Indonesia (Fig. 20.2). The increases in ticipants, suggesting that asymptomatic and mild human H5N1 cases have generally been correlated H5N1 LPAI or HPAI virus infections had occurred with seasonal increases in poultry outbreaks of following exposure to H5N1 virus-infected poultry H5N1. (12). The descriptive epidemiology of H5N1 cases In February 2003, two Hong Kong residents, a since early 2003 indicates that children and young 33-year-old man and his 9-year-old son, were hos- adults have been disproportionately affected. The pitalized and H5N1 virus was isolated from respira- median age of 256 H5N1 cases reported from 10 tory specimens from both cases (110). These two countries was 18 years (range, 3 months to 75 years) confi rmed H5N1 cases occurred among fi ve family (178). Most cases (89%) were younger than 40 members who traveled in late January 2003 from years. There were no statistically signifi cant differ- Hong Kong to Fujian Province, China. During their ences by sex across age groups. Overall mortality visit, one family member, a 7-year-old girl, devel- was 60%, with the highest case fatality in cases aged oped pneumonia and died. No H5N1 testing was 10 to 19 years (76%) and the lowest in cases aged performed. The remaining four surviving family 50 years or older (40%). For fatal cases, the median members returned to Hong Kong where the father duration from illness onset to death was 9 days and son became ill and were hospitalized. The man (range, 2 to 31 days). In Indonesia, the median age developed severe pulmonary disease, ARDS, and of 54 H5N1 cases was 18.5 years (range, 18 months died, while the son survived. The source of their to 45 years): 96.3% were younger than 40 years, H5N1 virus infections is unclear. 53% were younger than 20 years, and 24% were Widespread outbreaks of H5N1 HPAI among children younger than 10 years (126). Overall mor- domestic poultry occurred in Vietnam and Thailand tality in 54 Indonesian H5N1 cases was 76%, and and were associated with human H5N1 cases in mortality was higher in females than in males. these countries in what has been referred to as the Two analytical studies have confi rmed the obser- “fi rst wave” of H5N1 cases (November 2003 through vation from case investigations that direct contact March 2004) with 68% mortality. During this period, with sick or dead poultry is the major risk factor for Vietnam reported 22 cases and 15 deaths, and Thai- H5N1 and that H5N1 is primarily a zoonotic disease land reported 12 cases and 8 deaths. It should be (4, 34). Many cases have had direct contact with sick noted that a fatal H5N1 case in a 24-year-old man or dead backyard poultry, primarily chickens. One who was initially suspected to be a SARS case and observational study in Azerbaijan attributed trans- who died of respiratory failure in November 2003 mission of H5N1 virus to direct contact with dead in Beijing, China, was retrospectively confi rmed wild swans (defeathering) (46). An observational 468 Avian Influenza

30 100

90 25 80

70 20 60

15 50

40 No. of cases 10 30

20 5 10

0 0 y Jul Jul an Dec Feb Mar Apr May Jun Aug Sep OctNovDec Feb MarApr May Jun Aug Sep Oct Nov Dec FebMarAprMay JunJuly Aug Sep Oct NovDec Feb Mar Apr May JunJul 3-Nov 4-Jan 5-Jan 6-J 7-Jan Date of onset Viet Nam (N=93)* Thailand (N=25) Cambodia (N=7) Indonesia (N=94)** China (N=25) Azerbaijan (N=8) Egypt (N=29)** Turkey (N=12)*** Iraq (N=2)** Nigeria (N=0)** Djibouti (N=1) Lao PDR (N=2) CFR Trend****

Figure 20.2. Epidemic curve of human H5N1 HPAI cases by onset date and country from November 2003 to June 25, 2007. Source: World Health Organization, Western Paciﬁ c Regional Ofﬁ ce, Manila, Philippines.

study in China found that six H5N1 cases in urban and may pose a theoretical risk of H5N1 virus trans- areas had no known contact with poultry that were mission to humans in close contact with infected sick or died of illness, but had visited a live poultry animals, no human H5N1 cases have been linked to market prior to illness onset, suggesting that envi- mammalian animal exposures to date. Drinking, ronmental exposures associated with visiting live bathing in, or swimming in H5N1 virus-contaminated poultry markets may be risk factors for H5N1 virus water is likely to pose a low, but unknown, risk for infection (183). For example, fomite contact or H5N1 virus transmission to humans. The role of in halation of aerosolized fecal matter or material multiple exposures to H5N1 virus or dose-response on poultry feathers contaminated with H5N1 viruses in transmission of H5N1 HPAI viruses to humans is could occur during visits to live poultry markets. A unknown. study of 54 H5N1 cases in Indonesia reported that a Limited, nonsustained, human-to-human trans- source of infection or exposure to H5N1 virus could mission of H5N1 HPAI viruses has been observed not be identifi ed for 24% of cases (178). Consump- rarely or could not be excluded in some cases in tion of uncooked coagulated duck blood or under- which very close prolonged contact with a severely cooked poultry have also been implicated as possible ill case at home or in a hospital occurred. This has sources of transmission of H5N1 virus (6). Contact occurred primarily, but not exclusively, among with fertilizer containing fresh poultry feces, sur- blood-related family members. A seroepidemiologi- faces contaminated with poultry or other animal cal study of health care workers in Hong Kong iden- feces, and self-inoculation of the respiratory tract are tifi ed two persons who had contact with H5N1 plausible transmission risks. While H5N1 HPAI patients, but denied poultry contact, and had sero- virus infections of many nonavian species have been logical evidence of H5N1 virus infection in 1997 documented, including pigs (22, 79), dogs (133), (11). Nosocomial transmission of H5N1 virus from cats (73, 78, 118, 134, 182), stone martens (170), an H5N1 case to a nurse was reported in Vietnam Owsten’s civets (119), and tigers and leopards (69), (6). Limited, nonsustained human-to-human trans- 20 / Public Health Implications of Avian Influenza Viruses 469 mission of H5N1 virus could not be excluded in at for H5N1 testing that were classifi ed as probable least two clusters in Indonesia in 2005 (65). Proba- H5N1 cases (126). Clusters are signifi cant because ble nosocomial transmission of H5N1 virus from an the fi rst signs that H5N1 virus strains have changed 11-year-old girl to her 26-year-old mother and 32- to transmit more easily to and among people might year-old aunt likely occurred through very close be an increase in the size of family clusters, an unprotected bedside contact while the girl was increase in the frequency of clusters, and an increase severely ill (152). Limited, nonsustained human-to- in cases among close non–blood-related family human-to-human transmission also is believed to members. Such developments would prompt urgent have taken place in a family cluster of eight H5N1 concern about an increasing risk of an H5N1 pan- cases with seven deaths in North Sumatra, Indone- demic. While every suspected and confi rmed human sia, during 2006 (171). Transmission from the index H5N1 case should be investigated promptly, it is case to six blood-related family members is believed imperative that every suspected and confi rmed to have occurred through very close unprotected cluster of H5N1 cases is investigated thoroughly. contact at the case’s home while she was very sick, Rapid understanding of the epidemiology, clinical and with subsequent transmission from one case to characteristics, and virological fi ndings in such his son during very close unprotected contact in a case clusters are critical to facilitating rapid response hospital. Seroepidemiological studies conducted and early containment of a potential infl uenza among health care workers exposed to H5N1 patients pandemic. in 2004 reported no evidence of patient-to-health Few seroprevalence studies have been conducted care worker transmission of H5N1 virus (2, 82, since 1997 to assess the risk of human infection with 124). H5N1 HPAI viruses among persons exposed to Clusters of human H5N1 cases with at least two poultry. A cluster serosurvey found no evidence of epidemiologically linked confi rmed cases have been H5N1 virus–neutralizing antibodies among 351 par- identifi ed in several countries and account for more ticipants from 93 households in a rural Cambodian than 25% of all reported H5N1 cases. The earliest village where H5N1 poultry outbreaks and a human evidence of H5N1 clusters occurred in Hong Kong H5N1 case had occurred (157). The serosurvey was during the 1997 outbreak when two pediatric H5N1 conducted approximately 2 months after poultry cases were identifi ed among fi rst cousins who played outbreaks occurred and the human H5N1 case was together but did not live in the same household (20). identifi ed. A seroepidemiological study among 901 The next cluster was among family members that participants from four rural Thai villagers where at had traveled to Fujian Province, China, in 2003 in least one human H5N1 case was identifi ed found no which two confi rmed H5N1 cases and one probable evidence of H5N1-neutralizing antibodies (30). A H5N1 case were identifi ed (110). The majority of serosurvey of 110 poultry market workers in Guang- H5N1 cluster cases to date are believed to have dong, China, found only one person with evidence resulted through avian-to-human transmission after of H5N1-neutralizing antibodies (159), and a similar common exposures, such as to sick or dead poultry study of 295 poultry workers in northern Nigeria or dead wild birds (46, 65, 104). Most clusters have found no evidence of H5N1-neutralizing antibodies involved two or three cases; the largest to date was (106). In all of these studies, participants had sub- eight (seven confi rmed, one probable) with seven stantial exposure to poultry that were likely to be deaths (171). Limited, nonsustained human-to- infected with H5N1 HPAI viruses. human transmission could not be excluded in some These limited cross-sectional seroprevalence clusters as described above. studies and surveys suggest that human infection More than 90% of H5N1 cluster cases have with H5N1 viruses is very rare even among persons occurred among blood-related family members, sug- in direct contact with sick and dead poultry. Given gesting possible genetic susceptibility, although the likelihood that millions of people have been in exposure, age, immunological, or other factors may direct or close contact with sick and dead poultry infl uence susceptibility to H5N1 virus infection infected with H5N1 viruses in many countries, (64). It is highly likely that the incidence and size avian-to-human transmission of H5N1 viruses of some clusters have been underdetected because remains a rare event, even though it is likely that specimens were not available from some individuals human H5N1 cases have been underdetected in 470 Avian Influenza many countries. However, some limitations should interstitial infi ltrates, and multisegmental and lobular be recognized. Some H5N1 virus–infected individu- consolidation (6) (Figs. 20.3 and 20.4). Progression als might not develop detectable levels of H5N1- to bilateral pneumonia and respiratory failure requir- neutralizing antibodies and H5N1 antibody levels ing invasive mechanical ventilation are common. decline over time and might not always be an accu- Complications in H5N1 patients include ARDS, rate indicator of previous H5N1 virus infection. multiorgan dysfunction with renal and cardiac There is no internationally accepted standardized disease, and disseminated intravascular coagulation serological assay for detection of H5N1 antibodies. (DIC) and a septic-like shock syndrome. DIC and Understanding of the natural history of the immune multiorgan failure were reported in an H5N1 case in response is incomplete for severe cases of confi rmed a woman in the fourth month of pregnancy in China H5N1 HPAI virus infection that survived. Similarly, (129). the natural history of the immune response for clin- Prolonged shedding of H5N1 virus in the respira- ically mild cases or asymptomatic H5N1 virus in- tory tract has been reported to 16 days and most fection is unknown. Collection of serial serum H5N1 patients are likely to be contagious for at least specimens from surviving H5N1 cases will help to 2 weeks (6). H5N1 viral RNA or isolation of H5N1 defi ne the kinetics of the immune response to H5N1 virus has been reported from rectal swab and diar- virus infection over time and interpret the results of rheal stool specimens from fatal cases (14, 28). H5N1 seroprevalence studies. H5N1 virus has been isolated from cerebrospinal Limited clinical data on H5N1 cases are available fl uid (3), serum (3, 129), and plasma (24) from crit- as of mid-2007. Most H5N1 clinical data have been ically ill patients who died. These fi ndings indicate published in case reports or small case series. The that H5N1 viremia occurs in severely ill patients in estimated incubation period for H5N1 cases appears the late stages of H5N1 disease and that this may to be approximately 2 to 5 days, and generally 1 contribute to the pathogenesis of H5N1, including week or less following exposure to sick or dead central nervous system involvement. One autopsy poultry (6, 23, 57, 104). The estimated incubation study reported fi nding mRNA in intestinal tissue, period is 4 to 9 days for cases in Thailand in which suggesting that H5N1 viral replication may be limited, nonsustained human-to-human transmis- occurring in the gastrointestinal tract (150). Further sion of H5N1 virus is believed to have occurred studies are needed to understand the signifi cance of (152). Early symptoms observed in H5N1 cases detection of H5N1 virus in patients with diarrhea include high fever with signs and symptoms of and to elucidate the role of the gastrointestinal tract lower respiratory tract disease occurring within 1 to with H5N1 pathogenesis. 4 days, including cough, shortness of breath, While most H5N1 cases have had severe disease, dyspnea, and tachypnea. Other symptoms in the some clinically mild cases have been reported among early stages of H5N1 disease include headache, sore children. In the 1997 Hong Kong outbreak, 7 of 11 throat, diarrhea, vomiting, abdominal pain, myalgia, confi rmed pediatric H5N1 cases were clinically mild and rhinorrhea. (uncomplicated infl uenza), and 4 were severe, with While nearly all H5N1 cases have presented to 2 deaths (20). The extent and frequency of clinically hospital with fever, pneumonia and hypoxia, atypi- mild and asymptomatic H5N1 cases are unknown, cal presentations such as fever with diarrhea, nausea primarily because surveillance has not focused on and vomiting (3), and fever, diarrhea, and seizures people with mild illness. At least four clinically mild progressing to coma with a clinical diagnosis of H5N1 patients have been identifi ed during fi eld encephalitis have been reported (28). The median investigations of more severe index cases in Turkey duration from illness onset to hospital admission in and Indonesia (64, 104). However, limited cross- 194 H5N1 cases was 4 days (range, 0 to 18 days) sectional serosurveys have identifi ed little evidence (178). Common laboratory fi ndings at admission in for mild illness or asymptomatic H5N1 virus infec- H5N1 cases include leukopenia, lymphopenia, mild tion (30, 157, 159). Additional seroprevalence to moderate thrombocytopenia, and elevated trans- studies are needed among family members and close aminases (6). Hypoalbuminemia has been reported contacts of H5N1 cases and in people exposed to (64). Chest radiographic fi ndings in H5N1 patients H5N1 virus-infected poultry, such as live market include diffuse, multifocal, or patchy infi ltrates, poultry workers, commercial poultry workers, Figure 20.3. Chest radiographic fi ndings in a fatal case of clade 2.1 H5N1 HPAI virus infection in a 37-year-old woman. Bilateral lower lobe consolidation with patchy infi ltrates in the upper lung fi elds were evident at admission on illness day 7. Despite mechanical ventilation, the patient progressed to acute respiratory distress syndrome (ARDS) on day 10 and died on illness day 11. Source: T. Uyeki, Centers for Disease Control and Prevention.

Figure 20.4. Chest radiographic fi ndings in a surviving clade 2.1 H5N1 HPAI virus infection of a 21-year-old man. Infi ltrates are present in the left mid-lung fi eld at admission on illness day 5. One week later, consolidation and diffuse infi ltrates are present throughout all lung fi elds. The patient recovered fully without mechanical ventilation. Source: T. Uyeki, Centers for Disease Control and Prevention.

471 472 Avian Influenza persons involved in culling activities, and people in pected or confi rmed H5N1 patients. The WHO has contact with backyard poultry, especially as H5N1 published updated infection control guidelines (173). viruses continue to evolve. Studies are also needed All respiratory secretions and all bodily fl uids, to investigate whether genetic or other factors, such including blood and feces of H5N1 patients should as those infl uencing expression of the host infl am- be considered potentially infectious. matory response, might infl uence disease severity Because most H5N1 patients have been admitted following H5N1 virus infection. late in their illness with severe disease, most com- The WHO has published guidance for investiga- monly with pneumonia, supplemental oxygen should tions of suspected human H5N1 cases (179). Case- be administered along with other supportive mea- fi nding in most countries has focused on hospitalized sures, such as appropriate fl uid management and patients with severe respiratory disease who had a invasive mechanical ventilation for respiratory history of poultry contact. Collection of the appropri- failure. Although there are no data from controlled ate respiratory specimens from suspected cases is clinical trials, antiviral treatment with oseltamivir critical because throat swabs have been shown to is recommended for all cases, and treatment should have a higher yield for detection of H5N1 virus than be initiated as soon as possible (125). The optimal nasopharyngeal or nasal swabs. Lower respiratory dose and duration of oseltamivir treatment is tract specimens have the highest yield for H5N1 unknown and higher doses and longer duration can virus, and have higher viral load than nasal or throat be considered (172). Combination treatment with swabs (28). Collection of serial respiratory specimens oseltamivir and amantadine can be considered in from multiple sites on multiple days from suspected countries with known or likely amantadine-sensitive H5N1 patients will increase the chances of detecting H5N1 viruses. Resistance to amantadine and riman- H5N1 virus. Guidance on collection, transportation, tadine has been reported for clade 1 and 2.1 H5N1 and shipping of clinical specimens is available (177). viruses. Oseltamivir resistant H5N1 viruses have Confi rmation of H5N1 virus infection can be carried been documented in case reports (29, 77). Cortico- out as described in the Diagnosis section. Case defi ni- steroids are not recommended, except for persistent tions for classifi cation of H5N1 cases are available septic shock with adrenal insuffi ciency (125). Anti- (case under investigation; suspected; probable; and biotic chemoprophylaxis is not recommended and confi rmed), and WHO requests that probable and antibiotic treatment should be based on evidence- laboratory-confi rmed H5N1 cases should be reported based guidelines for community-acquired pneumo- (175). Human H5N1 cases are now required to be nia and guided by microbiological laboratory testing reported to the WHO within 24 hours of diagnosis results (125). The fi ndings of animal studies suggest under the International Health Regulations (174). that more research into treatment with H5N1-neu- Clinical management of suspected and confi rmed tralizing antibodies and passive immunotherapy is H5N1 patients is focused on medical care for the needed (130). Treatment of one H5N1 patient with patient and implementation of appropriate infection convalescent serum from a recovered H5N1 patient control procedures. Patients should be isolated has been reported (185). immediately and placed in a separate room. Infec- The pathogenesis of H5N1 virus infection appears tion control measures should be implemented to be driven by high viral replication and an abnor- promptly, including standard, contact, and droplet mal host infl ammatory response. H5N1 viruses bind precautions. This updated WHO guidance is based preferentially to cells bearing receptors with sialic on current understanding that human-to-human acid bound to galactose by α-2,3 linkages (SA α-2,3 transmission of H5N1 viruses is most likely via Gal) that are found predominantly in the lower respi- large droplets, and human-to-human transmission ratory tract on bronchiolar and alveolar cells (127, remains a very rare event to date. Airborne precau- 155). This may explain why most H5N1 patients tions should be followed for aerosol generating pro- initially develop signs and symptoms of lower respi- cedures. Personal protective equipment, including ratory tract disease and why nearly all H5N1 patients disposable gown, gloves, surgical mask, fi t-tested develop severe pulmonary disease. H5N1 viral load N95 or equivalent respirator for aerosol-generating is higher in lower respiratory tract specimens than in procedures, and eye goggles, should be worn by all upper respiratory tract specimens (28). However, health care workers and visitors in contact with sus- H5N1 virus has been isolated from upper respiratory 20 / Public Health Implications of Avian Influenza Viruses 473 tract specimens from some cases, usually in the late Antiviral chemoprophylaxis with oseltamivir stages of illness and H5N1 virus has been shown to (requires a prescription) is recommended up to 7 infect upper respiratory tract tissue (101). One obser- days after the last known exposure to poultry infected vational study found that high pharyngeal H5N1 with LPAI or HPAI viruses. Public health or medi- viral load was correlated with hypercytokinemia cal personnel should be responsible for active daily with proinfl ammatory cytokines and chemokines in monitoring of workers for compliance and adverse fatal H5N1 cases (28). High plasma levels of IL-6, events associated with oseltamivir chemoprophy- IL-8, and IL-10 and γ interferon were found in fatal laxis, and for any signs and symptoms of AI virus H5N1 cases compared to nonfatal cases or human infection, including ILI and conjunctivitis with infl uenza patients (28). This study suggested that LPAI viruses. Monitoring should be done up to 7 high replication of H5N1 viruses may trigger cyto- days after the last known exposure to infected kine dysregulation and that early antiviral treatment poultry. Self-monitoring by workers can also be may be essential to preventing hypercytokinemia. done if resources do not permit active monitoring of Extrapulmonary H5N1 viral dissemination into the all exposed workers by designated public health gastrointestinal tract (14, 28), cerebrospinal fl uid (3), staff. Public health offi cials should be informed of and blood (3, 24, 129) has been documented and may any illness, including minor signs and symptoms, be a factor in multiorgan dysfunction. Few H5N1 and appropriate clinical specimens (conjunctival, cases have had bacterial co-infections, although this nasal, throat specimens) collected for testing by real- could be due to empiric treatment with broad-spec- time RT-PCR should be performed for infl uenza A trum antibiotics and suboptimal microbiological (H1 and H3), and suspected AI subtypes at a quali- testing. Most of the pathogenesis with H5N1 appears fi ed laboratory. Paired acute and convalescent sera to be due directly to viral damage or a virus induced can also be collected for serological testing. abnormal host infl ammatory response. Hemophago- Responders should receive human infl uenza vaccine cytosis has been reported as a complication in some to decrease the risk of co-infection and possible H5N1 patients and may also be a result of hypercy- reassortment with human infl uenza A and AI viruses. tokinemia (144). The etiology of the marked lym- Interim guidance for responders is available (19, 33, phopenia observed in most H5N1 cases is not 107, 169). completely understood but could involve differential apoptosis induced by H5N1 virus. Further under- CONCLUSIONS standing of the pathogenesis of H5N1 virus infection AI viruses, both LPAI and HPAI viruses, have been may facilitate development of targeted therapies. transmitted to humans through direct poultry contact and pose ongoing threats to public health. Recent RECOMMENDED MEASURES FOR attention has focused on the widespread epizootic RESPONDERS TO LPAI AND HPAI and potential pandemic threat of H5N1 HPAI OUTBREAKS viruses, and this has resulted in advances in the Persons involved in culling and disinfection activi- understanding of the epidemiology, clinical aspects, ties and poultry workers involved in responding to and virology of human infections with H5N1 viruses. suspected poultry outbreaks of LPAI or HPAI should The fact that the H5N1 HPAI viruses have proved be equipped with appropriate personal protective to be very lethal, yet successful, pathogens in birds equipment (PPE) and educated about the signs and and are also lethal to a number of mammals, includ- symptoms of AI virus infection in poultry and in ing humans, has been devastating to economies and humans, biosecurity and infection control. The most families. This striking lethality of H5N1 infections important issues are to practice standard biosecurity for a variety of species and the spread of the HPAI practices and to be outfi tted with PPE (goggles, dis- to multiple continents have also provided a unique posable protective clothing and gloves, disposable window to view on an ongoing basis how a pan- fi t-tested N95 respirator or equivalent, boots that can demic virus might evolve from an AI virus that be disinfected), including following proper donning transmits rarely to humans. However, many unan- and removal, disinfection, and hand hygiene. Ideally, swered questions remain about avian-to-human a worker should be assigned to monitor compliance transmission of H5N1 viruses. Given that the past with PPE. two infl uenza pandemics were caused by novel 474 Avian Influenza infl uenza A viruses that arose through genetic reas- remain in our understanding of how interactions at sortment between LPAI and human infl uenza A the human-animal interface infl uence the risk of viruses, the potential public health impact of LPAI transmission of both HPAI and LPAI viruses to viruses must not be minimized, even though LPAI poultry workers and live poultry market workers. virus infections of humans have not been reported To better understand and monitor the public to cause severe or fatal illness. Furthermore, the health impact of AI viruses, a number of actions and continuing evolution of LPAI and HPAI viruses cir- activities are imperative. The key to reducing the culating among domestic poultry dictates that active public health risk and to prevent human infections surveillance of AI viruses infecting poultry, domes- with AI viruses is to prevent and control poultry ticated animals, wild birds, and people must be outbreaks of AI through improved biosecurity, ongoing to identify the emergence of other AI poultry vaccination, and rapid response to outbreaks, viruses that pose a pandemic threat. Improved viro- and to protect those at risk of exposure to infected logical surveillance will help to identify the emer- poultry. This requires improved collaboration, com- gence of novel infl uenza A virus subtypes of munication, and coordination between veterinary pandemic potential in humans, to facilitate antigenic health and public health authorities worldwide. and genetic characterization, and antiviral resistance Public health and animal health authorities must testing and to identify changes in the threat of these work closely side-by-side in outbreak investiga- viruses to public health. While H5N1 HPAI viruses tions. To facilitate these activities and to address a currently pose the greatest pandemic threat, other AI major gap in AI virus surveillance in many coun- viruses, including H7 and H9 LPAI viruses, and H7 tries, a long-term perspective is needed, and sus- HPAI viruses, have transmitted to humans, and tained funding and political commitment must be should be all considered pandemic threats. Although provided to develop and strengthen global epide- human infections with AI viruses appear to be rare, miological and laboratory capacity for infl uenza human infections with both LPAI and HPAI viruses viruses for both veterinary health and public health. in many countries have likely been underrecognized In addition, improving biosecurity and developing and undetected. and exercising national and local capacity to respond Much more research is needed to better under- rapidly to avian and human outbreaks and cases stand human infections with AI viruses, especially must remain very high priorities. While the surveil- how H5N1 HPAI virus infection of the respiratory lance gaps are greatest in developing countries, tract is initiated. Unanswered questions remain strengthening of surveillance and response capacity about the immune response to human infection with for AI viruses is needed worldwide. AI viruses and the most appropriate serological testing methods to ascertain prior or acute human REFERENCES infections with LPAI and HPAI viruses. The patho- 1. Air, G.M., A.A. Ghate, and S.J. Stray. 1999. genesis of HPAI virus infections of humans is not Infl uenza neuraminidase as target for antivirals. well understood, and whether there are genetic or Advances in Virus Research 54:375–402. other biological factors that can infl uence infection 2. Apisarnthanarak, A., S. Erb, I. Stephenson, J.M. or disease severity following infection need to be Katz, M. Chittaganpitch, S. Sangkitporn, R. Kitphati, investigated. There are no proven therapies for P. Thawatsupha, S. Waicharoen, U. Pinitchai, P. HPAI virus infections and development of new Apisarnthanarak, V.J. Fraser, and L.M. Mundy. treatments is urgently needed. For example, the 2005. Seroprevalence of anti-H5 antibody among benefi t of combination antiviral administration and Thai health care workers after exposure to avian passive immunotherapy for treatment of H5N1 infl uenza (H5N1) in a tertiary care center. Clini- cal Infectious Diseases 40(2):e16–e18. HPAI virus infections must be studied as soon as 3. Apisarnthanarak, A., R. Kitphati, K. Thongphu- suitable patients can be identifi ed. The most signifi - beth, P. Patoomanunt, P. Anthanont, W. Auwanit, cant scientifi c advancements have been in our under- P. Thawatsupha, M. Chittaganpitch, S. Saeng- standing of the molecular characteristics of AI Aroon, S. Waicharoen, P. Apisarnthanarak, G.A. viruses, but major gaps remain in our understanding Storch, L.M. Mundy, and V.J. Fraser. 2004. of the epidemiology and clinical aspects of human Atypical avian infl uenza (H5N1). Emerging infections with AI viruses. Furthermore, major gaps Infectious Diseases 10(7):1321–1324. 20 / Public Health Implications of Avian Influenza Viruses 475

4. Areechokchai, D., C. Jiraphongsa, Y. Laosirita- son, L. Conn, X. Lu, N.J. Cox, and J.M. Katz. worn, W. Hanshaoworakul, and M. O’Reilly. 2002. Risk of infl uenza A (H5N1) infection 2006. Investigation of avian infl uenza (H5N1) among poultry workers, Hong Kong, 1997–1998. outbreak in humans—Thailand, 2004. Morbidity Journal of Infectious Diseases 185(8):1005– and Mortality Weekly Report 55(Suppl. 1):3–6. 1010. 5. Banzhoff, A., E. Montomoli, A. Hilbert, F. Laghi- 13. Brydon, E.W., S.J. Morris, and C. Sweet. 2005. Pasini, P.L. Capecchi, C. Gentile, P. di Giovanni, Role of apoptosis and cytokines in infl uenza virus T. Staniscia, A. Podda, P. Durando, L. Sticchi, morbidity. Federation of European Microbiologi- and R. Gasparini. 2007. MF59-adjuvanted infl u- cal Societies Microbiological Reviews 29(4):837– enza vaccine is well tolerated and effective at 850. low doses, offering a suitable vaccine for pre- 14. Buchy, P., S. Mardy, S. Vong, T. Toyoda, J.T. pandemic vaccination programs. In: Abstracts for Aubin, M. Miller, S. Touch, L. Sovann, J.B. the Options for the Control of Infl uenza VI, Dufourcq, B. Richner, P.V. Tu, N.T. Tien, W. Toronto, Canada, 201. Lim, J.S. Peiris, and W.S. Van der. 2007. Infl enza 6. Beigel, J.H., J. Farrar, A.M. Han, F.G. Hayden, A/H5N1 virus infection in humans in Cambodia. R. Hyer, J. de, S. Lochindarat, T.K. Nguyen, T.H. Journal of Clinical Virology 39(3):164–168. Nguyen, T.H. Tran, A. Nicoll, S. Touch, and K.Y. 15. Butt, K.M., G.J. Smith, H. Chen, L.J. Zhang, Yuen. 2005. Avian infl uenza A (H5N1) infection Y.H. Leung, K.M. Xu, W. Lim, R.G. Webster, in humans. New England Journal of Medicine K.Y. Yuen, J.S. Peiris, and Y. Guan. 2005. 353(13):1374–1385. Human infection with an avian H9N2 infl uenza 7. Beveridge, W.I. 1991. The chronicle of infl uenza A virus in Hong Kong in 2003. Journal of Clini- epidemics. History and Philosophy of the Life cal Microbiology 43(11):5760–5767. Sciences 13(2):223–234. 16. Call, S.A., M.A. Vollenweider, C.A. Hornung, 8. Bhat, N., J.G. Wright, K.R. Broder, E.L. Murray, D.L. Simel, and W.P. McKinney. 2005. Does this M.E. Greenberg, M.J. Glover, A.M. Likos, D.L. patient have infl uenza? Journal of the American Posey, A. Klimov, S.E. Lindstrom, A. Balish, Medical Association 293(8):987–997. M.J. Medina, T.R. Wallis, J. Guarner, C.D. 17. Centers for Disease Control and Prevention. Paddock, W.J. Shieh, S.R. Zaki, J.J. Sejvar, D.K. 2004. Update: Infl uenza activity—United States Shay, S.A. Harper, N.J. Cox, K. Fukuda, and and worldwide, 2003–2004 season, and composi- T.M. Uyeki. 2005. Infl uenza-associated deaths tion of the 2004–05 infl uenza vaccine. Morbidity among children in the United States, 2003–2004. and Mortality Weekly Report 53:547–552. New England Journal of Medicine 353(24):2559– 18. Centers for Disease Control and Prevention. 2567. 2004. Update: Infl uenza activity—United States, 9. Brankston, G., L. Gitterman, Z. Hirji, C. Lemieux, 2003–2004 Season. Morbidity and Mortality and M. Gardam. 2007. Transmission of infl uenza Weekly Report 53:284–287. A in human beings. Lancet Infectious Diseases 19. Centers for Disease Control and Prevention. 7(4):257–265. 2007. Interim guidance for protection of persons 10. Bresson, J.L., C. Perronne, O. Launay, C. Gerdil, involved in U.S. avian infl uenza outbreak dis- M. Saville, J. Wood, K. Hoschler, and M.C. ease control and eradication activities. Available Zambon. 2006. Safety and immunogenicity of an at http://www.cdc.gov/fl u/avian/professional/ inactivated split-virion infl uenza A/ protect-guid.htm. Centers for Disease Control Vietnam/1194/2004 (H5N1) vaccine: phase I ran- and Prevention: Atlanta, GA. domised trial. Lancet 367(9523):1657–1664. 20. Chan, P.K. 2002. Outbreak of avian infl uenza A 11. Bridges, C.B., J. Katz, W.H. Seto, P.K. Chan, (H5N1) virus infection in Hong Kong in 1997. D.N. Tsang, W. Ho, K.H. Mak, W. Lim, J.S. Clinical Infectious Diseases 34(Suppl. 2):S58– Tam, M. Clarke, S.G. Williams, A.W. Mounts, S64. J.S. Bresee, L. Conn, T. Rowe, J. Hu-Primmer, 21. Chen, W., P.A. Calvo, D. Malide, J. Gibbs, U. R.A. Abernathy, X. Lu, N.J. Cox, and K. Fukuda. Schubert, I. Bacik, S. Basta, R. O’Neill, J. 2000. Risk of infl uenza A (H5N1) infection Schickli, P. Palese, P. Henklein, J.R. Bennink, among health care workers exposed to patients and J.W. Yewdell. 2001. A novel infl uenza A with infl uenza A (H5N1), Hong Kong. Journal of virus mitochondrial protein that induces cell Infectious Diseases 181:344–348. death. Nature Medicine 7(12):1306–1312. 12. Bridges, C.B., W. Lim, J. Hu-Primmer, L. Sims, 22. Choi, Y.K., T.D. Nguyen, H. Ozaki, R.J. Webby, K. Fukuda, K.H. Mak, T. Rowe, W.W. Thomp- P. Puthavathana, C. Buranathal, A. Chaisingh, 476 Avian Influenza

P. Auewarakul, N.T. Hanh, S.K. Ma, P.Y. Hui, C. Jiraphongsa. 2007. Seroprevalence of antibod- Y. Guan, J.S. Peiris, and R.G. Webster. 2005. ies to avian infl uenza A (H5N1) virus among Studies of H5N1 infl uenza virus infection of pigs residents of villages with confi rmed human H5N1 by using viruses isolated in Vietnam and Thailand cases, Thailand, 2004–05. In: Abstracts for the in 2004. Journal of Virology 79(16):10821– Options for the Control of Infl uenza VI, Toronto, 10825. Canada, p. 18. 23. Chotpitayasunondh, T., K. Ungchusak, W. Han- 31. DeLay, P.D., H.L. Casey, and H.S. Tubiash. shaoworakul, S. Chunsuthiwat, P. Sawanpany- 1967. Comparative study of fowl plague virus alert, R. Kijphati, S. Lochindarat, P. Srisan, P. and a virus isolated from man. Public Health Suwan, Y. Osotthanakorn, T. Anantasetagoon, S. Reports 82(7):615–620. Kanjanawasri, S. Tanupattarachai, J. Weerakul, 32. Department of Health Press Release. 2007. The R. Chaiwirattana, M. Maneerattanaporn, R. Pool- Government of the Hong Kong Special Adminis- savathitikool, K. Chokephaibulkit, A. Apisarn- trative Region, March 20, 2007. Press Release. thanarak, and S.F. Dowell. 2005. Human disease 33. Department of the Interior. 2007. Employee from infl uenza A (H5N1), Thailand, 2004. Emerg- health and safety guidance for avian infl uenza ing Infectious Diseases 11(2):201–209. surveillance and control in wild birds. Available 24. Chutinimitkul, S., P. Bhattarakosol, S. Srisu- at http://medical.smis.doi.gov/avian%20safety% ratanon, A. Eiamudomkan, K. Kongsomboon, S. 20and%20health.pdf. Department of the Interior: Damrongwatanapokin, A. Chaisingh, K. Suwan- Washington, D.C. nakarn, T. Chieochansin, A. Theamboonlers, and 34. Dinh, P.N., H.T. Long, N.T. Tien, N.T. Hien, Y. Poovorawan. 2006. H5N1 infl uenza A virus T.Q. Mai le, l.H. Phong, l. Tuan, V, T.H. Van, and infected human plasma. Emerging Infectious N.B. Nguyen, T.P. Van, and N.T. Phuong. 2006. Diseases 12(6):1041–1043. Risk factors for human infection with avian infl u- 25. Cox, N.J., G. Neumann, R.O. Donis, and Y. enza A H5N1, Vietnam, 2004. Emerging Infec- Kawaoka. 2005. Orthomyxoviruses: infl uenza. tious Diseases 12(12):1841–1847. In: B. Mahy and V. Meulen (eds.). Topley and 35. Du Ry van Beest Holle, A. Meijer, M. Koopmans, Wilson’s Microbiology and Microbial Infections, and C.M. de Jager. 2005. Human-to-human 10th ed. Hodder Arnold: London, pp. 634–688. transmission of avian infl uenza A/H7N7, The 26. Cox, N.J. and K. Subbarao. 1999. Infl uenza. Netherlands, 2003. European Surveillance 10(12): Lancet 354(9186):1277–1282. 264–268. 27. Dawson, E.D., C.L. Moore, D.M. Dankbar, M. 36. Edwards, L.E., P. Terebuh, A. Adija, T. Rowe, J. Mehlmann, M.B. Townsend, J.A. Smagala, C.B. Kleene, J. Hu-Primmer, S. Jenkins, K. Fukuda, N. Smith, N.J. Cox, R.D. Kuchta, and K.L. Rowlen. Cox, J. Katz, and C. Bridges. 2004. Serological 2007. Identifi cation of A/H5N1 infl uenza viruses diagnosis of human infection with avian infl uenza using a single gene diagnostic microarray. Ana- (H7N2) virus. In: Abstracts of the International lytical Chemistry 79(1):378–384. Conference on Emerging Infectious Diseases, 28. de Jong, M.D., V.C. Bach, T.Q. Phan, M.H. Vo, Atlanta, GA, Abstract 60. T.T. Tran, B.H. Nguyen, M. Beld, T.P. Le, H.K. 37. Ellis, J.S., J.W. Smith, S. Braham, M. Lock, K. Truong, V.V. Nguyen, T.H. Tran, Q.H. Do, and Barlow, and M.C. Zambon. 2007. Design and J. Farrar. 2005. Fatal avian infl uenza A (H5N1) validation of an H5 TaqMan real-time one-step in a child presenting with diarrhea followed by reverse transcription-PCR and confi rmatory coma. New England Journal of Medicine assays for diagnosis and verifi cation of infl uenza 352(7):686–691. A virus H5 infections in humans. Journal of Clin- 29. de, J., T.T. Tran, H.K. Truong, M.H. Vo, G.J. ical Microbiology 45(5):1535–1543. Smith, V.C. Nguyen, V.C. Bach, T.Q. Phan, Q. 38. Fedorko, D.P., N.A. Nelson, J.M. McAuliffe, and H. Do, Y. Guan, J.S. Peiris, T.H. Tran, and J. K. Subbarao. 2006. Performance of rapid tests for Farrar. 2005. Oseltamivir resistance during treat- detection of avian infl uenza A virus types H5N1 ment of infl uenza A (H5N1) infection. New and H9N2. Journal of Clinical Microbiology England Journal of Medicine 353(25):2667– 44(4):1596–1597. 2672. 39. Fiore, A.E., D.K. Shay, P. Haber, J.K. Iskander, 30. Dejpichai, R., Y. Laosititaworn, P. Phutha- T.M. Uyeki, G. Mootrey, J.S. Bresee, and N.J. vathana, T. Uyeki, M. O’Reilly, N. Yampikul- Cox. 2007. Prevention and control of infl uenza. sakul, S. Phurahong, P. Poorak, J. Prasertsopon, Recommendations of the Advisory Committee on R. Kularb, K. Nateerom, N. Sawanpanyalert, and Immunization Practices (ACIP), 2007. Morbidity 20 / Public Health Implications of Avian Influenza Viruses 477

and Morality Weekly Report Recommended American Journal of Epidemiology 111(1):13– Report 56(RR–6):1–54. 22. 40. Fouchier, R.A., P.M. Schneeberger, F.W. 49. Guo, Y., J. Li, and X. Cheng. 1999. Discovery of Rozendaal, J.M. Broekman, S.A. Kemink, V. men infected by avian infl uenza A (H9N2) virus. Munster, T. Kuiken, G.F. Rimmelzwaan, M. Chinese Journal of Experimental and Clinical Schutten, G.J. Van Doornum, G. Koch, A. Virology 13(2):105–108. Bosman, M. Koopmans, and A.D. Osterhaus. 50. Guo, Y., M. Xie, J. Wang, J. Dang, Y. Guo, Y. 2004. Avian infl uenza A virus (H7N7) associated Zhang, and K. Wu. 2000. A strain of infl uenza A with human conjunctivitis and a fatal case of H9N2 virus repeatedly isolated from human pop- acute respiratory distress syndrome. Proceedings ulation in China. Chinese Journal of Experimen- of the National Academy of Sciences U S A tal and Clinical Virology 14:209–212. 101(5):1356–1361. 51. Gursel, M., D. Verthelyi, I. Gursel, K.J. Ishii, and 41. Fox, J.P., C.E. Hall, M.K. Cooney, and H.M. Foy. D.M. Klinman. 2002. Differential and competi- 1982. Infl uenzavirus infections in Seattle fami- tive activation of human immune cells by distinct lies, 1975–1979. I. Study design, methods and classes of CpG oligodeoxynucleotide. Journal the occurrence of infections by time and age. Leukocyte Biology 71(5):813–820. American Journal of Epidemiology 116(2):212– 52. Hall, W.J., R.G. Douglas, Jr., R.W. Hyde, F.K. 227. Roth, A.S. Cross, and D.M. Speers. 1976. Pulmo- 42. Frank, A.L., L.H. Taber, W.P. Glezen, E.A. nary mechanics after uncomplicated infl uenza A Geyer, S. McIlwain, and A. Paredes. 1983. Infl u- infection. American Review of Respiratory Dis- enza B virus infections in the community and the eases 113(2):141–148. family. The epidemics of 1976–1977 and 1979– 53. Hayden, F.G., R. Fritz, M.C. Lobo, W. Alvord, 1980 in Houston, Texas. American Journal of W. Strober, and S.E. Straus. 1998. Local and sys- Epidemiology 118(3):313–325. temic cytokine responses during experimental 43. Frank, A.L., L.H. Taber, C.R. Wells, J.M. Wells, human infl uenza A virus infection. Relation to W.P. Glezen, and A. Paredes. 1981. Patterns of symptom formation and host defense. Journal of shedding of myxoviruses and paramyxoviruses in Clinical Investigation 101(3):643–649. children. Journal of Infectious Diseases 144(5): 54. Hehme, N., H. Engelmann, W. Kunzel, E. Neu- 433–441. meier, and R. Sanger. 2002. Pandemic prepared- 44. Gerhard, W. 2001. The role of the antibody ness: lessons learnt from H2N2 and H9N2 response in infl uenza virus infection. Current candidate vaccines. Medical Microbiology and Topics in Microbiology and Immunology 260: Immunology 191(3–4):203–208. 171–190. 55. Hehme, N.W. 2007. GSK’s pandemic vaccine 45. Gill, J.S., R. Webby, M.J. Gilchrist, and G.C. development from 1st to 2nd generation candi- Gray. 2006. Avian infl uenza among waterfowl date vaccines. World Health Organization: hunters and wildlife professionals. Emerging Geneva. Infectious Diseases 12(8):1284–1286. 56. Hers, J.F., and J. Mulder. 1961. Broad aspects of 46. Gilsdorf, A., N. Boxall, V. Gasimov, I. Agayev, the pathology and pathogenesis of human infl u- F. Mammadzade, P. Ursu, E. Gasimov, C. Brown, enza. American Review of Respiratory Disease S. Mardel, D. Jankovic, G. Pimentel, I.A. Ayoub, 83(2)Pt 2:84–97. E.M. Elassal, C. Salvi, D. Legros, S.C. Pessoa da, 57. Hien, T.T., N.T. Liem, N.T. Dung, L.T. San, P.P. A. Hay, R. Andraghetti, G. Rodier, and B. Ganter. Mai, N.N. Chau, P.T. Suu, V.C. Dong, L.T. Mai, 2006. Two clusters of human infection with infl u- N.T. Thi, D.B. Khoa, L.P. Phat, N.T. Truong, enza A/H5N1 virus in the Republic of Azerbai- H.T. Long, L.T. Giang, N.D. Tho, L.H. Nga, N.T. jan, February-March 2006. European Surveillance Tien, L.H. San, L.V. Tuan, C. Dolecek, T.T. 11(5):122–126. Thanh, M. De Jong, C. Schultsz, P. Cheng, W. 47. Glezen, W.P. and R.B. Couch. 1978. Interpan- Lim, P. Horby, J. Farrar, and K.F.T.U. WHO demic infl uenza in the Houston area, 1974–76. International Avian Infl uenza Investigative team New England Journal of Medicine 298(11):587– 2004. Avian infl uenza A (H5N1) in 10 patients 592. in Vietnam. New England Journal of Medicine 48. Glezen, W.P., R.B. Couch, L.H. Taber, A. 350:1179–1188. Paredes, J.E. Allison, A.L. Frank, and C. Aldridge. 58. Hilleman, M.R. 2002. Realities and enigmas of 1980. Epidemiologic observations of infl uenza B human viral infl uenza: pathogenesis, epidemiol- virus infections in Houston, Texas, 1976–1977. ogy and control. Vaccine 20(25–26):3068–3087. 478 Avian Influenza

59. Horimoto, T., and Y. Kawaoka. 2005. Infl uenza: primary chick kidney cells. Journal of General lessons from past pandemics, warnings from Virology 73(Pt 5):1159–1165. current incidents. Nature Reviews Microbiology 69. Keawcharoen, J., K. Oraveerakul, T. Kuiken, 3(8):591–600. R.A. Fouchier, A. Amonsin, S. Payungporn, S. 60. Horner, G.J., and F.D. Gray, Jr. 1973. Effect of Noppornpanth, S. Wattanodorn, A. Theamboo- uncomplicated, presumptive infl uenza on the dif- niers, R. Tantilertcharoen, R. Pattanarangsan, N. fusing capacity of the lung. American Review of Arya, P. Ratanakorn, D.M. Osterhaus, and Y. Respiratory Disease 108(4):866–869. Poovorawan. 2004. Avian infl uenza H5N1 in 61. Imai, M., A. Ninomiya, H. Minekawa, T. Notomi, tigers and leopards. Emerging Infectious Diseases T. Ishizaki, M. Tashiro, and T. Odagiri. 2006. 10(12):2189–2191. Development of H5-RT-LAMP (loop-mediated 70. Kistner, O. 2007. Phase 1/2 clinical study with isothermal amplifi cation) system for rapid diag- Baxters H5N1 vaccine. World Health Organiza- nosis of H5 avian infl uenza virus infection. tion: Geneva. Vaccine 24(44–46):6679–6682. 71. Koopmans, M., B. Wilbrink, M. Conyn, G. 62. Izurieta, H.S., W.W. Thompson, P. Kramarz, Natrop, N.H. van der, H. Vennema, A. Meijer, D.K. Shay, R.L. Davis, F. DeStefano, S. Black, S.J. van, R. Fouchier, A. Osterhaus, and A. H. Shinefi eld, and K. Fukuda. 2000. Infl uenza Bosman. 2004. Transmission of H7N7 avian and the rates of hospitalization for respiratory infl uenza A virus to human beings during a large disease among infants and young children. outbreak in commercial poultry farms in the New England Journal of Medicine 342(4):232– Netherlands. Lancet 363(9409):587–593. 239. 72. Kuhne, M., O. Morgan, J. Ellis, P. Nair, T.G. 63. Johansson, B.E., P.M. Price, and E.D. Kilbourne. Wreghitt, M.D. Curran, R.J. Manvell, B.Z. Londt, 1995. Immunogenicity of infl uenza A virus N2 I.H. Brown, and M. Zambon. 2007. Human anti- neuraminidase produced in insect larvae by bacu- body response to avian infl uenza A (H7N3) virus lovirus recombinants. Vaccine 13(9):841–845. during an outbreak in poultry in Norfolk, United 64. Kandun, I.N., E.R. Sedyaningsih, and T.M. Uyeki. Kingdom. In: Abstracts of the Options for the 2007. Human H5N1 infl uenza—Dr. Kandun and Control of Infl uenza VI, Toronto, Canada, p. colleagues reply. New England Journal of Medi- 137. cine 356(13):1376–1377. 73. Kuiken, T., G. Rimmelzwaan, R.D. van, A.G. 65. Kandun, I.N., H. Wibisono, E.R. Sedyaningsih, van, M. Baars, R. Fouchier, and A. Osterhaus. Yusharmen, W. Hadisoedarsuno, W. Purba, H. 2004. Avian H5N1 infl uenza in cats. Science Santoso, C. Septiawati, E. Tresnaningsih, B. Her- 306(5694):241. iyanto, D. Yuwono, S. Harun, S. Soeroso, S. 74. Kurtz, J., R.J. Manvell, and J. Banks. 1996. Avian Giriputra, P.J. Blair, A. Jeremijenko, H. Kosasih, infl uenza virus isolated from a woman with con- S.D. Putnam, G. Samaan, M. Silitonga, K.H. junctivitis. Lancet 348(9031):901–902. Chan, L.L. Poon, W. Lim, A. Klimov, S. Lind- 75. Lakey, D.L., J.J. Treanor, R.F. Betts, G.E. Smith, strom, Y. Guan, R. Donis, J. Katz, N. Cox, M. J. Thompson, E. Sannella, G. Reed, B.E. Wilkin- Peiris, and T.M. Uyeki. 2006. Three Indonesian son, and P.F. Wright. 1996. Recombinant bacu- clusters of H5N1 virus infection in 2005. New lovirus infl uenza A hemagglutinin vaccines are England Journal of Medicine 355(21):2186– well tolerated and immunogenic in healthy adults. 2194. Journal of Infectious Diseases 174(4):838–841. 66. Karron, R. 2007. Phase 1 evaluation of live atten- 76. Lamb, R.A., and R.M. Krug. 2001. Orthomyxo- uated H9N2 and H5N1 ca reassortant vaccines in viridae: the viruses and their replication. In: B.N. healthy adults. World Health Organization: Fields, D.M. Knipe, and P.M. Howley (eds.). Geneva. Fields Virology, 4th ed. Lippincott Williams & 67. Katz, J.M., A.I. Klimov, S.E. Lindstrom, and N.J. Wilkins: Philadelphia, PA, pp. 1487–1531. Cox. 2006. Infl uenza viruses. In: B. Detrick, R.G. 77. Le, Q.M., M. Kiso, K. Someya, Y.T. Sakai, T.H. Hamilton, and J.D. Folds (eds.). Manual of Nguyen, K.H. Nguyen, N.D. Pham, H.H. Ngyen, Molecular and Clinical Laboratory Immunology, S. Yamada, Y. Muramoto, T. Horimoto, A. 7th ed. ASM Press: Washington, D.C., pp. 691– Takada, H. Goto, T. Suzuki, Y. Suzuki, and Y. 699. Kawaoka. 2005. Avian fl u: isolation of drug- 68. Katz, J.M., and R.G. Webster. 1992. Amino acid resistant H5N1 virus. Nature 437(7062):1108. sequence identity between the HA1 of infl uenza 78. Leschnik, M., J. Weikel, K. Mostl, S. Revilla- A (H3N2) viruses grown in mammalian and Fernandez, E. Wodak, Z. Bago, E. Vanek, 20 / Public Health Implications of Avian Influenza Viruses 479

V. Benetka, M. Hess, and J.G. Thalhammer. 89. Monto, A.S., J.S. Koopman, and I.M. Longini, Jr. 2007. Subclinical infection with avian infl uenza 1985. Tecumseh study of illness. XIII. Infl uenza A (H5N1) virus in cats. Emerging Infectious Dis- infection and disease, 1976–1981. American eases 13(2):243–247. Journal of Epidemiology 121(6):811–822. 79. Li, H.Y., K. Yu, H. Yang, X. Xin, J. Chen, P. 90. Morens, D.M., and A.S. Fauci. 2007. The 1918 Zhao, Y. Bi, and H. Chen. 2004. Isolation and infl uenza pandemic: insights for the 21st century. characterization of H5N1 and H9N2 infl uenza Journal of Infectious Diseases 195(7):1018– viruses from pigs in China. Chinese Journal of 1028. Preventive Veterinary Medicine 26:1–6. 91. Mounts, A.W., H. Kwong, H.S. Izurieta, Y. Ho, 80. Li, J., S. Chen, and D.H. Evans. 2001. Typing T. Au, M. Lee, B.C. Buxton, S.W. Williams, K. and subtyping infl uenza virus using DNA micro- H. Mak, J.M. Katz, W.W. Thompson, N.J. Cox, arrays and multiplex reverse transcriptase PCR. and K. Fukuda. 1999. Case-control study of risk Journal of Clinical Microbiology. 39(2):696– factors for avian infl uenza A (H5N1) disease, 704. Hong Kong, 1997. Journal of Infectious Diseases 81. Li, S., C. Liu, A. Klimov, K. Subbarao, M.L. 180(2):505–508. Perdue, D. Mo, Y. Ji, L. Woods, S. Hietala, and 92. Munster, V.J., W.E. de, R.D. van, W.E. Beyer, M. Bryant. 1999. Recombinant infl uenza A virus G.F. Rimmelzwaan, A.D. Osterhaus, T. Kuiken, vaccines for the pathogenic human A/Hong and R.A. Fouchier. 2007. The molecular basis of Kong/97 (H5N1) viruses. Journal of Infectious the pathogenicity of the Dutch highly pathogenic Diseases 179(5):1132–1138. human infl uenza A H7N7 viruses. Journal of 82. Liem, N.T., WHO Team, and W. Lim. 2005. Infectious Diseases 196(2):258–265. Lack of H5N1 avian infl uenza transmission to 93. Myers, K.P., S.F. Setterquist, A.W. Capuano, and hospital employees, Hanoi, 2004. Emerging G.C. Gray. 2007. Infection due to 3 avian infl u- Infectious Diseases 11(2):210–215. enza subtypes in United States veterinarians. 83. Louria, D.B., H.L. Blumenfeld, J.T. Ellis, E.D. Clinical Infectious Diseases 45(1):4–9. Kilbourne, and D.E. Rogers. 1959. Studies on 94. Nair, P., H. Mahgoub, O. Morgan, S. Ibbotson, infl uenza in the pandemic of 1957–1958. II. Pul- C. Sellwood, and J.S. Nguyen Van Tam. 2007. monary complications of infl uenza. Journal of Public health response to an outbreak of low Clinical Investigation 38(1 Part 2):213–265. pathogenicity H7N3 avian infl uenza in UK, 84. Mackay, I.M., K.E. Arden, and A. Nitsche. 2002. including an associated case of human conjuncti- Real-time PCR in virology. Nucleic Acids vitis. In: Abstracts of the Options for the Control Research 30(6):1292–1305. of Infl uenza VI, Toronto, Canada, p. 130. 85. Martin, C.M., C.M. Kunin, L.S. Gottlieb, M.W. 95. Neumann, G., T. Watanabe, H. Ito, S. Watanabe, Barnes, C. Liu, and M. Finland. 1959. Asian H. Goto, P. Gao, M. Hughes, D.R. Perez, R. infl uenza A in Boston, 1957–1958. I. Observa- Donis, E. Hoffmann, G. Hobom, and Y. Kawaoka. tions in thirty-two infl uenza-associated fatal 1999. Generation of infl uenza A viruses entirely cases. Archives of Internal Medicine 103(4):515– from cloned cDNAs. Proceedings of the National 531. Academy of Sciences U S A 96(16):9345–9350. 86. Matrosovich, M., N. Zhou, Y. Kawaoka, and R. 96. Neuzil, K.M., B.G. Mellen, P.F. Wright, E.F. Webster. 1999. The surface glycoproteins of H5 Mitchel, and M.R. Griffi n. 2000. The effect of infl uenza viruses isolated from humans, chickens, infl uenza on hospitalizations, outpatient visits, and wild aquatic birds have distinguishable prop- and courses of antibiotics in children. New erties. Journal of Virology 73(2):1146–1155. England Journal of Medicine 342(4):225–231. 87. Matrosovich, M.N., T.Y. Matrosovich, T. Gray, 97. Newman, R.W., R. Jennings, D.L. Major, J.S. N.A. Roberts, and H.D. Klenk. 2004. Human and Robertson, R. Jenkins, C.W. Potter, I. Burnett, L. avian infl uenza viruses target different cell types Jewes, M. Anders, D. Jackson, and J.S. Oxford. in cultures of human airway epithelium. Proceed- 1993. Immune response of human volunteers and ings of the National Academy of Sciences U S A animals to vaccination with egg- grown infl uenza 101(13):4620–4624. A (H1N1) virus is infl uenced by three amino acid 88. Moldoveanu, Z., L. Love-Homan, W.Q. Huang, substitutions in the haemagglutinin molecule. and A.M. Krieg. 1998. CpG DNA, a novel Vaccine 11(4):400–406. immune enhancer for systemic and mucosal 98. Nguyen-Van-Tam, J.S. 2007. Oral presentation, immunization with infl uenza virus. Vaccine Options for the Control of Infl uenza VI, Toronto, 16(11–12):1216–1224. Canada. 480 Avian Influenza

99. Nguyen-Van-Tam, J.S., and A.W. Hampson. Health and Safety Administration: Washington, 2003. The epidemiology and clinical impact of D.C. pandemic infl uenza. Vaccine 21(16):1762–1768. 108. Palese, P. 2006. Making better infl uenza virus 100. Nguyen-Van-Tam, J.S., P. Nair, P. Acheson, A. vaccines? Emerging Infectious Diseases 12(1):61– Baker, M. Barker, S. Bracebridge, J. Croft, J. 65. Ellis, R. Gelletlie, N. Gent, S. Ibbotson, C. Joseph, 109. Peiris, J.S., et al. 2007. Avian infl uenza virus H. Mahgoub, P. Monk, T.W. Reghitt, T. Sund- (H5N1): a threat to human health. Clinical Micro- kvist, C. Sellwood, J. Simpson, J. Smith, J.M. biology Reviews 20(2):243–267. Watson, M. Zambon, and N. Lightfoot. 2006. 110. Peiris, J.S., W.C. Yu, C.W. Leung, C.Y. Cheung, Outbreak of low pathogenicity H7N3 avian infl u- W.F. Ng, J.M. Nicholls, T.K. Ng, K.H. Chan, enza in UK, including associated case of human S.T. Lai, W.L. Lim, K.Y. Yuen, and Y. Guan. conjunctivitis. European Surveillance 11(5): 2004. Re-emergence of fatal human infl uenza A E060504. subtype H5N1 disease. Lancet 363(9409):617– 101. Nicholls, J.M., M.C. Chan, W.Y. Chan, H.K. 619. Wong, C.Y. Cheung, D.L. Kwong, M.P. Wong, 111. Peiris, M., K.Y. Yuen, C.W. Leung, K.H. Chan, W.H. Chui, L.L. Poon, S.W. Tsao, Y. Guan, and P.L. Ip, R.W. Lai, W.K. Orr, and K.F. Shortridge. J.S. Peiris. 2007. Tropism of avian infl uenza A 1999. Human infection with infl uenza H9N2. (H5N1) in the upper and lower respiratory tract. Lancet 354(9182):916–917. Nature Medicine 13(2):147–149. 112. Poehling, K.A., K.M. Edwards, G.A. Weinberg, 102. Nicholson, K.G., A.E. Colegate, A. Podda, I. Ste- P. Szilagyi, M.A. Staat, M.K. Iwane, C.B. Bridges, phenson, J. Wood, E. Ypma, and M.C. Zambon. C.G. Grijalva, Y. Zhu, D.I. Bernstein, G. Herrera, 2001. Safety and antigenicity of non-adjuvanted D. Erdman, C.B. Hall, R. Seither, and M.R. and MF59-adjuvanted infl uenza A/duck/Singa- Griffi n. 2006. The underrecognized burden of pore/97 (H5N3) vaccine: a randomised trial of infl uenza in young children. New England Journal two potential vaccines against H5N1 infl uenza. of Medicine 355(1):31–40. Lancet 357(9272):1937–1943. 113. Poon, L.L., C.S. Leung, K.H. Chan, J.H. Lee, 103. O’Brien, M.A., T.M. Uyeki, D.K. Shay, W.W. K.Y. Yuen, Y. Guan, and J.S. Peiris. 2005. Detec- Thompson, K. Kleinman, A. McAdam, X.J. Yu, tion of human infl uenza A viruses by loop- R. Platt, and T.A. Lieu. 2004. Incidence of out- mediated isothermal amplifi cation. Journal of patient visits and hospitalizations related to infl u- Clinical Microbiology 43(1):427–430. enza in infants and young children. Pediatrics 114. Powers, D.C., G.E. Smith, E.L. Anderson, D.J. 113(3 Pt 1):585–593. Kennedy, C.S. Hackett, B.E. Wilkinson, F. Vol- 104. Oner, A.F., A. Bay, S. Arslan, H. Akdeniz, H.A. vovitz, R.B. Belshe, and J.J. Treanor. 1995. Infl u- Sahin, Y. Cesur, S. Epcacan, N. Yilmaz, I. Deger, enza A virus vaccines containing purifi ed B. Kizilyildiz, H. Karsen, and M. Ceyhan. 2006. recombinant H3 hemagglutinin are well tolerated Avian infl uenza A (H5N1) infection in eastern and induce protective immune responses in Turkey in 2006. New England Journal of Medi- healthy adults. Journal of Infectious Diseases cine 355(21):2179–2185. 171(6):1595–1599. 105. Ortiz, E.J., T.J. Kochel, A.W. Capuano, S.F. Set- 115. Puzelli, S., T.L. Di, C. Fabiani, L. Campitelli, terquist, and G.C. Gray. 2007. Avian infl uenza M.A. De Marco, I. Capua, J.F. Aguilera, M. and poultry workers, Peru, 2006. Infl uenza and Zambon, and I. Donatelli. 2005. Serological anal- Other Respiratory Diseases 1:65–69. ysis of serum samples from humans exposed to 106. Ortiz, J.R., M.A. Katz, M.N. Mahmoud, S. avian H7 infl uenza viruses in Italy between 1999 Ahmed, S.I. Bawa, E.C. Farnon, M.B. Sarki, A. and 2003. Journal of Infectious Diseases Nasidi, M.S. Ado, A.H. Yahaya, T.M. Joannia, 192(8):1318–1322. R.S. Akpan, J. Vertefeuille, J. Achenbach, R.F. 116. Reid, A.H., T.G. Fanning, T.A. Janczewski, R.M. Breiman, J.M. Katz, T.M. Uyeki, and S.S. Wali. Lourens, and J.K. Taubenberger. 2004. Novel 2007. Lack of evidence for avian-to-human trans- origin of the 1918 pandemic infl uenza virus mission of avian infl uenza A (H5N1) among nucleoprotein gene. Journal of Virology poultry workers, Kano Nigeria, 2006. Journal of 78(22):12462–12470. Infectious Diseases 196:1685–1691. 117. Reid, A.H., J.K. Taubenberger, and T.G. Fanning. 107. OSHA. 2004. Avian Infl uenza. Protecting 2004. Evidence of an absence: the genetic origins Workers at Risk. Available at http://www. of the 1918 pandemic infl uenza virus. Nature osha.gov/dts/shib/shib121304.html. Occupational Reviews Microbiology 2(11):909–914. 20 / Public Health Implications of Avian Influenza Viruses 481

118. Rimmelzwaan, G.F., R.D. van, M. Baars, T.M. 127. Shinya, K., M. Ebina, S. Yamada, M. Ono, N. Bestebroer, A.G. van, R.A. Fouchier, A.D. Oster- Kasai, and Y. Kawaoka. 2006. Avian fl u: infl u- haus, and T. Kuiken. 2006. Infl uenza A virus enza virus receptors in the human airway. Nature (H5N1) infection in cats causes systemic disease 440(7083):435–436. with potential novel routes of virus spread within 128. Shortridge, K.F. 1992. Pandemic infl uenza: a and between hosts. American Journal of Pathol- zoonosis? Seminar on Respiratory Infections ogy 168(1):176–183. 7(1):11–25. 119. Robertson, S.I. 2006. Avian infl uenza H5N1 in 129. Shu, Y., H. Yu, and D. Li. 2006. Lethal avian viverrids: implications for wildlife health and infl uenza A (H5N1) infection in a pregnant conservation. Proceedings Biological Sciences woman in Anhui Province, China. New England 273(1595):1729–1732. Journal of Medicine 354(13):1421–1422. 120. Rocha, E., N.J. Cox, R.A. Black, M.W. Harmon, 130. Simmons, C.P., N.L. Bernasconi, A.L. Suguitan, C.J. Harrison, and A.P. Kendal. 1991. Antigenic K. Mills, J.M. Ward, N.V. Chau, T.T. Hien, F. and genetic variation in infl uenza A (H1N1) virus Sallusto, d.Q. Ha, J. Farrar, A. Lanzavecchia, and isolates recovered from a persistently infected K. Subbarao. 2007. Prophylactic and therapeutic immunodefi cient child. Journal of Virology effi cacy of human monoclonal antibodies against 65(5):2340–2350. H5N1 infl uenza. Public Library of Science Med- 121. Rogers, G.N., and J.C. Paulson. 1983. Receptor icine 4(5):e178. determinants of human and animal infl uenza 131. Simonsen, L., K. Fukuda, L.B. Schonberger, and virus isolates: differences in receptor specifi city N.J. Cox. 2000. The impact of infl uenza epidem- of the H3 hemagglutinin based on species of ics on hospitalizations. Journal of Infectious Dis- origin. Virology 127(2):361–373. eases 181(3):831–837. 122. Rowe, T., R.A. Abernathy, J. Hu-Primmer, W.W. 132. Skowronski, D.M., S.A. Tweed, M. Petric, T. Thompson, X. Lu, W. Lim, K. Fukuda, N.J. Cox, Booth, Y. Li, and T. Tam. 2006. Human illness and J.M. Katz. 1999. Detection of antibody to and isolation of low-pathogenicity avian infl u- avian infl uenza A (H5N1) virus in human serum enza virus of the H7N3 subtype in British Colum- by using a combination of serologic assays. bia, Canada. Journal of Infectious Diseases Journal of Clinical Microbiology 37(4):937–943. 193(6):899–900. 123. Rudenko, L., and A. Katlinsky. 2007. Evaluation 133. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- of Russian Live Attenuated Vaccine H5N2 in Heng, N. Pariyothorn, S. Payungporn, A. Theam- Clinical Trials. World Health Organization: boonlers, S. Chutinimitkul, R. Thanawongnuwech, Geneva. and Y. Poovorawan. 2006. Fatal avian infl uenza 124. Schultsz, C., V.C. Dong, N.V. Chau, N.T. Le, W. A H5N1 in a dog. Emerging Infectious Diseases Lim, T.T. Thanh, C. Dolecek, J. de, T.T. Hien, 12(11):1744–1747. and J. Farrar. 2005. Avian infl uenza H5N1 and 134. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- healthcare workers. Emerging Infectious Dis- Heng, N. Meemak, N. Pariyothorn, S. Payung- eases 11(7):1158–1159. porn, A. Theamboonlers, and Y. Poovorawan. 125. Schunemann, H.J., S.R. Hill, M. Kakad, R. 2006. Avian infl uenza H5N1 in naturally infected Bellamy, T.M. Uyeki, F.G. Hayden, Y. Yazdan- domestic cat. Emerging Infectious Diseases panah, J. Beigel, T. Chotpitayasunondh, M.C. 12(4):681–683. Del, J. Farrar, T.H. Tran, B. Ozbay, N. Sugaya, 135. Stephenson, I., R. Bugarini, K.G. Nicholson, A. K. Fukuda, N. Shindo, L. Stockman, G.E. Vist, Podda, J.M. Wood, M.C. Zambon, and J.M. Katz. A. Croisier, A. Nagjdaliyev, C. Roth, G. Thomson, 2005. Cross-reactivity to highly pathogenic avian H. Zucker, and A.D. Oxman. 2007. WHO Rapid infl uenza H5N1 viruses after vaccination with Advice Guidelines for pharmacological manage- nonadjuvanted and MF59-adjuvanted infl uenza ment of sporadic human infection with avian A/duck/Singapore/97 (H5N3) vaccine: a potential infl uenza A (H5N1) virus. Lancet Infectious Dis- priming strategy. Journal of Infectious Diseases eases 7(1):21–31. 191(8):1210–1215. 126. Sedyaningsih, E.R., S. Isfandari, V. Setiawaty, L. 136. Stephenson, I., K.G. Nicholson, A. Colegate, A. Rifati, S. Harun, W. Purba, S. Imari, S. Giriputra, Podda, J. Wood, E. Ypma, and M. Zambon. 2003. P.J. Blair, S.D. Putnam, T.M. Uyeki, and T. Soen- Boosting immunity to infl uenza H5N1 with doro. 2007. Epidemiology of cases of H5N1 virus MF59-adjuvanted H5N3 A/duck/Singapore/97 infection in Indonesia, July 2005–June 2006. vaccine in a primed human population. Vaccine Journal of Infectious Diseases 196(4):522–527. 21(15):1687–1693. 482 Avian Influenza

137. Stephenson, I., K.G. Nicholson, R. Gluck, R. infl uenza vaccine in young and elderly adults. Mischler, R.W. Newman, A.M. Palache, N.Q. Journal of Infectious Diseases 173(6):1467– Verlander, F. Warburton, J.M. Wood, and M.C. 1470. Zambon. 2003. Safety and antigenicity of whole 147. Treanor, J.J., J.D. Campbell, K.M. Zangwill, T. virus and subunit infl uenza A/Hong Kong/1073/99 Rowe, and M. Wolff. 2006. Safety and immuno- (H9N2) vaccine in healthy adults: phase I ran- genicity of an inactivated subvirion infl uenza A domised trial. Lancet 362(9400):1959–1966. (H5N1) vaccine. New England Journal of Medi- 138. Subbarao, K., H. Chen, D. Swayne, L. Mingay, cine 354(13):1343–1351. E. Fodor, G. Brownlee, X. Xu, X. Lu, J. Katz, N. 148. Treanor, J.J., B.E. Wilkinson, F. Masseoud, J. Cox, and Y. Matsuoka. 2003. Evaluation of a Hu-Primmer, R. Battaglia, D. O’Brien, M. Wolff, genetically modifi ed reassortant H5N1 infl uenza G. Rabinovich, W. Blackwelder, and J.M. Katz. A virus vaccine candidate generated by plasmid- 2001. Safety and immunogenicity of a recombi- based reverse genetics. Virology 305(1):192– nant hemagglutinin vaccine for H5 infl uenza in 200. humans. Vaccine 19(13–14):1732–1737. 139. Taubenberger, J.K. 2007. Discovery and charac- 149. Tweed, S.A., D.M. Skowronski, S.T. David, A. terization of the 1918 pandemic infl uenza virus in Larder, M. Petric, W. Lees, Y. Li, J. Katz, M. historical context. Antiviral Therapy 12:581– Krajden, R. Tellier, C. Halpert, M. Hirst, C. 591. Astell, D. Lawrence, and A. Mak. 2004. Human 140. Taubenberger, J.K., A.H. Reid, R.M. Lourens, R. illness from avian infl uenza H7N3, British Wang, G. Jin, and T.G. Fanning. 2005. Charac- Columbia. Emerging Infectious Diseases 10(12): terization of the 1918 infl uenza virus polymerase 2196–2199. genes. Nature 437(7060):889–893. 150. Uiprasertkul, M., P. Puthavathana, K. Sangsiri- 141. Thomas, P.G., R. Keating, D.J. Hulse-Post, and wut, P. Pooruk, K. Srisook, M. Peiris, J.M. Nich- P.C. Doherty. 2006. Cell-mediated protection in olls, K. Chokephaibulkit, N. Vanprapar, and P. infl uenza infection. Emerging Infectious Diseases Auewarakul. 2005. Infl uenza A H5N1 replication 12(1):48–54. sites in humans. Emerging Infectious Diseases 142. Thompson, W.W., D.K. Shay, E. Weintraub, L. 11(7):1036–1041. Brammer, C.B. Bridges, N.J. Cox, and K. Fukuda. 151. Ulmer, J.B. 2002. Infl uenza DNA vaccines. 2004. Infl uenza-associated hospitalizations in the Vaccine 20 (Suppl. 2):S74–S76. United States. Journal of the American Medical 152. Ungchusak, K., P. Auewarakul, S.F. Dowell, R. Association 292(11):1333–1340. Kitphati, W. Auwanit, P. Puthavathana, M. 143. Thompson, W.W., D.K. Shay, E. Weintraub, L. Uiprasertkul, K. Boonnak, C. Pittayawonganon, Brammer, N. Cox, L.J. Anderson, and K. Fukuda. N.J. Cox, S.R. Zaki, P. Thawatsupha, M. Chitta- 2003. Mortality associated with infl uenza and ganpitch, R. Khontong, J.M. Simmerman, and respiratory syncytial virus in the United States. S. Chunsutthiwat. 2005. Probable person-to- Journal of the American Medical Association person transmission of avian infl uenza A (H5N1). 289(2):179–186. New England Journal of Medicine 352(4):333– 144. To, K.F., P.K. Chan, K.F. Chan, W.K. Lee, W.Y. 340. Lam, K.F. Wong, N.L. Tang, D.N. Tsang, R.Y. 153. Uyeki, T.M. 2003. Infl uenza diagnosis and treat- Sung, T.A. Buckley, J.S. Tam, and A.F. Cheng. ment in children: a review of studies on clinically 2001. Pathology of fatal human infection associ- useful tests and antiviral treatment for infl uenza. ated with avian infl uenza A H5N1 virus. Journal Pediatric Infectious Diseases Journal 22(2):164– of Medical Virology 63(3):242–246. 177. 145. Townsend, M.B., E.D. Dawson, M. Mehlmann, 154. Uyeki, T.M., Y.H. Chong, J.M. Katz, W. Lim, J.A. Smagala, D.M. Dankbar, C.L. Moore, C.B. Y.Y. Ho, S.S. Wang, T.H. Tsang, W.W. Au, S.C. Smith, N.J. Cox, R.D. Kuchta, and K.L. Rowlen. Chan, T. Rowe, J. Hu-Primmer, J.C. Bell, W.W. 2006. Experimental evaluation of the FluChip Thompson, C.B. Bridges, N.J. Cox, K.H. Mak, diagnostic microarray for infl uenza virus surveil- and K. Fukuda. 2002. Lack of Evidence for lance. Journal of Clinical Microbiology Human-to-Human Transmission of Avian Infl u- 44(8):2863–2871. enza A (H9N2) Viruses in Hong Kong, China 146. Treanor, J.J., R.F. Betts, G.E. Smith, E.L. Ander- 1999. Emerging Infectious Diseases 8(2):154– son, C.S. Hackett, B.E. Wilkinson, R.B. Belshe, 159. and D.C. Powers. 1996. Evaluation of a recombi- 155. van Riel D., V.J. Munster, W.E. de, G.F. Rim- nant hemagglutinin expressed in insect cells as an melzwaan, R.A. Fouchier, A.D. Osterhaus, and 20 / Public Health Implications of Avian Influenza Viruses 483

T. Kuiken. 2006. H5N1 virus attachment to lower in humans from national reference laboratories. respiratory tract. Science 312(5772):399. Available at http://www.who.int/entity/csr/ 156. Verthelyi, D., R.T. Kenney, R.A. Seder, A.A. disease/avian_infl uenza/whoacceptancecriteria. Gam, B. Friedag, and D.M. Klinman. 2002. CpG pdf. World Health Organization: Geneva. oligodeoxynucleotides as vaccine adjuvants in 167. World Health Organization. 2005. World Health primates. Journal of Immunology 168(4):1659– Organization global infl uenza preparedness 1663. plan. Available at http://www.who.int/entity/csr/ 157. Vong, S., B. Coghlan, S. Mardy, D. Holl, H. resources/publications/influenza/GIP_2005_ Seng, S. Ly, M.J. Miller, P. Buchy, Y. Froehlich, 5Eweb.pdf. World Health Organization: Geneva. J.B. Dufourcq, T. Uyeki, W. Lim, and T. Sok. 168. World Health Organization. 2007. World Health 2006. Low frequence of poultry-to-human H5N1 Organization global infl uenza surveillance virus transmission, Southern Cambodia, 2005. network. Available at http://www.who.int/csr/ Emerging Infectious Diseases 12(10):1542– disease/infl uenza/surveillance/en/. World Health 1547. Organization: Geneva. 158. Wagner, R., M. Matrosovich, and H.D. Klenk. 169. World Health Organization/WPRO. 2004. World 2002. Functional balance between haemaggluti- Health Organization interim recommendations nin and neuraminidase in infl uenza virus infec- for the protection of persons involved in the mass tions. Reviews in Medical Virology 12(3): slaughter of animals potentially infected with 159–166. highly pathogenic avian infl uenza viruses. Avail- 159. Wang, M., B. Di, D.H. Zhou, B.J. Zheng, H. Jing, able at http://www.who.int/entity/csr/disease/ Y.P. Lin, Y.F. Liu, X.W. Wu, P.Z. Qin, Y.L. avian_infl uenza/guidelines/interim_recommen- Wang, L.Y. Jian, X.Z. Li, J.X. Xu, E.J. Lu, T.G. dations/en/index.html. World Health Organiza- Li, and J. Xu. 2006. Food markets with live birds tion/Western Pacifi c Regional Offi ce: Manila. as source of avian infl uenza. Emerging Infectious 170. World Health Organization. 2006. Avian infl u- Diseases 12(11):1773–1775. enza—H5N1 infection found in a Stone Marten 160. Webby, R.J., D.R. Perez, J.S. Coleman, Y. Guan, in Germany, March 9, 2006. Available at http:// J.H. Knight, E.A. Govorkova, L.R. Clain-Moss, www.who.int/csr/don/2006_03_09a/en/. World J.S. Peiris, J.E. Rehg, E.I. Tuomanen, and R.G. Health Organization: Geneva. Webster. 2004. Responsiveness to a pandemic 171. World Health Organization. 2006. Avian infl u- alert: use of reverse genetics for rapid develop- enza—situation in Indonesia—update 16. May ment of infl uenza vaccines. Lancet 363(9415): 31, 2006. Available at http://www.who.int/csr/ 1099–1103. don/2006_05_31/en/. World Health Organiza- 161. Webster, R.G., J. Geraci, G. Petursson, and K. tion: Geneva. Skirnisson. 1981. Conjunctivitis in human beings 172. World Health Organization. 2007. Clinical caused by infl uenza A virus of seals. New England management of human infection with avian infl u- Journal of Medicine 304(15):911. enza A (H5N1) virus. Available at http://www. 162. Webster, R.G., and E.A. Govorkova. 2006. H5N1 who.int/csr/disease_avian_infl uenza/guidelines/ infl uenza—continuing evolution and spread. New Clinical Management07.pdf. England Journal of Medicine 355(21):2174– 173. World Health Organization. 2007. Avian infl u- 2177. enza, including infl uenza A (H5N1) in humans: 163. Webster, R.G., M. Peiris, H. Chen, and Y. Guan. World Health Organization interim infection 2006. H5N1 outbreaks and enzootic infl uenza. control guideline for health care facilities. Avail- Emerging Infectious Diseases 12(1):3–8. able at http://www.who.int/csr/disease/avian_ 164. World Health Organization. 2007. Acute respira- infl uenza/guidelines/infectioncontrol1/en/. World tory infections: infl uenza. Available at http:// Health Organization: Geneva. www.who.int/vaccine_research/diseases/ari/en/. 174. World Health Organization. 2007. WHA58.3 World Health Organization: Geneva. Revision of the international health regulations. 165. World Health Organization. 2007. Recommenda- Available at http://www.who.int/gb/ebwha/pdf_ tions for infl uenza vaccines. Available at http:// fi les/WHA58/WHA58_3-en.pdf. World Health www.who.int/csr/disease/influenza/vaccinerec- Organization: Geneva. ommendations/en/. World Health Organization: 175. World Health Organization. 2006. World Health Geneva. Organization case defi nitions for human infec- 166. World Health Organization. 2006. WHO criteria tions with infl uenza A(H5N1) virus. Available at for accepting positive results of H5N1 infection http://www.who.int/csr/disease/avian_infl uenza/ 484 Avian Influenza

guidelines/case_definition2006_08_29/en/. Roizman, S.E. Straus (eds.). Fields Virology, World Health Organization: Geneva. 4th ed. Lippincott: Philadelphia, PA, pp. 1533– 176. World Health Organization. 2006. World Health 1579. Organization reference laboratories for diagnosis 181. Yeldandi, A.V., and T.V. Colby. 1994. Patho- of infl uenza A/H5 infection. Available at http:// logic features of lung biopsy specimens from www.who.int/csr/disease/avian_infl uenza/guide- infl uenza pneumonia cases. Human Pathology lines/referencelabs/en/. World Health Organiza- 25(1):47–53. tion: Geneva. 182. Yingst, S.L., M.D. Saad, and S.A. Felt. 2006. 177. World Health Organization. 2006. Collecting, Qinghai-like H5N1 from domestic cats, northern preserving and shipping specimens for the diag- Iraq. Emerging Infectious Diseases 12(8):1295– nosis of avian infl uenza A(H5N1) virus infection 1297. guide for fi eld operations. Available at http:// 183. Yu, H.E.A. 2007. Human infl uenza A (H5N1) www.who.int/entity/csr/resources/publications/ cases, urban areas of People’s Republic of China, surveillance/World Health Organization_CDS_ 2005–2006. Emerging Infectious Diseases 13: EPR_ARO_2006_1/en/index.html. World Health 1061–1064. Organization: Geneva. 184. Yuanji, G. 2002. Infl uenza activity in China: 178. World Health Organization. 2007. Update: World 1998–1999. Vaccine 20(Suppl. 2):S28–S35. Health Organization-confi rmed human cases of 185. Zhou, B., Zhong N., and Y. Guan. 2007. Treat- avian infl uenza A(H5N1) infection, 25 November ment with convalescent plasma for infl uenza A 2003–24 November 2006. Weekly Epidemio- (H5N1) infection. New England Journal of Med- logical Record 82:41–47. icine. 357:1450–1451. 179. World Health Organization. 2007. World Health 186. Zhu, Q.Y., E.D. Qin, W. Wang, J. Yu, B.H. Liu, Organization guidelines for investigation of Y. Hu, J.F. Hu, and W.C. Cao. 2006. Fatal infec- human cases of avian infl uenza A(H5N1). Avail- tion with infl uenza A (H5N1) virus in China. New able at http://www.who.int/entity/csr/resources/ England Journal of Medicine 354(25):2731– publications/infl uenza/World Health Organiza- 2732. tion_CDS_EPR_GIP_2006_4/en/index.html. 187. Ziegler, T., H. Hall, A. Sanchez-Fauquier, W.C. World Health Organization: Genva. Gamble, and N.J. Cox. 1995. Type- and subtype- 180. Wright, P.F., and R.G. Webster. 2001. Ortho- specifi c detection of infl uenza viruses in clinical myxoviruses. In: D.M. Knipe, P.M. Howley, specimens by rapid culture assay. Journal of D.E. Griffi n, R.A. Lamb, M.A. Martin, B. Clinical Microbiology 33(2):318–321. 21 The Role of Educational Programs in the Control of Avian Influenza

Nathaniel L. Tablante

INTRODUCTION tions. For example, an educational program designed The prevention and control of avian infl uenza (AI) for a United States vertically integrated broiler oper- involve a broad spectrum of activities, including ation may not be applicable to a village type of basic research, monitoring and surveillance, biose- operation in Southeast Asia. Nonetheless, an effec- curity, prompt and accurate diagnosis, containment, tive educational program on the control of AI must vaccination, communication, and education. While contain key components such as basic information most of these activities require technical expertise, on AI virus. sophisticated equipment, and a considerable amount of labor and materials, educational programs do not. KEY COMPONENTS Yet, educational programs can have a signifi cant The fi rst step in developing an educational program impact on the control of AI. on the control of AI is to conduct an evaluation of An AI control program cannot succeed without an the educational needs of the target audience. Based educational component. An educational program on on the results of this evaluation, an appropriate the control of AI is necessary because understanding outline or syllabus may be created. The outline must the nature of the disease and how it spreads are vital have an introductory part, which should cover edu- to its successful prevention, control, and eradication. cational objectives followed by general information Personnel involved in prevention, preparedness, and about AI virus and the subtypes that have the great- response activities related to outbreaks of AI must est impact on poultry and human health. This intro- be trained using a comprehensive educational ductory section must include a discussion of the program. Likewise, producers, various sectors of the structure and characteristics of the AI virus, natural poultry industry, and the general public must be reservoirs and susceptible hosts, modes of trans- properly educated on the characteristics of AI so that mission, incubation period, clinical signs and they can take the necessary precautions to prevent lesions, public health implications, diagnosis, sur- outbreaks or respond appropriately when one veillance and monitoring, eradication (including occurs. proper methods of depopulation and disposal of infected fl ocks), decontamination, and vaccination. BASIC PROGRAM REQUIREMENTS The second part should focus on biosecurity guide- An educational program on the control of AI must lines for specifi c groups or sectors of the poultry be presented in clear, simple, practical, and easy-to- industry. understand language. It must take into account local conditions such as the type of poultry operations, EVALUATING EDUCATIONAL NEEDS geographical location, level of biosecurity and man- In order to be effective, an educational program on agement, poultry production and marketing systems, AI prevention and control must fi rst determine the cultural practices, resources, and economic condi- needs of the target audience. Although a basic

Avian Influenza Edited by David E. Swayne 485 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 486 Avian Influenza knowledge of the principles of biosecurity is essen- days). This must be followed by a detailed descrip- tial, each sector will have specifi c educational needs. tion and visual presentation of clinical signs and An educator or trainer must ask the following lesions of LPAI and HPAI. Lecture slide presenta- questions: tions should contain images of AI clinical signs and lesions. These may be obtained from several hard- 1. Who is the target audience? The target audience copy or Web-based sources (3, 17). For handouts, could be farmers or producers (commercial or use available information on AI that is organized, backyard), emergency response personnel clear and easily available such as the U.S. Depart- (federal, state, university, or industry), poultry ment of Agriculture (USDA) Question and Answer company personnel (including veterinarians and Fact Sheet on AI (24). Other good sources of infor- technical service personnel), live haul crews, pro- mation on AI for education and training purposes cessing plant employees, breeder farm, hatchery, are publications and other media formats that are and feed delivery workers, vaccination crews, readily available through the Web sites of the USDA, utility company personnel, suppliers (of equip- Centers for Disease Control and Prevention (CDC), ment, medications, disinfectants, sawdust, etc.), and United Nations agencies like the Food and Agri- backyard and specialty fl ock owners (including culture Organization (FAO) or the World Health game bird producers), and live bird auctions, Organization (WHO). These sources of information retail markets, bird suppliers, and dealers. on AI are listed in Table 21.1. 2. What are the activities of the target audience that may contribute to the spread of AI? Human Health Implications 3. Based on the activities and characteristics of the Although AI viruses are highly species specifi c and target audience, what type of information must primarily cause infection in domestic birds, sporadic be presented? cases of transmission to humans have been reported (15). In 1997, an H5N1 HPAI virus affected 18 DEVELOPING AN EDUCATIONAL people in Hong Kong, resulting in 6 deaths (17). The OUTLINE OR SYLLABUS AI viruses isolated from the Hong Kong patients The outline or syllabus must contain the training were linked to close contact of these patients with or educational objectives, topics to be covered, a live chickens infected with H5N1 HPAI viruses, summary of the topics covered, a conclusion and suggesting direct transmission of the virus from acknowledgment section, and a program evaluation. chicken to human rather than person-to-person The latter is used for feedback and program improve- spread (14). According to the WHO, a second wave ment purposes. of H5N1 HPAI outbreaks began in December 2003 and has killed 169 of 280 infected humans in 12 BASIC INFORMATION countries worldwide as of March 19, 2007, mostly General information on AI virus must focus on the in Southeast Asia (28). Similar to the 1997 Hong H5 and H7 subtypes, which have the greatest poten- Kong outbreak, the most recent H5N1 HPAI cases tial to mutate into a highly pathogenic form and in China, Indonesia, and Turkey have identifi ed infect domestic poultry and humans (17). A brief direct contact with infected birds as the most likely description of the structure and characteristics of the source of exposure (27). An educational program in virus (an RNA virus containing two surfaces anti- countries currently experiencing outbreaks of HPAI gens: hemagglutinin [HA] and neuraminidase [NA]), should therefore focus on biosecurity practices the major subtypes based on antigenic make-up (144 among high-risk populations such as people who possible combinations arising from 16 hemaggluti- have close contact with live poultry either on village nins, H1–H16 and 9 neuraminidases, N1–N9), clin- farms or live bird markets (LBMs). Individuals who ical signs, pathotypes (low [LP] and high are involved in emergency response (surveillance, pathogenicity [HP]), natural reservoirs (wild water- depopulation, carcass disposal, sample collection, fowl such as ducks and geese), susceptible hosts and diagnosis) must also be educated on the proper (domestic poultry), modes of transmission (direct handling of H5N1 HPAI virus–infected birds, contact through feces or respiratory secretions or including the use of personal protective equipment aerosol generation), and incubation period (3 to 14 (PPE), vaccination with the current seasonal infl u- Table 21.1. Sources of background information on avian infl uenza. Title Contents Type Source

Questions and Answers: Biology, history, and Fact sheet (USDA USDA Ofﬁ ce of Communications, 1400 Independence Ave Avian Inﬂ uenza (March prevention of AI Release No. SW, Washington, DC 20250 or www.aphis.usda.gov 2007) 0458.05)

Avian Inﬂ uenza (“Bird Description and clinical Fact sheet (WHO http://www.who.int/mediacentre/factsheets/avian_inﬂ uenza/en/

487 Flu”) Fact Sheet signs of AI Web site) index.html

Key Facts About Avian General information about Fact sheet (CDC http://www.cdc.gov/fl u/avian/gen-info/pdf/avian_facts.pdf Infl uenza (Bird Flu) and avian infl uenza and bird Web site) Avian Infl uenza A fl u (H5N1) Virus

Avian Infl uenza: Questions General information about Fact sheet (FAO http://www.fao.org/AG/AGAInfo/subjects/en/health/ and Answers avian infl uenza, human Web site) diseases-cards/avian_qa.html and poultry health, biosecurity 488 Avian Influenza enza vaccine, antiviral medication, and health mon- (13). itoring (26). In addition to the WHO guidelines, the The U.S. Departments of Health and Human Ser- CDC provides a comprehensive guide for human vices, Agriculture, and Interior are currently involved protection (4). The guidelines focus on key areas in an interagency effort to detect H5N1 HPAI in such as basic infection control, the use of personal wild birds (22). The initiative is divided into two protective equipment, vaccination with seasonal phases: (1) the initial phase addresses early detection infl uenza vaccine, administration of antiviral drugs activities in Alaska, and in particular, coastal areas for prophylaxis, surveillance and monitoring of that have the most potential for contact among Asian workers, and evaluation of ill workers. and North American birds, and (2) the second phase addresses subsequent HPAI detection activities in Diagnosis and Surveillance four major North American fl yways. The plan for Early detection is vital to the control of AI. Methods wild bird surveillance includes several interrelated of surveillance (active and passive) as well as target components, including the investigation of wild bird sectors and species must also be covered in an edu- deaths or sickness, the sampling of live-captured cational program. In the United States, the USDA– birds, the deployment of sentinel species, environ- Animal and Plant Inspection Service (APHIS) mental sampling, and sampling hunter-harvested conducts AI surveillance in domestic poultry (com- birds. All fi eld personnel involved in these efforts mercial and backyard fl ocks) through the National must be trained on tracheal and cloacal sample col- Poultry Improvement Plan (NPIP), LBM system, lection, storage, and transport procedures as outlined and wild bird populations (23). Custom-made edu- by USDA/APHIS (22). cational programs must be designed for people On the international front, FAO has established involved in each key area. guiding principles for HPAI surveillance in Asia (8). The USDA/APHIS supports a control and preven- For infected countries or compartments not practic- tion program in the LBM system through regular ing vaccination, FAO recommends that surveillance monitoring and surveillance of all facilities by vol- be targeted to the following high-risk areas and untarily participating states (21). The USDA/APHIS populations using methods described in Annex 1 of also encourages backyard and small poultry produc- the guiding principles: (1) domestic waterfowl, (2) ers to strengthen biosecurity practices and report unusual mortality in wild birds, (3) LBMs, and (4) sick birds promptly through its “Biosecurity for the sentinel villages. FAO recommends that molecular Birds” program, which includes information on AI characterization of all isolates and molecular epide- and biosecurity practices in a lay worded fact sheet miological studies be performed, drawing on the (20). The USDA/APHIS has developed an H5/H7 expertise available in the regional network and in LPAI Prevention and Control Program for commer- international reference laboratories as required. In cial poultry and the LBM system. This voluntary addition, FAO requires that virus isolates be pro- and cooperative state/federal/industry program vided to international reference laboratories and includes a commercial poultry segment that requires molecular data uploaded to international gene monitoring of broiler, commercial egg, and turkey sequence databases (e.g., GenBank). In countries/ fl ocks for H5 and H7 LPAI under the NPIP (9). The compartments that are endemically affected and LBM segment of the program is being addressed practicing vaccination, the main objectives of sur- through a Uniform Standards document that defi nes veillance are to ensure that vaccinated birds achieve uniform guidelines for LBM as well as producers protective levels of immunity and that fi eld viruses and distributors that supply those markets (9). The in these countries/compartments are detected and National Chicken Council (NCC), a nonprofi t trade fully characterized per FAO guidelines. An educa- association for the U.S. broiler chicken industry, tional program for surveillance personnel in coun- initiated on-farm preslaughter testing of all poultry tries where H5N1 HPAI is endemic or poses a high fl ocks for H5 and H7 LPAI in 2006. Under the NCC risk to poultry and human populations should include program, participating poultry companies will familiarization with the FAO guiding principles for promptly and humanely destroy all fl ocks that are HPAI surveillance. found positive for H5 and H7 AI and dispose of the Because of the various international, national, carcasses in an environmentally acceptable manner federal, state, and industry surveillance programs for 21 / The Role of Educational Programs in the Control of Avian Influenza 489

AI, fi eld personnel from poultry companies, state, response—this level of response requires the com- and federal agencies must be trained on disease rec- bined efforts of local, state, industry, and federal ognition, reporting, and proper methods of sample agricultural offi cials as well as nonagricultural gov- collection, including what samples to collect and ernment personnel (e.g., the Federal Emergency how to collect and transport them to a diagnostic Management Agency) and the private sector in laboratory. Field personnel should know that AI national-level crisis management, response coordi- virus replicates in the respiratory and intestinal tracts nation, consultation, and consequence management. (17); therefore, they must properly collect tracheal Topics covered in the NAHEMS guidelines include or cloacal swabs from live or dead birds. The USDA (1) fi eld investigations of animal health emergen- recommends that samples be taken by swabbing the cies, (2) implementation of an animal emergency mucus that coats the throat of live birds (24). In wild response using the ICS, (3) disease control and birds, a fecal sample can be taken instead. These eradication strategies and policies, (4) operational samples must be placed in sealed tubes and shipped procedures for disease control and eradication, (5) or transported to USDA-approved laboratories site-specifi c emergency management strategies for where a real-time reverse transcriptase–polymerase various types of facilities, (6) administrative and chain reaction (RRT-PCR) test must be performed. resources management, and (7) educational Detailed protocols for the collection of tracheal resources. and cloacal swabs are available from several sources, Emergency response training may vary outside most notably WHO (25). Specimens for virus isola- the United States depending on the program needs, tion must be kept at 4º C immediately at collection local regulations, cultural practices, and available and tested within 48 hours after collection or frozen resources of individual nations. at −50º C or colder for storage until virus isolation Outlined in Table 21.2 are good sources is attempted (17). In addition, fi eld personnel must of information of emergency preparedness and be trained on proper collection of blood samples for response. serologic testing using educational materials showing detailed step-by-step procedures (10, 17). Depopulation An educational program on AI control must include Emergency Preparedness and Response a discussion, slide presentation, and demonstration Training (if possible) of humane methods of mass euthanasia In the United States, emergency responders must be or depopulation of fl ocks infected with AI. Accord- trained on the principles of the National Response ing to FAO, destruction of infected and at-risk Plan and the National Incident Management System poultry (stamping-out) has long been the accepted (23). The USDA/APHIS has established the National method of control for HPAI in the face of a disease Animal Health Emergency Management System outbreak (8). Under a stamping-out policy, poultry (NAHEMS), which provides an operational frame- on infected premises are destroyed. The FAO rec- work for responding to a foreign animal disease ommends that policy on the culling of “at-risk” emergency. The NAHEMS guidelines are designed poultry as part of a stamping-out policy be risk for use at any of three levels of response commen- based, taking into account the likelihood that the surate with the severity of the outbreak: (1) a local/ birds are infected. Infected poultry should be culled limited response—this level of response is managed as quickly as possible and preferably within 24 by local, State, Federal, and industry offi cials, with hours of detecting infection to prevent further spread response coordination provided primarily at the of the virus. State and regional levels and with national-level In the United States, federal and state regulations consultation and consequence management (e.g., also require depopulation and subsequent disposal trade issues); (2) a regional response—a regional of poultry fl ocks infected with H5 or H7 HPAI and response is managed by local, State, Federal, and most poultry fl ocks infected with H5 or H7 LPAI. industry offi cials, and in some cases includes the Currently, carbon dioxide is the preferred method of involvement of the appropriate State emergency mass poultry euthanasia in the United States (16). management agency as specifi ed in State animal Humane standards set by the 2000 Report of the health emergency response plans); and (3) a national American Veterinary Medical Association (AVMA) Table 21.2. Emergency preparedness and response information. Title Contents Type Source

Preparing for Pandemic Human health guidelines Fact sheet (CDC http://www.wvfl u.org/PDFs/preparingforpandemicfl u.pdf Infl uenza to prevent pandemic fl u Web site) Responding to the AVIAN Recommended activities to WHO report http://www.who.int/csr/resources/publications/infl uenza/ INFLUENZA PANDEMIC prepare for pandemic fl u (WHO Web site) WHO_CDS_CSR_GIP_05_8-EN.pdf THREAT: RECOMMENDED STRATEGIC ACTIONS 490 Safeguarding the United Summary of U.S. measures Fact sheet (USDA/ http://www.aphis.usda.gov/lpa/pubs/fsheet_faq_notice/fs_ States From Highly- to address bird fl u threat APHIS Web ahhpaiplan.pdf Pathogenic Avian Infl uenza site) (HPAI): USDA Actions, Plans, and Capabilities for Addressing the Bird Flu Threat Summary of the National National response plan for Report (USDA/ http://aphisweb.aphis.usda.gov/newsroom/hot_issues/ Highly Pathogenic Avian quick and effective APHIS Web avian_infl uenza/contents/printable_version/ Infl uenza Response Plan response to AI in the site) DraftSummaryNationalHPAIresponseplan08-31-06.pdf United States 21 / The Role of Educational Programs in the Control of Avian Influenza 491

Panel on Euthanasia must be utilized to depopulate developed an innovative mass depopulation system fl ocks. Per AVMA guidelines, mass euthanasia of using fi re fi ghting foam that is applied on infected meat-type poultry has been limited to inhalant gases, birds resulting in a rapid physically induced hypoxia particularly carbon dioxide (1). In other countries, via airway obstruction (2). The foam method is cur- asphyxiation using carbon dioxide is also the method rently a viable technology for fl oor-reared turkeys of choice for destruction of chickens and is most and broilers but is not feasible for caged layers (2). effective where birds are reared on the fl oor (8). For In caged layers, the “modifi ed atmosphere kill houses with open wire mesh walls or where birds (MAK) cart,” a carbon dioxide chamber, has been are kept in cages, the birds must be removed from used for euthanizing spent hens and is approved by the shed or cages before destruction. This may the United Egg Producers (19). However, the MAK involve placing poultry in large containers or skips, cart has limited capacity for large-scale depopula- smaller garbage bins or even strong plastic bags into tion of caged layers. A larger version of this system which carbon dioxide is added. The containers must called the Modifi ed Atmosphere Chamber (MAC) be free from leaks. Asphyxiation by carbon dioxide developed by Alberta Agriculture, Food and Rural is not as effective in ducks and geese as in chickens. Development can euthanize 30,000 spent hens in an Physical methods such as cervical dislocation using 8- to 10-hour day (5). Large steel trash containers cattle castration forceps are preferable for the covered and sealed with tarp and fi lled with carbon humane destruction of waterfowl (8). dioxide have also been used for mass euthanasia of Mass euthanasia of poultry fl ocks infected with caged layers (16). It is important to consider local AI is practiced to prevent spread of the disease. conditions such as available resources and regu- However, the procedure must be performed by qual- lations when deciding which method of depopula- ifi ed personnel and performed in the quickest, safest, tion to use. Regardless of the method used, state, and most humane way possible (23). Personnel must federal, and industry emergency response personnel be trained in key concepts, including euthanasia, must be trained on the method that best fi ts local handling of birds, safety, and biosecurity. Birds are conditions. usually euthanized by releasing carbon dioxide into Sources of information on methods of depopula- a poultry house that has been appropriately condition are listed in Table 21.3. tioned to contain the gas while the process is taking place. The euthanasia team will determine the Carcass Disposal number of birds to be euthanized, the size of the Emergency response personnel must also be trained enclosure, and the amount of carbon dioxide to be on biosecure and environmentally acceptable used. In addition to receiving training regarding methods of disposal of carcasses infected with AI. biosecurity issues, the euthanasia team must be The methods used must prevent spread of infection, trained in safety measures for handling carbon must have minimal impact on the local environment, dioxide. and must be acceptable to environmental protection Four methods of mass euthanasia using carbon agencies (8). During animal health emergencies, dioxide that have been used in previous AI out- “disposal” focuses on evaluation of disposal sites, breaks in the United States are whole house and selection of optimal disposal procedures, and dis- partial house (for breeders), portable panels with posal of miscellaneous materials. Common methods tarp (for turkeys), live haul cage cabinet (for broil- used to dispose of carcasses and materials include ers), and polyethylene tent (for broilers and breed- burial, incineration (including diagnostic laboratory ers) (11). A national training program on mass incineration), air-curtain incineration, landfi lling, poultry euthanasia/depopulation and carcass dis- rendering, composting, and alkaline hydrolysis (23). posal has been conducted by Malone and Tablante In countries outside the United States, the main in several poultry producing states since May 2005. methods used for disposal of infected carcasses and Materials used (step-by-step procedures with pic- other materials are burial, burning, and composting tures and video clips) in this training program must (8). It is preferable to dispose of carcasses on-farm, be included in an educational program on the control providing that there is a suitable site for burial or of AI. composting (8). Off-farm disposal or relocation of Researchers at the University of Delaware have carcasses to another site creates an additional risk to Table 21.3. Information sources on methods of depopulation. Title Contents Type Source

2000 Report of the AVMA Panel Euthanasia guidelines for Monograph Journal of the American Veterinary Medical Association on Euthanasia various animal species 2001;218(5):669–696. Foam-Based Mass Emergency Information and guidelines on Monograph Poultry Science 2007;86(2):219–224 Depopulation of Floor-Reared the use of water-based foam Meat-Type Poultry Operations for poultry depopulation

492 Use of Water-Based Foam for AVMA policy on the use of Web site http://www.avma.org/issues/policy/poultry_depopulation.asp Depopulation of Poultry water-based foam for poultry depopulation USDA/APHIS Performance USDA/APHIS policy and Web site http://www.avma.org/issues/policy/poultry_depopulation.asp Standards for the Use of guidelines on the use of Water-Based Foam as a water-based foam for poultry Method of Mass Depopulation depopulation of Domestic Poultry Euthanasia of Poultry: Guidelines for euthanasia of Web site http://animalwelfare.ucdavis.edu/publication/poultryeuth.html Considerations for Producers, poultry Transporters, and Veterinarians 21 / The Role of Educational Programs in the Control of Avian Influenza 493 farms along the route between the infected farm and USDA cleaning and disinfection recommendations the disposal site. If off-farm disposal is the only outlined in the 2006 Interim AI Response Plan must option available, personnel must be trained on the be included in an educational program on the control proper handling and transport of infected carcasses. of AI. The USDA plan involves humane destruction These people must be advised that, when transport- of residual birds; removal of dead birds or carcasses ing contaminated material from affected premises to remaining from depopulation on the fl oor or in the off-site locations, special procedures must be fol- litter; removal of free-fl ying wild birds in the house, lowed to prevent the spread of disease agents. Such insect and rodent control; cleaning of equipment; procedures include the use of disinfectants appropri- high-pressure washing of houses, thorough disinfec- ate for the pathogen, leak-proof transportation, and tion of houses, vehicles, and equipment; and ade- polyethylene plastic sheets. The transport vehicle(s) quate downtime (at least three times the longest must be accompanied by one or more designated expected incubation time of the disease) before government representatives for biosecurity reasons. repopulation (23). The training should emphasize The designated government representative(s) should the importance of starting with an initial dry clean- bring an appropriate disinfectant and liquid-absorb- ing (scraping and carting away feces, litter, feed, and ing material in addition to other tools or equipment other organic material) followed by preliminary dis- needed to clean up any spills occurring on the way infection (e.g., by spraying an appropriate liquid to the destination (23). disinfectant) followed by more thorough cleaning Composting of carcasses in combination with and a second round of disinfection. Personnel must feces and litter is another acceptable means of dis- know that AI viruses can survive for some time in posal (8). Extension specialists from the University organic material; therefore, thorough cleaning with of Maryland and the University of Delaware refi ned detergents is an important step in decontamination. an in-house composting technique for catastrophic Personnel must also be reminded that all organic poultry mortalities, utilizing existing poultry litter in matter must be removed from poultry houses with commercial broiler farms in the United States (18). no visible feathers or feces remaining. In addition, The technique, which is available as a slide presen- cleaning and disinfection crews must know the types tation on a compact disk, was successfully used to of disinfectants that are effective against AI viruses, control the 2004 outbreak of H7N2 LPAI on the including detergents, hypochlorites, alkalis, glutar- Delmarva Peninsula. Although this technique works aldehyde, and peroxide-based disinfectants (e.g., best in clear-span single-story poultry houses with Virkon; Dupont Animal Health Solutions, Nutley, fl oor litter, it may be used for on-farm outside com- New Jersey). A list of Web sites for disinfectants posting utilizing locally available carbon materials. approved for AI is listed in Table 21.5 and Chapter This technique must defi nitely be included in any 18. training program on the control of AI. Regardless of geographical location, personnel must receive train- Vaccination ing on each method of carcass disposal as well as If applicable, that is, permitted by federal and state on biosecurity and personal safety. Sources of infor- authorities, vaccination strategies must be included mation on methods of disposal of AI-infected poultry in an educational program on the control of AI. Vac- are listed in Table 21.4. cination with homologous hemagglutinin AI vaccines has been shown to decrease susceptibility of Cleaning and Disinfection poultry to infection by AI viruses. However, vac- After depopulation of infected birds, careful and cines do not completely prevent infection, especially thorough cleaning and disinfection ensures that AI in the fi eld; thus, biosecurity practices are essential does not reemerge on the premises (23). However, to prevent spread (16). personnel must be trained on proper cleaning and Vaccination has been used successfully in Hong disinfection procedures. Cleaning and disinfecting Kong and other regions to control AI viruses, includ- are critical in the prevention of the spread of disease ing in the United States and in Italy, demonstrating through movement of fomites that have been in that strategic use of vaccine in conjunction with contact with live animals, animal products, or areas other control measures can be effective in control- where they have lived or been stored (23). The ling or eliminating AI viruses (8). A joint World Table 21.4. Sources of information on carcass disposal. Title Contents Type Source

Disposal of Domestic Birds EPA guidelines on disposal Web site http://www.epa.gov/epaoswer/homeland/fl u.pdf Infected by Avian options for AI-infected Infl uenza: An Overview Of fl ocks Considerations and Options Carcass Disposal: A Carcass disposal options for Web site http://fss.k-state.edu/research/books/carcassdispfi les/ Comprehensive Review various animal species Carcass%20Disposal.html Guidelines for In-House Detailed step-by-step CD N. Tablante, University of Maryland, 8075 Greenmead Composting Of Poultry procedures on mass in- Drive, College Park, MD 20742, [email protected], or G. Mortalities Due to house composting of Malone, University of Delaware, 16483 County Seat Hwy Catastrophic Disease poultry mortalities Georgetown, DE 19947, [email protected] Guidelines for In-House Detailed step-by-step Web site http://www.agnr.umd.edu/MCE/Publications/PDFs/FS801.pdf

494 Composting of Catastrophic procedures on mass in- Poultry Mortality house composting of poultry mortalities

Table 21.5. Sources of Information on Approved Disinfectants for AI Viruses. Title Contents Type Source

Avian Infl uenza Q&A FAO-recommended Web site http://www.fao.org/ag/againfo/subjects/en/health/diseases-cards/avian_qa.html#8 disinfectants against AI Registered antimicrobial EPA-recommended Web site http://www.epa.gov/pesticides/factsheets/avian_fl u_products.htm products with label disinfectants claims for avian (bird) against AI fl u disinfectants 21 / The Role of Educational Programs in the Control of Avian Influenza 495

Health Organization (WHO)/World Organization of 2. Protect poultry fl ocks from coming into contact Animal Health (Offi ce Internationale des Epizooties with wild or migratory birds. Keep poultry away [OIE])/FAO/WHO meeting held in Rome in Febru- from any source of water that could have been ary 2004 concluded that while stamping-out is still contaminated by wild birds. the preferred option for an outbreak of HPAI and 3. Permit only essential workers and vehicles to should be used in all fl ocks exhibiting clinical enter the farm. disease, it may not be either desirable or feasible to 4. Provide clean clothing and disinfection facilities proceed with massive culling in some situations, in for employees. which case vaccination is considered a suitable 5. Thoroughly clean and disinfect equipment and option (8). According to FAO, vaccination can be vehicles (including tires and undercarriage) used either as a tool to support eradication or as a entering and leaving the farm. tool to manage the disease and reduce the viral load 6. Do not loan to, or borrow equipment or vehicles in the environment. FAO further recommends that from, other farms. vaccination teams (made up of veterinarians, techni- 7. Change footwear and clothing before working cians, and assistants) be trained in both the vacci- with your own fl ock after visiting another farm nation procedure and appropriate public health or live-bird market or avoid visiting another bird measures including the correct use of PPE. FAO farm if possible. urges team members to follow manufacturers’ rec- 8. Do not bring birds from slaughter channels, espe- ommendations on the storage and delivery of vaccine cially those from live-bird markets, back to the and ensure that detailed records of vaccination farm. (number and species vaccinated, location, date, identifi cation numbers of sentinel birds, etc.) are Biosecurity Risk Assessment recorded and entered in relevant databases. Vaccina- Designated personnel must be trained on biosecurity tion team members should also be well trained in risk assessment techniques (6). Risk assessment biosecurity measures to ensure they follow appropri- quantifi es the level of risk by assigning points for ate cleaning and disinfection procedures that mini- compliance or noncompliance with management mize any risk of their spreading AI viruses or other and biosecurity measures. The greater the number of poultry pathogens between fl ocks (8). points, the higher is the risk of a disease outbreak. Improvements to this system, including mapping of Biosecurity Training farms using GPS coordinates can assist the poultry An educational program on biosecurity must fi rst industries in coordinating regional control programs focus on the farmer or producer who is undoubtedly (7). the fi rst responder in any disease outbreak. Training growers or producers to implement strict biosecurity CONCLUSION measures at all times is a critical step toward AI An AI control program cannot succeed without an prevention and control. Several comprehensive educational component. An educational program on books, manuals, Web sites, and a CD-ROM on AI control must be comprehensive, yet simple, prac- poultry biosecurity are available (see Table 21.6). tical, and focused on the target audience. The When conducting an educational program, spe- program must be tailored to fi t local needs and con- cifi c guidelines for each poultry industry sector or ditions, cultural practices, regulations, and available compartment must be used. All biosecurity pro- resources. Regardless of the target audience and grams must contain the basic elements outlined by local conditions, an educational program on AI USDA (24) as follows: control must include basic information on AI virus and how it spreads, human health and safety impli- 1. Keep an “all-in, all-out” philosophy of fl ock cations, diagnosis and surveillance techniques, management. Avoid skimming fl ocks—birds left emergency preparedness and response guidelines, behind are exposed to work crews and equipment depopulation and carcass disposal options, cleaning that could carry poultry disease viruses. Process and disinfection procedures, biosecurity guidelines, each lot of birds separately, and clean and disin- vaccination programs, biosecurity measures, and fect poultry houses between fl ocks. risk assessment methods. Every educational program Table 21.6. Sources of information on poultry biosecurity. Title Contents Type Source

Biosecurity for the Birds Biosecurity guidelines for Brochure USDA/APHIS, 4700 River Road, Riverdale, MD 20737 (also backyard poultry ﬂ ock available at http://www.aphis.usda.gov/vs/birdbiosecurity/) owners in lay language

Biosecurity in the Poultry Biosecurity guidelines for Book American Association of Avian Pathologists, 953 College

496 Industry various sectors of the Station Road, Athens, GA 30602 poultry industry

Poultry Disease Risk Poultry biosecurity principles CD U.S. Poultry and Egg Association, 1530 Cooledge Road, Management: Practical and guidelines for various Tucker, GA 30084 Biosecurity Resources sectors of the poultry (CD) industry

Biosecurity for the Live Bird Biosecurity principles and CD USDA/APHIS, 4700 River Road, Riverdale, MD 20737 Marketing System: guidelines for the live bird Stopping Disease Before It marketing system Gets Started 21 / The Role of Educational Programs in the Control of Avian Influenza 497 must be evaluated regularly and constantly improved 10. Ison. A.J., S.J. Spiegle, and T.Y. Morishita. 2005. and updated to adapt to local needs, new technology, Poultry blood collection. In: Ohio State University and changing conditions. Extension Fact Sheet VME-23-05. Available at http://ohioline.osu.edu/vme-fact/0023.html. The REFERENCES Ohio State University: Columbus, OH. Accessed 1. AVMA. 2001. 2000 report of the AVMA panel on October 12, 2006. euthanasia. Journal of the American Veterinary 11. Malone, G.W., and N.L. Tablante. 2006. National Medical Association 218(5):669–696. training program on euthanasia and disposal pro- 2. Benson, E.R. 2006. Foam depopulation for emer- cedures for catastrophic poultry disease events. gency disease depopulation: science and practical In: Proceedings of the Poultry Science Associa- applications. In: Proceedings of the 41st National tion Annual Meeting, Alberta, Canada, pp. 176– Meeting on Poultry Health and Processing, Ocean 177. City, MD, pp. 19–26. 12. Mid-Atlantic Cooperative Extension. Undated. 3. Capua, I., and F. Mutinelli. 2001. A Colour Atlas Biosecurity for Poultry Training Manual. Mid- and Text on Avian Infl uenza. Papi Editore: Atlantic Cooperative Extension: College Park, Bologna, Italy, pp. 1–73. MD. 4. Centers for Disease Control and Prevention. 2006. 13. National Chicken Council. 2006. Testing Program Interim Guidance for Protection of Persons Launched to Ensure Chicken Products Are Free of Involved in USA AI Outbreak Disease Control and Avian Infl uenza. Available at http://www.national- Eradication Activities. Available at http://www. chickencouncil.com/fi les/AIProgram.pdf. National cdc.gov/flu/avian/professional/protect-guid.htm. Chicken Council: Washington, D.C. Accessed on Centers for Disease Control and Prevention: 10/12/06. Atlanta, GA. Accessed October 11, 2006. 14. Snacken, R., A.P. Kendal, L.R. Haaheim, and J.M. 5. Church S., S. Gal, and J. Church. 2005. Alberta Wood. 1999. The next infl uenza pandemic: lessons Champions Spent Hen Welfare With New Eutha- from Hong Kong, 1997. Emerging Infectious Dis- nasia System. Available at http://www.eggs.ab. eases 5(2):195–203. ca/news/eggindustry.html. Alberta Egg Producers: 15. Swayne, D.E. 2000. Understanding the ecology Calgary, Alberta, Canada. Accessed October 11, and epidemiology of avian infl uenza viruses: 2006. implications for zoonotic potential. In: C.C. Brown 6. Dekich, M.A. 1995. Principles of disease preven- and C.A. Bolin (eds.). Emerging Diseases of tion in commercial integrated broiler operations. Animals. ASM Press: Washington, DC, pp. 101– In: S.M. Shane, D. Halvorson, D. Hill, P. Villegas, 130. and D. Wages (eds.). Biosecurity in the Poultry 16. Swayne, D.E., and B. Akey. 2005. Avian infl uenza Industry. American Association of Avian Patholo- control strategies in the United States of America. gists: Athens, GA, pp. 85–94. In: R.S. Schrijver and G. Koch (eds.). Avian Infl u- 7. Delmarva Poultry Industry, Inc. 2006. Delmarva enza Prevention and Control, Wageningen UR Procedure Manual on Emergency Poultry Diseases. Frontis Series, 8th ed. Springer: Dordrecht, the Delmarva Poultry Industry, Inc.: Georgetown, DE. Netherlands, pp. 113–130. 8. Food and Agriculture Organization. 2004. Guiding 17. Swayne, D.E., and D.A. Halvorson. 2003. Infl u- principles for highly pathogenic AI surveillance enza. In: Y.M. Saif (ed.). Diseases of Poultry. and diagnostic networks in Asia. In: Proceedings Iowa State University Press: Ames, IA, pp. 135– of the FAO Expert Meeting on Surveillance and 160. Diagnosis of Avian Infl uenza in Asia, Bangkok, 18. Tablante, N.L., and G.W. Malone. 2005. Guide- Thailand. Available at http://www.fao.org/docs/ lines for in-house composting of catastrophic eims/upload//210749/Gui_principlesHPAI_ poultry mortalities. PowerPoint slide presentation july04_en.pdf. Food and Agriculture Organization: on CD-ROM. University of Maryland College Rome, Italy. Accessed October 12, 2006. Park, College Park, MD, and University of Dela- 9. Hegngi, F.N., A. Rhorer, P. Klein, K. Grogan, B. ware, Georgetown, DE. Carter, and T.J. Myers. 2006. Overview of the 19. United Egg Producers. 2006. Animal Husbandry USDA H5/H7 low pathogenicity avian infl uenza Guidelines for U.S. Egg Laying Flocks. Available program in the live bird marketing system. In: Pro- at http://www.uepcertifi ed.com/docs/2006_UEP- ceedings of the Annual Meeting of the American animal_welfare_guidelines.pdf. United Egg Pro- Association of Avian Pathologists, Honolulu, ducers: Alpharetta, GA. Accessed October 12, Hawaii, p. 75. 2006. 498 Avian Influenza

20. U.S. Department of Agriculture. 2005. Biosecurity 24. U.S. Department of Agriculture. 2006. Questions for the Birds: A National Campaign to Promote and Answers: Avian Infl uenza. Fact Sheet Release Avian Health Through Biosecurity. Available at No. 0458.05. Available at http://www.usda.gov/ http://www.aphis.usda.gov/vs/birdbiosecurity/ wps/portal/!ut/p/_s.7_0_A/7_0_1OB?contentidonl hpai.html. and http://www.usda.gov/documents/ y=true&contentid=2005/10/0458.xml. U.S. Depart- AvianFluBrochure.pdf. U.S. Department of Agri- ment of Agriculture Offi ce of Communications: culture: Washington, DC. Accessed October 13, Washington, DC. Accessed October 13, 2006. 2006. 25. World Health Organization. 2005. WHO labora- 21. U.S. Department of Agriculture/Animal and Plant tory guidelines for the collection of animal speci- Health Inspection Service. 2004. Prevention and mens for diagnosis of infl uenza infection. Available Control of H5 and H7 low Pathogenicity Avian at http://www.who.int/csr/disease/avian_infl uenza/ Infl uenza in the Live Bird Marketing System: guidelines/animalspecimens/en/index.html. World Uniform Standards for a State-Federal-Industry Health Organization: Geneva. Accessed October Cooperative Program. U.S. Department of Agri- 14, 2006. culture Animal and Plant Health Inspection 26. World Health Organization. 2005. WHO Guidance Service: Washington, DC. on Public Health Measures in Countries Experi- 22. U.S. Department of Agriculture/Animal and Plant encing Their First Outbreaks of H5N1 Avian Health Inspection Service. 2006. An Early Detec- Infl uenza. Available at http://www.who.int/csr/ tion System for Highly Pathogenic Avian Infl u- disease/avian_infl uenza/guidelines/fi rstoutbreak/ enza in wild migratory birds: U.S. Interagency en/. World Health Organization: Geneva. Accessed strategic plan. Available at http://www.usda.gov/ October 14, 2006. documents/wildbirdstrategicplanpdf_seg0.pdf. 27. World Health Organization. 2006. Avian Infl uenza U.S. Department of Agriculture Animal and Plant (Bird Flu) Fact Sheet. Available at http://www. Health Inspection Service: Washington, DC. who.int/mediacentre/factsheets/avian_influenza/ Accessed October 13, 2006. en/index.html. World Health Organization: 23. U.S. Department of Agriculture/Animal and Plant Geneva. Accessed October 14, 2006. Health Inspection Service. 2006. Available at 28. World Health Organization. 2007. Cumulative http://aphisweb.aphis.usda.gov/newsroom/hot_ number of confi rmed human cases of avian infl u- issues/avian_infl uenza/contents/printable_version/ enza A/(H5N1) reported to WHO. Available at DraftSummaryNationalHPAIresponseplan08-31- http://www.who.int/csr/disease/avian_influenza/ 06.pdf. U.S. Department of Agriculture Animal country/cases_table_2007_03_19/en/index.html. and Plant Health Inspection Service, Washington, World Health Organization: Geneva. Accessed DC. Accessed October 13, 2006. March 19, 2007. 22 Trade and Food Safety Aspects for Avian Influenza Viruses

David E. Swayne and Colleen Thomas

GLOBAL PRODUCTION AND TRADE OF ing to the Food and Agriculture Organization (FAO) POULTRY in 2004 indicated that chicken meat and eggs were Poultry are the most frequently raised farm animals ranked within their top fi ve agricultural import cat- and, on a global basis, birds are the major source of egories (22). In 2004, the top 10 importers of chicken animal protein in the human diet through both meat meat were the Russian Federation, Japan, the Euro- and eggs. The principal poultry species raised is the pean Union, Saudi Arabia, Mexico, Ukraine, Hong chicken, but signifi cant numbers of turkey, duck, Kong, China, the United Arab Emirates, and South goose, Japanese quail, guinea fowl, and various Africa (39). ratite species are also raised depending on culture, In addition to importation, domestic production customs, national production system, and markets. and distribution systems are critical for meeting the In developed countries, most production and con- culinary demands of consumers as well as supplying sumption is through specialized, integrated commer- the global market with other products, such as live cial farms and cold chain distribution. In addition, birds (domestic poultry and other birds), hatching there is a smaller contribution from poultry raised eggs, and fi ber (i.e., feathers). These systems not through rural (village), organic, and live poultry only include the economically viable production of market systems that supply some consumers with poultry but also contribute to the control of poultry specialty products such as live or fresh-killed birds. diseases and the prevention of disease spread. This In contrast, in many developing countries, the inte- is achieved by implementing sanitary standards and grated commercial poultry production sectors with effective disease control programs. Under the Agree- cold chain distribution are smaller. The majority of ment on the Application of Sanitary and Phytosani- poultry are raised in village or semicommercial tary Measures (the so-called SPS Agreement) of the sectors, and live poultry markets supply the local World Trade Organization (WTO), the World Orga- population with poultry meat and eggs. nization of Animal Health (Offi ce Internationale des Global production of chicken meat exceeded 55.8 Epizooties [OIE]) is responsible for establishing million metric tons in 2004, of which 6.7 million science-based standards for sanitary safety in inter- metric tons were exported worldwide (39). The top national trade of terrestrial animals and their prod- exporting countries, in decreasing order, were Brazil, ucts including poultry (42). This is achieved by the United States, the European Union, China, Thai- developing and adopting health measures to be used land, Canada, Argentina, the United Arab Emirates, by the veterinary authorities of importing and ex- Australia, and Saudi Arabia (39). Smaller quantities porting countries. The goal of these measures is to of turkey, goose, duck, and other poultry meat and prevent the transfer of agents pathogenic for animals eggs are also exported each year. The economic or humans while avoiding unjustifi ed trade barriers. importance of poultry product importation is illus- The most frequent sanitary restrictions to trade of trated by the fact that 32 of the 178 countries report- poultry and poultry products have been related to the

Avian Influenza Edited by David E. Swayne 499 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 500 Avian Influenza risk of Newcastle disease and, more recently, notifi - Infection in susceptible hosts is initiated through able avian infl uenza (NAI) as either high pathoge- inhalation of the virus or viral contact with the nicity notifi able avian infl uenza (HPNAI) or low mucous membranes, especially those of the upper pathogenicity notifi able avian infl uenza (LPNAI). respiratory tract. The unique anatomy of the bird HPNAI includes all H5 and H7 high pathogenicity contributes to upper respiratory exposure because avian infl uenza (HPAI). LPNAI includes all H5 and the middle nasal cavity is directly connected to the H7 low pathogenicity avian infl uenza (LPAI) but oropharynx through the choanal opening (31), thus excludes H1–H4, H6, and H8–H16 LPAI. Because permitting upper respiratory exposure during drink- this chapter deals with trade issues, HP NAI will be ing and eating activities. used throughout this chapter and is synonymous Recently, natural and experimental infections with HPAI. with the Euraslan lineage of H5N1 HPNAI viruses have been reported in various carnivores including Risks for Transmission of Avian Infl uenza to house cats, tigers, leopards, stone martens, and dogs Animals (30, 35, 50, 57, 76). Many of these infections were When chickens are inoculated by the intranasal associated with consumption of infected poultry or route, HPNAI causes a systemic infection which wild birds, but in most cases the mode of entry was simulates natural exposure. Virus is present in the not determined. The infections could have resulted respiratory and alimentary tracts, visceral organs, from gastrointestinal and/or oral mucous membrane brain, skin, skeletal muscle (meat), bone, and blood, exposure, or virus-containing droplets generated and virus is shed in nasal excretions, saliva, and during the tearing of carcasses while feeding could feces (62). Similar HPNAI systemic infection has have infected the conjunctiva and/or upper respira- also been commonly demonstrated in other gallina- tory mucosa. Pigs have been infected naturally and ceous poultry such as turkeys, quail, pheasants, par- experimentally with the Dutch H7N7 and the Eur- tridges, and guinea fowl (5, 6, 48). More recently, asian H5N1 lineages of HPNAI viruses (16). During the Eurasian lineage of H5N1 HPNAI viruses has the 2003 Dutch outbreak, infections in pigs occurred been shown to cause systemic infection in nongal- on farms with infected poultry, and in some cases linaceous poultry such as ducks, geese, and emus the pigs had been fed broken eggs obtained from the (11, 33, 49, 70). Previously, ducks and geese had infected chickens (36). This suggests transmission been shown to be resistant to infections by other through close contact with infected poultry or by HPNAI viruses (5, 64). In contrast, infection by the consumption of raw infected materials. For the LPNAI viruses in chickens is localized to respiratory latter method of transmission, scarifi cation of oral and alimentary tracts without the virus being present mucosa by broken egg shells may have been the in other organ systems (62). Similar restricted infec- portal of entry. Natural infections of swine with tions by LPNAI viruses have been reported in other H5N1 HPNAI virus have been rare (15), and in most poultry species (6, 64). The HPNAI and LPNAI studies experimental inoculation has inconsistently viruses do not cause persistent infections. Thus, the resulted in asymptomatic infections of swine (16, infection period is typically limited to 7 to 10 days 29, 56). but can be as long as 21 days. The modes of AI virus transmission to poultry and Risk of Transmission of Avian Infl uenza Virus other birds were presented in Chapter 4 (Epidemiol- to Humans ogy of Avian Infl uenza in Agricultural and Other AI infections have occurred sporadically in humans, Manomade Systems). Clearly, the movement of with less than 1500 documented cases over the past infected birds creates the highest risk for transmis- 50 years (12, 47). In contrast, hundreds of millions sion because the virus is replicating in and being of human cases of infl uenza A virus infection are shed from the respiratory and alimentary systems caused by endemic H1N1 and H3N2 human infl u- into the environment. In addition, feces, feathers, enza A viruses worldwide each year (65, 66). The and any item within that environment, such as equip- majority of the human AI virus infections have ment and supplies, clothes, and shoes, can be con- resulted from two HPNAI virus lineages: the 1996– taminated with the virus and become an effi cient 2007 Eurasian H5N1 lineage and the 2003 Dutch means for transporting the virus between premises. H7N7 lineage. Even fewer human infections have 22 / Trade and Food Safety Aspects for Avian Influenza Viruses 501 been caused by LPAI viruses and other HPNAI dent upon the AI virus strain. Although some AI viruses (9, 20), suggesting that infectivity of AI strains are more likely than others to infect humans, viruses for humans is at least in part strain depen- the risk of human infection from any AI strain is dent. All except one of the human fatalities has been low. In addition, various undefi ned host factors, associated with infection by the recent Eurasian such as young age and the presence of secondary lineage of H5N1 HPNAI virus. disease conditions, may increase susceptibility to AI Suffi cient epidemiological data are available from virus infections (13, 17, 69). The zoonotic aspects the two larger HPNAI epizootics with accompany- of AI viruses are discussed in Chapter 20 (Public ing human infections to draw some important con- Health Implications of Avian Infl uenza Viruses). clusions concerning human exposure risks and portals of virus entry that have resulted in human TRANSMISSION RISK THROUGH TRADE infections. The Dutch H7N7 HPNAI epizootic Human endeavors are the most frequent means of resulted predominately in clinical cases of conjunc- spreading NAI viruses. This has occurred through tivitis, with fl u-like illness reported infrequently movement of infected birds and their products or (34). This suggests that the portal of entry was most AI virus–contaminated equipment and supplies frequently through the conjunctival mucosa. For the between premises, compartments, regions, and clinical cases, individuals on depopulation crews countries. Within individual countries, the state/pro- and poultry veterinarians had higher infection rates vincial and national veterinary authorities regulate (41.2% and 26.3%, respectively) than poultry movement, in relation to animal health and products farmers and their families (14.7%) and other persons derived from such animals, to minimize the spread (7.7%), suggesting that the risk of human H7N7 of animal and zoonotic disease agents such as NAI. infection during the outbreak was associated with The OIE Terrestrial Animal Health Code (42) pro- the amount of exposure to infected poultry. During vides the sanitary recommendations for safe inter- the ongoing H5N1 HPNAI virus epizootic, human national trade and emphasizes safety and risk infections have resulted from very close direct or assessment for importation of animals and animal indirect exposure to H5N1 HPNAI-infected poultry products. The goal is to prevent unacceptable risks (live or dead) in the household, village, or live to animal and human health while avoiding unjusti- poultry market (46, 55), with presumed entry of the fi ed or politically motivated trade barriers. The virus through respiratory and/or oropharyngeal development of transparent, objective, and defensi- tissues (21). However, one case was linked to pre- ble risk analyses for international commerce are sumed virus exposure via consumption of uncooked essential for the prevention of NAI introduction and duck blood and organs, and another case was linked for the protection of animal health, and potentially to defeathering of H5N1 HPNAI virus–infected human health, while allowing fair and safe trade to dead swans (74, 75). Although the uncooked duck continue. blood consumption case could suggest exposure via the gastrointestinal tract after ingestion of the virus Levels of Risk for Avian Infl uenza Through in the raw food, proof that HPNAI virus replicates Trade in the human intestinal tract is lacking, and entry The level of risk in spreading NAI virus through could have been through contact with the oropha- trade is dependent upon several factors including the ryngeal mucosa (21). However, exposure to H5N1 following: (1) the ability to demonstrate freedom HPNAI-infected poultry does not usually produce from NAI in the country, zone, or compartment human infections. A recent study in Cambodia (CZC) through adequate surveillance and diagnos- described the lack of H5N1 HPNAI infections tics; (2) the type of NAI virus present, such as among villagers that had frequent close contact with LPNAI versus HPNAI; (3) the specifi c type of prod- H5N1 HPNAI-infected poultry, suggesting the ucts traded; and (4) the use of any type of treatments transmission potential from poultry to individual for NAI virus inactivation. Proper guidelines for humans is very low (73). These data suggest that conducting an importation risk analysis are con- human infections with AI viruses require exposure tained within Chapter 1.3.2 of the Terrestrial Animal to large quantities of virus via direct or indirect Health Code (40). Furthermore, in the recent revi- exposure to infected poultry, and infection is depen- sion of the AI code chapter (2.7.12.1), the OIE dif- 502 Avian Influenza ferentiates risk levels for NAI when importing accessible to susceptible wild birds). Two recently various poultry, other birds, or products derived completed risk analyses have determined that the from such birds (43). If the exporting CZC is free probability of introducing LPNAI through chicken from NAI based on appropriate test methods and meat imports was insignifi cant to negligible (51, adequate surveillance sampling, and the CZC has 77). been consistently transparent in reporting animal health issues to the OIE member countries, trade in Avian Infl uenza Virus Detection in Poultry, poultry and poultry products should not be prohib- Other Birds, and Their Products From Trade ited based on AI. If the exporting CZC is affected Cross-border transfer of AI viruses has occurred by NAI, the importation risk from HPNAI is greater from both legal and illegal trade of live poultry, than that from LPNAI due to the systemic nature of other live birds, and avian-derived products. Prior to HPNAI virus infections in poultry (the virus is the 1970s, the lack of consistent restrictions on bird present throughout the bodies of infected birds) and imports resulted in accidental introduction of exotic the greater economic impact of HPNAI compared to avian diseases such as Newcastle disease and AI. LPNAI. For example, due to shedding of LPNAI The linkage of the 1972 Newcastle disease outbreak from the cloaca (the common exit chamber for the in poultry of Southern California to unrestricted digestive and reproductive tracts), LPNAI virus can movement of exotic birds from Central America into be found on the surface of eggs laid by acutely the United States led to the development and aug- infected hens (61). However, the isolation of LPNAI mentation of quarantine and testing requirements for from the internal contents of eggs has not been imported pet birds and poultry in the United States reported to date. As a result, eggs produced in an (1974), the United Kingdom (1976), and other coun- LPNAI-affected CZC could be imported into an tries (1, 71). Following the implementation of justi- NAI-free country if the eggshell surface were sani- fi ed import restrictions and the quarantine and testing tized to eliminate any LPNAI virus and the eggs of imported birds, AI viruses have been isolated on were transported in new packing materials (40). infrequent occasions from smuggled or illegally However, sanitized eggs from an HPNAI-affected imported birds and include the following examples: CZC should not be imported into an NAI-free (1) H5N1 HPNAI virus from a Crested Hawk Eagle country because the virus is present not only on the (Spizaetus cirrhatus) smuggled from Thailand to surface of eggshells but could also be within the Belgium (2004) (72); (2) H5N1 HPNAI virus from internal contents of eggs laid by HPNAI-infected ducks and other birds smuggled from China to hens before they die (61). Taiwan (2003 and 2006) (14, 28); (3) H5N1 HPNAI With the specifi c commodities, the importation virus from several birds within a private Essex quar- risk varies with the specifi c product and are listed antine station in the United Kingdom (2005) (24); from highest to lowest as follows: (1) live poultry (4) H7N1 LPNAI from an Asian Glossy Starling (other than day-old poultry); (2) live birds other than (Aplonis panayensis) imported in a United Kingdom poultry; (3) day-old live poultry; (4) hatching eggs; quarantine station (1979) (1); (5) H5N2 LPNAI (5) eggs for human consumption; (6) egg products; from ducks in a Singapore quarantine station (1998 (7) products derived from poultry such as semen, and 2004) (3, 4); (6) H7N1 HPNAI from a Pekin raw meat, and other untreated products; and (8) Robin (Leiothrix lutea) imported from China in a poultry products which have been treated to inacti- U.S. quarantine station (45); (7) H7N1 LPNAI from vate NAI viruses. If the product is from an NAI- conures, parrots, and parakeets in the United affected CZC, treatment to inactivate NAI can be Kingdom and the Netherlands (1994) (2); and (8) used to eliminate the risk provided that the exporter H5N2 LPNAI from a Red-Lored Amazon Parrot has taken appropriate steps to prevent recontamina- (Amazona autumnalis autumnalis) smuggled into tion of the fi nal product, as recommended by the the United States (2005) (27). In contrast, AI virus OIE. Furthermore, importation of HPNAI-infected isolations from poultry products have been reported raw meat or other products may not result in infec- less frequently. In the past decade, AI viruses have tions unless the product is fed to susceptible hosts been isolated from poultry carcasses and meat (i.e., feeding raw scraps to backyard poultry or including H5N1 HPNAI virus from legal imports of placing garbage containing raw scraps in an area frozen duck meat from China into South Korea 22 / Trade and Food Safety Aspects for Avian Influenza Viruses 503

(2001) (70) and Japan (2003) (37), and H10N7 animal diseases to the OIE. Freedom from NAI can LPAI virus isolated from lungs and tracheas in be demonstrated for a country or parts of a country, chicken and duck carcasses illegally imported from such as a zone (i.e., a region with defi nable geo- China into Italy (2006) (10). In 2007, an outbreak graphic features) or a compartment (i.e., a functional of H5N1 HPNAI occurred on a single turkey farm unit separated from other units by biosecure man- in Suffolk, United Kingdom (19). The source of the agement practices) (52). However, under the SPS introduction was not clearly evident, but epidemio- agreement, the importing country cannot impose logical evidence indicated that the introduction was upon the exporting country sanitary measures that most likely from importation of infected fresh turkey are not based on international standards. If a country meat from Hungary that originated from a subclini- chooses to apply more stringent measures (i.e., cally infected fl ock. The study proposed that trans- above and beyond international standards), these mission followed a series of low probability events, should be supported by a scientifi cally valid risk with gulls consuming infected scraps discarded at assessment. Additionally, if an importing country is the processing plant and roosting on a neighboring affected by a disease it can only require measures turkey house that had poor biosecurity, leading to an that are equivalent to those applied nationally. HPNAI breakout in the turkey house. Various types of mitigation strategies can reduce A variety of H5N1 HPNAI viruses isolated from importation risks for NAI viruses and are discussed birds and humans between 2003 and 2004 have been later. demonstrated in meat obtained from both naturally and experimentally infected chickens, Japanese quail, Vaccination ducks, and geese (32, 60, 70). In one experimental The HPNAI viruses produce systemic infection in study, A/chicken/South Korea/ES/2003 (Korea/03) a variety of poultry species, and thus meat or other H5N1 HPNAI virus was transmitted to naïve broi- products from infected birds could contain the virus lers by feeding breast meat from infected chickens (7, 38, 48, 62, 70). Vaccination has been used as a (Table 22.1), but A/chicken/Pennsylvania/1370/1983 tool to prevent illness and death, reduce infection (PA/83) H5N2 HPNAI virus was not transmitted by rates, reduce shedding of virus from respiratory and feeding similar infected chicken meat (62). This dis- alimentary systems, prevent contact transmission, crepancy in transmission was the result of differences and prevent systemic infection (58, 63). In a recent in the challenge dose. Chickens fed Korea/03 H5N1 study, high levels of the virus were isolated from HPNAI virus-infected meat received 107.8 embryo breast meat of nonvaccinated chickens after intrana- infectious doses (EID50) of virus, while chickens fed sal challenge with a Korea/03 H5N1 HPNAI virus, PA/83 H5N2 HPNAI virus-infected meat received but no virus was recovered from breast or thigh meat 3.5−3.6 10 EID50 of virus. The probability of imported of chickens vaccinated at 1 day of age with either a raw product initiating HPNAI outbreaks via con- traditional inactivated AI vaccine or a recombinant sumption by a susceptible host is low. However, the fowl pox virus vaccine containing an AI virus H5 incident in the United Kingdom, along with experi- gene insert and intranasally challenged 3 weeks later mental feeding studies in chickens, emphasizes the with a high dose of the Korea/03 H5N1 HPNAI need for sanitary standards that prevent accidental virus (see Table 22.1) (62). Similarly, HPNAI virus importation of HPNAI virus–infected products that was not recovered from meat or viscera of domestic could possibly lead to HPNAI outbreaks. ducks vaccinated at 1 day of age and again at 4 weeks of age with a commercial inactivated H5N1 MITIGATION OF TRADE RISKS HPNAI virus and intranasally challenged with a The best method to ensure safe and fair trade for high dose of a 2005 Vietnam H5N1 HPNAI virus NAI-free countries is importation from other (11). Nonvaccinated ducks had virus in meat and NAI-free countries (44). Freedom from NAI can be visceral organs following challenge. Virus was not demonstrated only for countries that conduct sero- recovered from blood or meat of turkeys vaccinated logical and virological surveillance utilizing sensi- with commercial inactivated H7N1/H5N9 bivalent tive testing and targeted or random statistically vaccine and challenged with H7N1 HPNAI virus, based sampling methods (41) and that have his- but nonvaccinated turkeys had virus in meat and torically demonstrated transparency in reporting blood (68). These studies demonstrated that proper 504 Avian Influenza

Table 22.1. Effect of vaccination on transmission of HPAI virus through meat. Speciﬁ c-pathogen–free chickens vaccinated subcutaneously at 1 day of age with either a recombinant fowl pox virus containing an H5 AI gene insert (rFP-AI-H5) or inactivated H5N9 AI oil-emulsiﬁ ed vaccine (A/turkey/Wisconsin/68) and challenged intranasally 3 weeks later with H5N1 HPNAI virus (A/chicken/South Korea/ES/03) (62). Virus isolation from a b meat (log10 EID50/g) Feeding Study

Group Breast Thigh Virus dose/bird (log10 EID50) Mortality rFP-AI-H5 vaccine — — ND ND Inactivated vaccine — — ND ND Sham 7.3 ND 7.8a 9/10 a Meat samples were from day 2 after inoculation from euthanized (vaccine) or dead (sham) chickens. b Feeding the meat from the sham chickens was fed to naïve 3- to 4-week-old chickens. ND-not done. c —, No virus isolated; ND, not done.

vaccination can reduce the risk of HPNAI virus in reproducible microassay method for measuring poultry meat and viscera. virus inactivation in meat samples from chickens infected intranasally with HPNAI (59). Using a Inactivation Methods for Avian Inﬂ uenza minimal cooking temperature of 70º C, infectious Viruses in Poultry Products Korea/03 H5N1 HPAI virus was detected in infected A variety of treatment methods can be applied to meat at 1 second after treatment but not after 5 products from NAI-infected CZC to inactivate the seconds (59). Recently, a detailed thermal inactiva- NAI virus and render the products safe. Goose feath- tion study for Korea/03 H5N1 HPNAI virus estab- ers (down) are a common exported commodity from lished inactivation line equations, Dt values (time

Asia and are used as insulation or ﬁ ller for pillows, required to reduce infectious titer by 1 log10 at a quilts, sleeping bags, coats, and other apparel. speciﬁ c temperature), the Z value (the increase in

Because NAI viruses are very susceptible to heat, temperature required to reduce the Dt value by 1 detergents and a variety of chemicals (see Chapter log10), minimum cooking times at various tempera- 18, Methods for Inactivation of Avian Inﬂ uenza tures, and comparisons with minimum U.S. Depart- Virus in the Environment), feathers or other prod- ment of Agriculture (USDA) Food Safety and ucts can be treated with steam or detergents to kill Inspection Service (FSIS) time-temperature guide- any NAI virus that might be present. However, such lines for a 107 reduction of Salmonella (Table 22.2) treatments must follow standardized processes that (67). This study estimated a maximum titer of 108.7 provide uniform treatment to the product and kill the mean chicken EID50 of H5N1 HPNAI virus per gram virus without altering the physical qualities of the of chicken meat (breast or thigh). As shown in Table feathers. 22.2, the inactivation line equation for HPNAI in Heat is also commonly used to inactivate a variety chicken breast and thigh meat predicts inactivation of pathogenic and nonpathogenic viruses, bacteria, of HPNAI in a 100-g sample after a holding time of fungi, and protozoa in food products derived from 5.5 seconds at 70º C (158º F) or a holding time of poultry. Such heat applications are typically accom- 0.80 second at the standard USDA cooking tem- plished using pasteurization or cooking processes. perature of 73.9º C (165º F). In terms of the USDA FSIS guidelines for Salmonella reduction, the Poultry meat established time-temperature combinations exceed Initial thermal inactivation studies of AI viruses those predicted to inactivate H5N1 HPNAI virus in in poultry meat focused on the development of a chicken meat. 22 / Trade and Food Safety Aspects for Avian Influenza Viruses 505

Table 22.2. Time predicted for an 11-log10 reduction of Korea/03 virus titer in chicken meat at a given internal temperature, and number of log10 reductions of Korea/03 virus titer achieved in chicken meat cooked according to minimum current USDA FSIS time-temperature guidelines for a 7-log10 reduction of Salmonella (67). 95% PI upper Time predicted for an Minimum FSIS Predicted number of Temperature limit for Dt value 11-log10 EID50 time-temperature log10 EID50 reductions º C º F (seconds)a reduction of Korea/03 guidelineb of Korea/03 achievedc

57.8 136 215.8 39.6 min 63.3 min 17.6 58.9 138 125.0 22.9 min 39.7 min 19.1 60.0 140 72.4 13.3 min 25.2 min 20.9 61.1 142 41.9 7.7 min 16.1 min 23.1 70.0 158 0.50 5.5 sec 21.9 sec 43.8 73.9 165 0.073 0.80 sec <10 secd 13.7 per second

a D t values with upper limit of the 95% prediction interval were calculated from combined breast and thigh meat model line equation (y = log10 D t value), y = [(−0.2157) (temp. º C)] + 14.6773 + (2 × RMSE), where the RMSE = 0.0621 (RMSE = root-mean-square error). b From the time-temperature table for chicken meat with 1% fat. c Assuming that the required internal temperature is maintained for the length of time speciﬁ ed in the FSIS time-temperature table. d Required lethality is achieved instantly at this internal temperature.

Egg products for the ability to inactivate any theoretical LPNAI HPNAI virus has been isolated from the internal virus (Table 22.4). The thermal inactivation data contents of eggs laid by infected hens, while LPNAI from the egg product study predicts that the standard or LPAI viruses have not been demonstrated in the commercial pasteurization processes would inacti- 2.3 internal contents of eggs laid by acutely infected vate greater than 10 EID50 of LPNAI virus per ml hens (61). Pasteurization processes, which use lower of egg product, and seven of the nine processes temperatures than those used for cooking, are typi- would inactivate the theoretically impossible 1017.6 cally used to inactivate microorganisms in egg prod- or greater EID50 of LPNAI virus per milliliter of egg ucts. This allows retention of egg functional product. properties such as albumen and yolk color and In comparison to the precise meat cooking study viscosity traits. The maximum reported titer of (67), the initial pasteurization studies were not

HPNAI virus in the internal contents of chicken eggs designed to provide precise and complete Dt and Z 4.9 laid by infected hens is 10 EID50/ml (61). A recent values and inactivation line equations for NAI virus study of thermal inactivation of HPNAI virus in inactivation in egg products, but provided only liquid and dried egg products predicted that seven initial conservative guidelines based on approximate of nine standard commercial pasteurization time- inactivation temperatures that favored overestima- temperature combinations would effectively inacti- tion. Additional studies are needed to establish more 4.9 vate 10 EID50/ml of HPNAI virus and that six of precise inactivation times and temperatures for the nine processes would provide an extra safety HPNAI and LPNAI virus in egg products. The 2 margin of 10 or greater EID50/ml (Table 22.3). OIE’s Terrestrial Animal Health Code provides an Although LPNAI virus has not been demonstrated appendix with the summary of the recommended in the internal contents of eggs laid by acutely time and temperature combinations to render various infected hens, pasteurization processes were assessed poultry products safe (40). 506 Avian Influenza

Table 22.3. Estimated pasteurization times for eggs contaminated with HPNAI and estimated number of log10 reductions of HPNAI achieved by industry pasteurization standards.

Estimated time for Industry pasteurization standards inactivationb Estimated number of

Temperature 4.9 log10 6.9 log10 Holding HPNAI log10 EID50 a c Egg product (º C) Dt value EID50/ml EID50/ml time reductions

Dried egg white 54.4 3.1 days 15.2 days 21.4 days 7–10 days 2.3 to 3.2 Dried egg white 67.0 0.12 day 0.59 days 0.83 days 15 days 125 Liquid egg white 55.6 124.3 sec 609.1 sec 857.7 sec 372 sec 3.0 Liquid egg white 56.7 32.9 sec 161.2 sec 227.0 sec 210 sec 6.4 Whole egg 60.0 27.1 sec 132.8 sec 187.0 sec 210 sec 7.7 Whole egg blends 60.0 27.1 sec 132.8 sec 187.0 sec 372 sec 13.7 Whole egg blends 61.1 13.5 sec 66.2 sec 93.2 sec 210 sec 15.6 10% Salted yolk 62.2 <20 sec <98 sec <138 sec 372 sec >18.6 10% Salted yolk 63.3 <20 sec <98 sec <138 sec 210 sec >10.5

a D t values calculated from line equations (y = log10 D t value). Dried egg white: y = [(−0.114) (temp º C)] + 6.5560. Liquid egg white: y = [(−0.5248) (temp º C)] + 31.2732. Whole egg: y = [(−0.2740) (temp º C)] +

17.8730. For 10% salted yolk, 20 seconds is the estimated maximum Dt value. Dt value data and line equations adapted from Swayne and Beck (2004) (61). b 4.9 log10 EID50/ml is the highest virus titer reported in eggs laid by hens infected with HPAI (M. Brugh, unpublished data). c Dried egg white at 67º C [Baron et al. (2003) (8)]. All other pasteurization standards from Froning et al. (2002) (23). Pasteurization standards for whole egg blends are for whole egg products with less than 2% nonegg ingredients added. Modiﬁ ed from Swayne and Beck (2004) (61).

FOOD SAFETY RISKS? cells. AI virus replication stops after the host animal Natural and experimental NAI cases have demon- dies, limiting the possible viral load in the food strated the systemic nature of HPNAI infections in product. Second, the receptors needed for attach- poultry, including the presence of virus in meat and ment and replication of AI viruses are present pre- eggs (5, 6, 11, 33, 48, 49, 62, 70). Most human dominantly in the lungs and have not been described infections with HPNAI virus have resulted from in the human digestive tract. This suggests that AI close contact with live or dead HPNAI virus infected is more likely to infect humans via the respiratory birds (47). Consumption of an infected food (raw system. Finally, proper cooking will inactivate any duck blood pudding) has been associated with one HPNAI virus that might be present (21, 67). Taken human case of H5N1 HPNAI infection (75), but together, these data suggest that HPNAI is not cur- epidemiological data were insuffi cient to confi rm rently a signifi cant food safety issue for humans. that consumption of the infected product was the However, because most cases of HPNAI infection transmission route (21). Several factors limit the in humans have been linked to direct contact with potential impact of AI viruses on food safety. First, infected birds, known HPNAI-infected fl ocks should unlike free-living bacteria such as Salmonella not be processed for food in the home setting, live typhimurium and Escherichia coli, which can con- poultry markets, or slaughter plants. Human expo- tinue to grow in food products postharvest or post- sure could occur when catching, handling, transport- slaughter, AI viruses can only grow in living animal ing, or slaughtering diseased birds. Human infections 22 / Trade and Food Safety Aspects for Avian Influenza Viruses 507

Table 22.4. Estimated pasteurization times for eggs artiﬁ cially contaminated with LPNAI a virus and estimated number of log10 reductions of LPNAI virus achieved by industry pasteurization standards. Industry pasteurization standards Estimated number of LPNAI b c Egg product Temperature (º C) Dt value Holding time log10 EID50 reductions

Dried egg white 54.4 0.5 day 7 to 10 days 14 to 20 Dried egg white 67.0 <0.4 day 15 days >37.5 Liquid egg white 55.6 163.9 sec 372 sec 2.3 Liquid egg white 56.7 32.8 sec 210 sec 6.4 Whole egg 60.0 11.9 sec 210 sec 17.6 Whole egg blends 60.0 11.9 sec 372 sec 31.3 Whole egg blends 61.1 5.3 sec 210 sec 39.6 10% Salted yolk 62.2 4.4 sec 372 sec 84.5 10% Salted yolk 63.3 3.2 sec 210 sec 65.6 a LPNAI virus has not been demonstrated in the internal contents of eggs laid by acutely infected hens. All data are based on artiﬁ cial contamination of egg products with LPNAI virus. b D t values calculated from line equations (y = log10 D t value). Liquid egg white: y = [(−0.6347) (temp º C)] + 37.5039. Whole egg: y = [(−0.3162) (temp º C)] + 20.0473. 10% salted yolk: y = [(−0.1341) (temp º C)] +

8.9872. For dried egg white, 0.5 day is the estimated Dt value for 54.4º C, and 0.4 day is the estimated maximum Dt value for 57º C. Dt values for temperatures above 57º C were not determined. Dt value data and line equations adapted from Swayne and Beck (2004) (61). c Dried egg white at 67º C [Baron et al. (2003) (8)]. All other pasteurization standards from Froning et al. (2002) (23). Pasteurization standards for whole egg blends are for whole egg products with less than 2% nonegg ingredients added. Modifi ed from Swayne and Beck (2004) (61). could presumably occur through generation and fi nancial losses while ensuring farmers, workers, inhalation of small droplets, dust, or aerosols con- and consumers of safety (25, 26). Clearance of AI taining the virus or by touching the nasal, conjunc- virus infection can be confi rmed in the fl ock by tival, or oral mucous membranes with contaminated testing for AI virus in the normal daily fl ock mortal- hands. ity by real-time reverse transcription polymerase LPNAI/LPAI and HPNAI infections differ in chain reaction or pen-side antigen capture tests on both birds and humans. In birds, LPNAI and LPAI oropharyngeal or tracheal swabs before marketing produce limited respiratory and gastrointestinal and processing the birds (18, 53, 54). In addition, infections, and virus is not detected in poultry meat the processed carcasses should not contain respira- or the internal contents of eggs (61, 62). In humans, tory or digestive tissues. there have only been 11 documented LPNAI/LPAI infections in the past 30 years, none of which were CONCLUSIONS fatal (47). All of the human cases presented with Poultry are the most frequently raised farm animals conjunctivitis and/or respiratory infections. These and, on a global basis, birds are the major source of data indicate that LPNAI/LPAI is an even lower animal protein in the human diet. Domestic produc- food safety risk for humans than HPNAI. Because tion and distribution systems, as well as imported the poultry infections last only 7 to 10 days, LPNAI/ products, are critical to meet the culinary demands LPAI virus–infected fl ocks have been safely mar- of consumers and to supply markets with other prod- keted after recovery from infection Controlled mar- ucts, such as live birds, hatching eggs, and feathers. keting of recovered fl ocks allows farmers to recoup The OIE Terrestrial Animal Health Code provides 508 Avian Influenza sanitary standards for international trade and empha- REFERENCES sizes science-based risk assessment for safe impor- 1. Alexander, D.J. 1981. Isolation of infl uenza A tation of animals and animal products. The goal is viruses from exotic birds in Great Britain. In: R.W. to prevent unacceptable risks to animal and human Bankowski (ed.). Proceedings of the First Interna- health while avoiding unjustifi ed or politically moti- tional Symposium on Avian Infl uenza, April 1981, vated trade barriers. Beltsville, Maryland. Symposium on Avian Infl u- Human endeavors are the most frequent means of enza, US Animal Health Association: Richmond, spreading NAI viruses. This has occurred through VA, pp. 79–92. movement of infected birds and their products or AI 2. Alexander, D.J. 1998. Avian infl uenza in the virus–contaminated equipment and supplies between Eastern Hemisphere (excluding the Pacifi c Basin) during 1992–1997. In: D.E. Swayne and R.D. premises, compartments, regions, and countries. The Slemons (eds.). Proceedings of the Fourth Interna- level of risk in spreading NAI virus through trade is tional Symposium on Avian Infl uenza, May 29–31, dependent upon several factors, including (1) the 1997, Athens, Georgia. Symposium on Avian presence or absence of NAI in a CZC as demon- Infl uenza, US Animal Health Association: Rich- strated through adequate surveillance and diagnos- mond, VA, pp. 9–13. tics, as well as transparent reporting; (2) the type of 3. Alexander, D.J. 2003. Report on avian infl uenza in NAI virus present, such as LPNAI versus HPNAI; the Eastern Hemisphere during 1997–2002. Avian (3) the specifi c type of products traded; and (4) the Diseases 47(Suppl.):792–797. use of any type of treatments for NAI virus inactiva- 4. Alexander, D.J. 2007. Summary of avian infl uenza tion. Because HPNAI viruses cause systemic infec- activity in Europe, Asia, Africa and Australasia, tion in poultry, the risk for transfer through trade 2002–2006. Avian Diseases 51(Suppl.):792– 797. is greater than for that for LPNAI viruses. Cross- 5. Alexander, D.J., W.H. Allan, D.G. Parsons, and G. border transfer of NAI viruses has occurred from Parsons. 1978. The pathogenicity of four avian both legal and illegal trade of live poultry, other live infl uenza viruses for fowls, turkeys and ducks. birds, and avian-derived products. These incidences Research in Veterinary Science 24:242–247. emphasize the need for sanitary standards that 6. Alexander, D.J., G. Parsons, and R.J. Manvell. prevent accidental importation of HPNAI virus that 1986. Experimental assessment of the pathogenic- potentially could lead to HPNAI outbreaks. Various ity of eight infl uenza A viruses of H5 subtype for mitigation strategies can be used to reduce risk, such chickens, turkeys, ducks and quail. Avian Pathol- as vaccination of poultry in an NAI-affected CZC or ogy 15:647–662. using inactivation processes such as cooking or pas- 7. Antarasena, C., R. Sirimujalin, P. Prommuang, teurization of products obtained from an affected S.D. Blacksell, N. Promkuntod, and P. Prom- muang. 2006. Tissue tropism of a Thailand strain CZC. of high-pathogenicity avian infl uenza virus (H5N1) To date, the total number of confi rmed human AI in tissues of naturally infected native chickens virus infections has been relatively small, with the (Gallus gallus), Japanese quail (Coturnix coturnix majority caused by only two lineages: the H5N1 japonica) and ducks (Anas spp.). Avian Pathology Eurasian lineage (1996–2007), and the H7N7 Dutch 35(3):250–253. lineage (2003). Because currently circulating HPNAI 8. Baron, F., F. Nau, C. Guerin-Dubiard, F. Gonnet, strains are not easily transmitted to humans, human J.J. Dubois, and M. Gautter. 2003. Effect of dry infection has required exposure to large quantities heating on the microbiological quality, functional of virus to the respiratory tract. Most cases have properties, and natural bacteriostatic ability of egg occurred following direct or indirect exposure to white after reconstitution. Journal of Food Protec- infected poultry. Taken together, current data indi- tion 66(5):825–832. 9. Beare, A.S., and R.G. Webster. 1991. Replication cate that HPNAI is primarily an animal health issue of avian infl uenza viruses in humans. Archives of rather than a human health or food safety issue. Virology 119:37–42. 10. Beato, M.S., C. Terregino, G. Cattoli, and I. Capua. ACKNOWLEDGMENTS 2006. Isolation and characterization of an H10N7 C. Zepeda and A. Thiermann are thanked for critical avian infl uenza virus from poultry carcasses smug- review of the manuscript and suggestions for gled from China into Italy. Avian Pathology improvement. 35(5):400–403. 22 / Trade and Food Safety Aspects for Avian Influenza Viruses 509

11. Beato, M.S., A. Toffan, R. De Nardi, A. Cristalli, in mice as compared to other highly pathogenic H5 C. Terregino, G. Cattoli, and I. Capua. 2007. A avian infl uenza viruses. Journal of Virology conventional, inactivated oil emulsion vaccine 74(3):1443–1450. suppresses shedding and prevents viral meat colo- 21. EFSA. 2006. Food as a possible source of infection nisation in commercial (Pekin) ducks challenged with highly pathogenic avian infl uenza viruses for with HPAI H5N1. Vaccine 25(20):4064–4072. humans and other mammals. The EFSA journal 12. Bosman, A., Y.M. Mulder, J.R.J. de Leeuw, A. 74:1–29. Meijer, M. Du Ry van Beest Holle, R. A. Kamst, 22. FAO. 2007. FAO Statistical Yearbook. 2005–2006. P. G. van der Velden, M.A. E. Conyn-Van Spae- http://www.fao.org/statistics/yearbook/vol_1_1/ ndonck, M.P.G. Koopmans, and M.W.M.M. index.asp. Accessed April 14, 2007. Ruijten. 2003. Avian fl u epidemic 2003: Public 23. Froning, G.W., D. Peters, P. Muriana, K. Eskridge, health consequences. RIVM report 2003:1–35. D. Travnicek, and S.S. Sumner. 2002. International 13. Centers for Disease Control and Prevention. 2005. Egg Pasteurization Manual. United Egg Associa- Avian Infl uenza Infection in Humans. Available at tion: Alpharetta, GA, pp. 1–67. http://www.cdc.gov/fl u/avian/gen-info/avian-fl u- 24. Gauthier-Clerc, M., C. Lebarbenchion, and F. humans.htm. Accessed April 7, 2007. Thomas. 2007. Recent expansion of highly patho- 14. Chang, T.J. 2004. Avian Infl uenza—Taiwan: Not. genic avian infl uenza H5N1: a critical review. Ibis PROMED. 20040114.0148. Available at http:// 149:202–214. www.promedmail.org/pls/promed/f?p=2400:1202 25. Halvorson, D.A. 1998. Epidemiology and control :3489339336032481318::NO::F2400_P1202_ of avian infl uenza in Minnesota. In: Proceedings CHECK_DISPLAY,F2400_P1202_PUB_MAIL_ of the 47th New England Poultry Health Confer- ID:X,24053. ProMed. Accessed on 4/23/07. ence, New England Poultry Association: Ports- 15. Chen, H., H. Li, H. Yang, C. Qiao, K. Yu, A. mouth, NH, pp. 5–11. Schudel, and M. Lombard. 2006. Swine infl uenza 26. Halvorson, D.A. 2002. The control of H5 or in China. Development in Biologicals 124:73. H7 mildly pathogenic avian infl uenza: a role for 16. Choi, Y.K., T.D. Nguyen, H. Ozaki, R.J. Webby, inactivated vaccine. Avian Pathology 31(1):5– P. Puthavathana, C. Buranathal, A. Chaisingh, P. 12. Auewarakul, N.T.H. Hanh, S.K. Ma, P.Y. Hui, Y. 27. Hawkins, M.G., B.M. Crossley, A. Osofsky, R.J. Guan, J. Peiris, Sr., and R.G. Webster. 2005. Webby, C. Lee, D.L. Suarez, and S.K. Hietala. Studies of H5N1 infl uenza virus infection of pigs 2006. Avian infl uenza A virus subtype H5N2 by using viruses isolated in Vietnam and Thailand in a red-lored Amazon parrot. Journal of the in 2004. Journal of Virology 79(16):10821– American Veterinary Medical Association 228(2): 10825. 236–241. 17. Chotpitayasunondh, T., S. Lochindarat, P. Srisan, 28. Huang, T.M. 2005. Taiwan detects avian fl u virus K. Chokepaibulkit, J. Weerakul, M. Maneeratta- (H5N1) on smuggled birds from China. naporn, and P. Sawanpanyalert. 2004. Cases of PROMED 20051020.3055. Available at http:// infl uenza A (H5N1)—Thailand, 2004 (Reprinted www.promedmail.org/pls/promed/f?p=2400:1001 from MMWR 53:100–103, 2004). Journal of the :3489339336032481318::::F2400_P1001_BACK_ American Medical Association 291(9):1059– PAGE, F2400_P1001_ARCHIVE_ 1060. NUMBER,F2400_P1001_USE_ARCHIVE:1202, 18. Davison, S., R.J. Eckroade, and A.E. Ziegler. 20051020.3055,Y. ProMed. Accessed April 23, 2003. A review of the 1996–98 nonpathogenic 2007. H7N2 avian infl uenza outbreak in Pennsylvania. 29. Isoda, N., Y. Sakoda, N. Kishida, G.-R. Bai, K. Avian Diseases 47:823–827. Matsuda, T. Umemura, and H. Kida. 2006. Patho- 19. DEFRA. 2007. Outbreak of highly pathogenic genicity of a highly pathogenic avian infl uenza H5N1 avian infl uenza in Suffolk in January 2007. virus, A/chicken/Yamaguchi/7/04 (H5N1) in dif- Report of the Epidemiological Findings by the ferent species of birds and mammals. Archives of National Emergency Epidemiology Group. 5 April Virology 151(7):1267–1279. 2007. http://www.defra.gov.uk/animalh/diseases/ 30. Keawcharoen, J., K. Oraveerakul, T. Kuiken, A.M. notifi able/disease/ai/pdf/epid_fi ndings050407.pdf. Fouchier Ron, A. Amonsin, S. Payungporn, S. Accessed April 25, 2007. Noppornpanth, S. Wattanodorn, A. Theamboo- 20. Dybing, J.K., S. Schultz Cherry, D.E. Swayne, niers, R. Tantilertcharoen, R. Pattanarangsan, N. D.L. Suarez, and M.L. Perdue. 2000. Distinct Arya, P. Ratanakorn, D.M.E. Osterhaus, and Y. pathogenesis of Hong Kong-origin H5N1 viruses Poovorawan. 2004. Avian infl uenza H5N1 in tigers 510 Avian Influenza

and leopards. Emerging Infectious Diseases mcode/en_chapitre_3.8.9.htm. Offi ce Internatio- 10(12):2189–2191. nale des Epizooties: Paris. Accessed May 8, 31. King, A.S., and J. McLelland. 1984. Respiratory 2007. system. In: Birds. Their Structure and Function. 42. Offi ce Internationale des Epizooties. 2006. Ter- Bailliere Tindall: London, pp. 110–144. restrial Animal Health Code. Available at http:// 32. Kishida, N., Y. Sakoda, N. Isoda, K. Matsuda, M. www.oie.int/eng/normes/mcode/en_sommaire.htm. Eto, Y. Sunaga, T. Umemura, and H. Kida. 2005. Accessed April 14, 2007. Pathogenicity of H5 infl uenza viruses for ducks. 43. Offi ce Internationale des Epizooties. 2006. Zoning Archives of Virology 150(7):1383–1392. and compartmentalisation. In: Terrestrial Animal 33. Kishida, N., Y. Sakoda, N. Isoda, K. Matsuda, M. Health Standards Commission Report 2006. Offi ce Eto, Y. Sunaga, T. Umemura, and H. Kida. 2005. Internationale des Epizooties: Paris, pp. 1–6. Pathogenicity of H5 infl uenza viruses for ducks. 44. Offi ce Internationale des Epizooties. 2007. Avian Archives of Virology 150(7):1383–1392. Infl uenza. Terrestrial Animal Health Code. Article 34. Koopmans, M., B. Wilbrink, M. Conyn, G. Natrop, 2.7.12.1. Available at http://www.oie.int/eng/ H. van der Nat, H. Vennema, A. Meijer, J. van normes/mcode/en_chapitre_2.7.12.htm. Offi ce Steenbergen, R. Fouchier, A. Osterhaus, and A. Internationale des Epizooties: Paris, France. Bosman. 2004. Transmission of H7N7 avian infl u- Accessed April 24, 2007. enza A virus to human beings during a large out- 45. Pearson, J.E., D.A. Senne, and B. Panigrahy. 1998. break in commercial poultry farms in the Avian infl uenza in the Western Hemisphere includ- Netherlands. Lancet 363(9409):587–593. ing the Pacifi c Basin 1992–1996. In: D.E. Swayne 35. Kuiken, T., G. Rimmelzwaan, D. van Riel, G. van and R.D. Slemons (eds.). Proceedings of the Fourth Amerongen, M. Baars, R. Fouchier, and A. Oster- International Symposium on Avian Infl uenza, May haus. 2004. Avian H5N1 infl uenza in cats. Science 29–31, 1997, Athens, Georgia. Symposium on 306(5694):241. Avian Infl uenza, US Animal Health Association: 36. Loeffen, W.L.A., and G. Koch. 2003. Avian Richmond, VA, pp. 14–17. Infl uenza (H7N7) in Dutch pigs. PROMED. 46. Perdue, M.L., D.L. Suarez, and D.E. Swayne. 20030520.1240. http://www.promedmail.org/pls/ 1999. Avian Infl uenza in the 1990’s. Poultry and promed/f?p=2400:1202:4445814957589253518:: Avian Biology Reviews 11(1):1–20. NO::F2400_P1202_CHECK_DISPLAY,F2400_ 47. Perdue, M.L., and D.E. Swayne. 2005. Public P1202_PUB_MAIL_ID:X,21644. CIDC. Accessed health risk from avian infl uenza viruses. Avian April 23, 2007. Diseases 49(3):317–327. 37. Mase, M., M. Eto, N. Tanimura, K. Imai, K. Tsu- 48. Perkins, L.E.L., and D.E. Swayne. 2001. Pathobi- kamoto, T. Horimoto, Y. Kawaoka, and S. Yama- ology of A/chicken/Hong Kong/220/97 (H5N1) guchi. 2005. Isolation of a genotypically unique avian infl uenza virus in seven gallinaceous species. H5N1 infl uenza virus from duck meat imported Veterinary Pathology 38:149–164. into Japan from China. Virology 339(1):101–109. 49. Perkins, L.E.L., and D.E. Swayne. 2002. Pathoge- 38. Mo, I.P., M. Brugh, O.J. Fletcher, G.N. Rowland, nicity of a Hong Kong-origin H5N1 highly patho- and D.E. Swayne. 1997. Comparative pathology of genic avian infl uenza virus for emus, geese, ducks, chickens experimentally inoculated with avian and pigeons. Avian Diseases 46(1):53–63. infl uenza viruses of low and high pathogenicity. 50. Roberton, S., I, D.J. Bell, G.J.D. Smith, J.M. Nich- Avian Diseases 41:125–136. olls, K.H. Chan, D.T. Nguyen, P.Q. Tran, U. 39. NASS. 2006. Dairy and Poultry Statistics. Agricul- Streicher, L.L.M. Poon, H. Chen, P. Horby, M. tural Statistics. Available at http://www.nass.usda. Guardo, Y. Guan, and J.S.M. Peiris. 2006. Avian gov/Publications/Ag_Statistics/agr06/. Accessed infl uenza H5N1 in viverrids: implications for wild- April 14, 2007. life health and conservation. Proceedings Biologi- 40. Offi ce International des Epizooties. 2007. Terres- cal Sciences 273(1595):1729–1732. trial Animal Health Code, 2007. Available at 51. Sabirovic, M., S. Hall, and A. Paterson. 2004. http://www.oie.int/eng/normes/en_mcode.htm. Quantitative risk assessment: low pathogenic noti- Offi ce International des Epizooties: Paris. Accessed fi able avian infl uenza (H5 and H7) in poultry meat. March 28, 2007. International Disease Monitoring—Quantitative 41. Offi ce Internationale des Epizooties. 2006. Guide- Risk Assessments. Available at http://www.defra. lines for the Surveillance of Avian Infl uenza. Ter- gov.uk/animalh/diseases/monitoring/pdf/lpai- restrial Animal Health Code 2006. Appendix 3.8.9. poultrymeat.pdf. DEFRA. Accessed April 24, Available at http://www.oie.int/eng/normes/ 2007. 22 / Trade and Food Safety Aspects for Avian Influenza Viruses 511

52. Scott, A., C. Zepeda, L. Garber, J. Smith, D. pathogenicity avian infl uenza viruses can be Swayne, A. Rhorer, J. Kellar, A. Shimshony, H. present in chicken breast and thigh meat following Batho, V. Caporale, and A. Giovannini. 2006. The intranasal virus inoculation. Avian Diseases 49(1): concept of compartmentalisation. Revue Scientifi - 81–85. que et Technique Offi ce International des Epizoo- 63. Swayne, D.E., J.R. Beck, and T.R. Mickle. 1997. ties 25(3):873–879. Effi cacy of recombinant fowl pox vaccine in pro- 53. Senne, D.A., J.C. Pedersen, and B. Panigrahy. tecting chickens against highly pathogenic 2005. Live-bird markets in the Northeastern United Mexican-origin H5N2 avian infl uenza virus. Avian States: a source of avian infl uenza in commercial Diseases 41:910–922. poultry. In: R.S. Schrijver and G. Koch (eds.). 64. Swayne, D.E., and D.A. Halvorson. 2008. Infl u- Avian Infl uenza. Prevention and Control, Springer: enza. In: Y.M. Saif, J.R. Glisson, A.M. Fadly, L.R. Dordrecht, pp. 19–24. McDougald, and L. Nolan (eds.). Diseases of 54. Senne, D.A., T.J. Holt, and B.L. Akey. 2005. An Poultry, 12th ed. Blackwell: Ames, IA in press. overview of the 2002 outbreak of low-pathogenic 65. Swayne, D.E., and D.J. King. 2003. Avian H7N2 avian infl uenza in Virginia, West Virginia infl uenza and Newcastle disease. Journal of the and North Carolina. In: R.S. Schrijver and G. Koch American Veterinary Medical Association 222(11): (eds.). Avian Infl uenza. Prevention and Control, 1534–1540. Springer: Dordrecht, pp. 41–47. 66. Teates, K., L. Brammer, A. Balish, T. Wallis, 55. Shortridge, K.F. 1999. Poultry and the infl uenza H. Hall, A. Klimov, K. Fukuda, N. Cox, and M. H5N1 outbreak in Hong Kong, 1997: abridged Katz. 2004. Update: Infl uenza activity—United chronology and virus isolation. Vaccine 17:S26– States and worldwide, May-October 2004. Mor- S29. bidity and Mortality Weekly Report 53(42):993– 56. Shortridge, K.F., N. Zhou, G. Yi, G. Peng, T. Ito, 995. Y. Kawaoka, S. Kodihalli, S. Krauss, D. Markwell, 67. Thomas, C., and D.E. Swayne. 2007. Thermal K.G. Murti, M. Norwood, D. Senne, L. Sims, A. inactivation of H5N1 high pathogenicity avian Takada, R.G. Webster, N.N. Zhou, Y. Guan, and infl uenza virus in naturally infected chicken meat. P. Gao. 1998. Characterization of avian H5N1 Journal of Food Protection 70(3):674–680. infl uenza viruses from poultry in Hong Kong. 68. Toffan, A., M. S. Beato, R. De Nardi, E. Bertoli, Virology 252(2):331–342. G. Cattoli, C. Terregino, and I. Capua. 2007. Vac- 57. Songserm, T., A. Amonsin, R. Jam-on, N. Sae- cination prevents viral colonization of muscles in Heng, N. Pariyothorn, S. Payungpom, A. Theam- experimentally infected turkeys challenged with booniers, S. Chutinimitkul, R. Thanawongnuwech, highly pathogenic and low pathogenicity avian and Y. Poovorawan. 2006. Fatal avian infl uenza A infl uenza viruses of the H7N1 subtype. Abstracts H5N1 in a dog. Emerging Infectious Diseases of OIE/FAO Conference on Vaccination: A Tool 12(11):1744–1747. for the Control of Avian Infl uenza 20–22 March 58. Swayne, D.E. 2004. Application of new vaccine 2007, Verona, Italy, p. 64. technologies for the control of transboundary 69. Tran, T.H., T.L. Nguyen, T.D. Nguyen, T.S. diseases. Developments in biologicals 119:219– Luong, P.M. Pham, v. Nguyen, V, T.S. Pham, C.D. 228. Vo, Q.M. Le Thi, T.T. Ngo, B.K. Dao, P. Le Phuc, 59. Swayne, D.E. 2006. Microassay for measuring T.T. Nguyen, T.L. Hoang, V.T. Cao, G. Le Truong, thermal inactivation of H5N1 high pathogenicity D.T. Nguyen, N. Le Hong, K.T. Nguyen Thi, S. avian infl uenza virus in naturally-infected chicken Le Hoang, T. Le Van, C. Dolecek, T.T. Tran, M. meat. International Journal of Food Microbiology d. Jong, C. Schultsz, and P. Cheng. 2004. Avian 108(2):268–271. infl uenza A (H5N1) in 10 patients in Vietnam. 60. Swayne, D.E. 2008. Current developments in New England Journal of Medicine 350:1179– avian infl uenza vaccines including food safety 1188. aspects in vaccinated birds. Development in 70. Tumpey, T.M., D.L. Suarez, L.E.L. Perkins, D.A. Biologicals, in press. Senne, J.G. Lee, Y.J. Lee, I.P. Mo, H.W. Sung, and 61. Swayne, D.E., and J.R. Beck. 2004. Heat inactiva- D.E. Swayne. 2002. Characterization of a highly tion of avian infl uenza and Newcastle disease pathogenic H5N1 avian infl uenza a virus isolated viruses in egg products. Avian Pathology 33(5): from duck meat. Journal of Virology 76(12):6344– 512–518. 6355. 62. Swayne, D.E., and J.R. Beck. 2005. Experimental 71. United States Animal Health Association. 1974. study to determine if low pathogenicity and high VVND found in birds intended for importation into 512 Avian Influenza

the United States. Proceedings of the United States 74. WHO. 2005. Avian infl uenza—situation in Viet Animal Health Association 78:284–285. Nam—update 5. WHO Disease Outbreak News. 72. Van Borm, S., I. Thomas, G. Hanquet, B. Lambre- http://www.who.int/csr/don/2005_01_21/en/ cht, M. Boschmans, G. Dupont, M. Decaestecker, index.html. Accessed on 9/13/06. R. Snacken, and B.T. van den. 2005. Highly patho- 75. WHO. 2006. Human avian infl uenza in Azerbai- genic H5N1 infl uenza virus in smuggled Thai jan, February-March 2006. WHO Weekly Epide- eagles, Belgium. Emerging Infectious Diseases miological Record 81(18):183–188. 11(5):702–705. 76. Yingst, S.L., M.D. Saad, and S.A. Felt. 2006. 73. Vong, S., B.M.S. Coghlan, D. Holl, H. Seng, Qinghai-like H5N1 from domestic cats, northern S. Ly, M.J. Miller, P. Buchy, Y. Froehlich, Iraq. Emerging Infectious Diseases 12(8):1295– J.B. Dufourcq, T.M. Uyeki, W. Lim, and T. Sok. 1297. 2006. Low frequency of poultry-to-human H5N1 77. Zepeda, C., and M.D. Salman. 2006. Assessing the virus tranmsission, Southern Cambodia, 2005. probability of the presence of low pathogenicity Emerging Infectious Diseases 12(10):1542– avian infl uenza virus in exported chicken meat. 1547. Avian Diseases 51(Suppl.):344–351. 23 Control of Low Pathogenicity Avian Influenza

David A Halvorson

INTRODUCTION mercial poultry in the entire country. However, The control of animal diseases is a responsibility of swine infl uenza in turkey breeders continues to be a animal owners, veterinarians, and regulatory offi - problem where both are reared in the same area. cials. Other stakeholders provide necessary tools for Four key factors have been pivotal in preventing animal disease control that include vaccines and and controlling AI: (1) keeping commercial poultry pharmaceuticals. Several poultry diseases have been in confi nement (no contact with the external envi- successfully controlled or eliminated through various ronment) to avoid contact with wild birds; (2) main- strategies based on understanding of the pathogen- taining separation between different types and eses for the individual disease and poultry produc- groups of commercial poultry; (3) maintaining sep- tion management systems such as (1) eliminating aration between poultry populations and live poultry vertical transmission has led to successful elimina- markets, auctions, shows, and backyard birds; and tion of pullorum, fowl typhoid and Mycoplasma (4) avoiding or controlling movement of poultry. infection in most commercial production systems; Throughout this chapter, “AI” will refer to the (2) immunizing the host by vaccination has been “low pathogenicity” (LP) pothotype, unless “high successful for controlling fowl pox and Marek’s pathogenicity” (HP) in specifi cally named, although disease; (3) medicating fl ocks with anti-coccidials many of the control principles apply to both LP and has been successful for coccidiosis control; and (4) HPAI. preventing exposure by isolation and sanitation (biosecurity) has been successful in the control of Avian Infl uenza Virus and Natural Hosts turkey coronaviral enteritis and infectious coryza of The AI virus has an envelope that accounts for its chickens. In each case, control strategies were susceptibility to detergents and disinfectants. Addi- designed to take advantage of characteristics of the tionally, it is susceptible to heat and drying; it is pathogen as well as characteristics of the host and generally inactivated within 1 week at 21º C but may its environment. survive for 5 weeks at 4º C (48). Moist feces col- Avian infl uenza (AI) is also a poultry disease that lected from 1983 Pennsylvania H5N2 HPAI infected has been successfully controlled in poultry by taking hens infected SPF chicks placed on the feces 2 days, advantage of characteristics of infl uenza A virus as but not 4 days, after collection (6). The Pennsylvania well as characteristics of the host and environment. H7N2 LPAI virus from 1997 was inactivated in Because the disease is not vertically transmitted, chickens manure in less than 1 week at 15º to 20º C biosecurity alone has been highly successful in the (33). United States as a prevention and control strategy The known natural reservoir for AI viruses is wild for AI, and in 2006 for the fi rst year since AI was waterfowl and shorebirds (orders Anseriformes and detected in the United States, there was not a single Charadriiformes) where these AI viruses primarily outbreak of avian-origin infl uenza reported in com- cause an enteric infection and rarely a respiratory

Avian Influenza Edited by David E. Swayne 513 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 514 Avian Influenza infection. A duck can shed virus for 30 days and can infl uenza [HPAI]) outbreak that affected poultry in excrete 108 mean embryo infectious doses per nine states during 1924–1925. It began in the live milliliter (EID50/ml) of feces (55), contaminating poultry marketing system in New York and spread surface water, sloughs, and shorelands. In its natural to New Jersey and Pennsylvania markets. Infected reservoir, waterborne virus is transmitted most effi - poultry were ultimately found at farms or markets ciently via the fecal-oral route. Thus, water con- in Connecticut, Illinois, Indiana, Michigan, Mis- tamination, fecal-oral transmission, and movement souri, and West Virginia (48). Authorities docu- of wild waterfowl and shore birds are the mecha- mented the lack of transmission through the air and nisms that provide for AI virus survival and disper- the role of contaminated crates and railcars in sal in nature. From available information, it appears spreading the disease (31). In the absence of knowl- that other orders of birds may be infected, but the edge of the etiologic agent and without modern diag- AI virus is not maintained in them in nature; however, nostic tools, computers, and global information it is important to recognize that these birds may act systems, the disease was controlled and eradicated as both biological and mechanical vectors to move successfully. This multistate outbreak was detected the AI virus between the waterfowl reservoir and in the fall of 1924 and was eradicated by the spring domestic birds. Thus, the waterfowl-shorebird res- of 1925 following the application of quarantine, ervoir, the environment these birds occupy, and any- depopulation, and sanitation procedures. thing or anyone sharing that environment may be a In 1964, low pathogenicity AI (LPAI) virus was source of AI virus for domestic poultry. isolated from turkeys in California followed by isolations from turkeys in Massachusetts and in Wis- Unnatural Hosts of Avian Infl uenza consin in 1965 (2). During the early 1960s, the The most important man-made reservoirs of AI Minnesota turkey industry was experiencing respira- viruses are live poultry markets. Live poultry markets tory problems in the fall of each year (M. C. Kumar, coupled with a village poultry production system personal communication), which were recognized to with multispecies and nonconfi nement are particu- be due to H6N2 AI in 1966 through hemagglutina- larly likely to maintain AI viruses. The infection is tion inhibition (HI) serological testing and through both enteric and respiratory in gallinaceous birds. virus isolation in 1967. Introductions of LPAI virus Although virus titer in respiratory secretions and have been detected in Minnesota every year but one excretions is likely to be higher, much more virus is since 1966. found in the feces because of the greater mass of intestinal excretions. MINNESOTA SITUATION: A MICROCOSM In poultry, AI virus transmission is probably not FOR AVIAN INFLUENZA IN THE UNITED as effi cient as in water birds, but access to shared STATES feeders and waterers, where there is opportunity for Minnesota is a major brooding area for wild water- contamination from both feces and respiratory secre- fowl and is a major staging area for the fall migration tions, facilitates transmission within poultry fl ocks. south. This results in a high density of susceptible Gallinaceous birds are not natural hosts and the AI juvenile waterfowl, especially mallards, between virus does not ordinarily maintain itself in gallina- July and November each year. Sentinel studies have ceous poultry. In order for a host-adapted virus to shown that these juvenile waterfowl undergo multi- thrive in gallinaceous poultry, a high density of sus- ple infections with LPAI viruses, sometimes simul- ceptible individuals is required along with the virus taneously, during this period of time (22). These being moved from place to place by human activity, juveniles have a seasonal infection with LPAI viruses conditions that are often met in live poultry markets. that occurs in conjunction with hatching, brooding, As with the natural reservoir of AI, anything or fl edging, and staging for migration of susceptible anyone sharing the man-made reservoir environment juveniles. Infl uenza virus has been recovered directly may be a source of virus for domestic poultry. from lake and pond water utilized by infected wild ducks (22). Thus, the waterfowl and any creature History of Early Avian Infl uenza Outbreaks in sharing that waterfowl environment (such as free- the United States fl ying birds, small mammals, and humans) may The fi rst avian infl uenza (AI) outbreak in the United spread the virus. This source of infection may result States was the fowl plague (highly pathogenic avian in seasonal LPAI outbreaks in poultry. 23 / Control of Low Pathogenicity Avian Influenza 515

Minnesota is also a major turkey producing state, through prevention of exposure to infl uenza viruses ranking fi rst or second in the United States for the by avoiding direct or indirect contact with waterfowl past 50 years in the number of turkeys raised each and shorebirds and their environment. year. Much of the turkey production is in the same The repeated LPAI outbreaks in Minnesota area where wild waterfowl nest, rear their young, turkeys resulted in interest in sampling of waterfowl and gather for the fall migration; some of the turkey in the state. Two studies that addressed AI virus in production involved range rearing until 1998. As a Minnesota waterfowl illustrate the breadth of infec- result of contact between wild birds and turkeys, tion (22, 26). These studies, 1980–1983 and 1998– LPAI outbreaks have been documented since 1966. 2000, identifi ed hemagglutinins H1 through H12 in In the past 40 years, there have been 135 introduc- ducks. During the same years of sampling, infl uenza tions of AI into the Minnesota poultry industry was also recorded in turkeys. The H subtypes (Table 23.1). Three time periods are of interest. detected in turkeys and ducks are shown in Table There is the time period from 1966 through 1977 23.2. Although H3, H4, and H6 were predominant when virus isolation and HI serological tests were in ducks in both studies, no correlation was seen the predominant tools available for diagnosis. During with subtypes involved in turkey outbreaks. This this time period, H6 and H5 AI viruses were detected lack of correlation could be due to sampling, to host a total of 16 times, but the number of fl ocks infected susceptibility to certain subtypes, or to other was not recorded. In the period from 1978 to 1997, unknown factors. with additional capability at the National Veterinary The economic impact of LPAI on the Minnesota Services Laboratories (NVSL) to identify specifi c H turkey industry has been described (38). In a 3-year and N antibodies in sera from convalescent poultry period, losses due to mortality, morbidity, condem- (2), 100 introductions were recorded, which involved nation, medication, clean up, and other costs were 1058 fl ocks of turkeys, three fl ocks of chicken $2.00 per market turkey in infected fl ocks. An addi- layers, one fl ock of broiler breeders, and three fl ocks tional indirect cost of $2.50 per market turkey was of game birds. This record of LPAI in Minnesota calculated to cover the costs of clean up, downtime, turkeys raises the question: Why would the industry and rescheduling. For infected turkey breeder fl ocks, persist in range production? The answer is found the calculated costs were $8.39 per bird. The eco- in the historical market prices for turkeys that were nomic losses associated with LPAI virus infection consistently higher in the fall of the year because in Minnesota turkeys as well as the 1983–1984 of the Thanksgiving and Christmas holidays. The HPAI virus outbreak in Pennsylvania poultry led to favorable market in the fall resulted in turkeys being the development of the Minnesota Cooperative grown on range so that more birds could be raised Avian Infl uenza Control Program (18, 39). This with a fi xed number of buildings. This seasonal cooperative program is the basis for the control dis- price differential gradually declined as turkey pro- cussion that follows a review of biosecurity appro- duction and marketing became more of a year-round priate to LPAI. activity, and with the decline in price differential came a decline in range rearing from over 50% of BIOSECURITY: THE FIRST LINE OF turkey fl ocks in the 1960s to about 10% of fl ocks in DEFENSE the early 1990s. Biosecurity, the prevention of exposure to disease In response to the 1997 human infection with agents thorough management practices, is the H5N1 in Hong Kong, range production in Minne- primary component of all AI control plans (13). The sota essentially ceased in 1998 and subsequent years, physical and functional separation in the poultry accounting for less than 0.5% of the turkey fl ocks in industries is a large part of the conceptual biosecu- the state. While swine infl uenza (H1 and H3 sub- rity (42) that has resulted in much of the poultry types) continues to infect turkeys, only three avian disease prevention success in the United States. subtypes (H6, H9, and H10) have been detected Conceptual biosecurity includes the separation of since 1998 and they involved a total of fi ve turkey breeder, hatchery, grow-out, and processing facili- fl ocks. It appears very likely that the cessation of ties and functions. It also involves location of farms, range turkey production in 1998 has had a large with particular attention to poultry density, process- impact on the decline in cases of LPAI in Minnesota ing and public roads used to transport poultry and turkeys. In Minnesota, prevention of LPAI is largely poultry-related products. Conceptual biosecurity, 516 Avian Influenza

Table 23.1. Forty years of avian inﬂ uenza introductions in Minnesota (1966–2005). Year Flocks Subtypes

1966 NA H6N2, H6N? 1967 NA H6N? 1968 NA H6N? 1969 NA H6N? 1970 NA H6N? 1971 NA H6N? 1972 NA H5N?, H6N8 1973 NA H6N2 1974 NA H5N1, H6N8 1975 NA H6N2 1976 NA H6N? 1977 NA H6N2, H6N8 1978 141 H1N1, H4N8, H6N1, H6N2, H6N8, H9N2 1979 30 H4N1, H6N1, H6N2, H9N2, H10N7 1980 22 H4N2, H4N6, H4N8, H7N3, H10N7 1981 50 H5N2, H6N8, H10N7 1982 59 H1N1, H3N2, H4N2, H4N8, H5N2, H6N1, H6N2, H6N8, H6N?, H9N2, H?N2 1983 2 H5N? 1984 13 H1N1, H2N3, H4N6, H6N8, H8N4 1985 73 H1N1, H2N7, H4N2, H4N6, H4N8, H5N2, H5N6, H6N8, H7N3 1986 20 H1N1, H4N3, H4N6, H4N8, H6N5, H9N9 1987 38 H1N1, H3N8, H5N2, H5N8, H7N7, H9N5 1988 258 H2N2, H4N6, H5N6, H7N9, H8N4, H9N2 1989 16 H1N1, H4N3, H4N8, H9N2, H10N7 1990 14 H1N1, H6N2, H10N7, H13N2 1991 110 H1N1, H4N2, H4N6, H4N8, H5N2, H5N3, H6N1, H6N2, H6N8, H7N3 1992 17 H1N1, H4N2, H6N8, H7N3 1993 4 H1N1, H4N6, H5N9, H9N2 1994 8 H5N2, H6N?, H7N1 1995 178 H1N1, H6N8, H9N2, H10N7 1996 5 H2N2, H9N2, H?N2 1997 0 1998 1 H1N1, (H5N2 pheasant) 1999 24 H1N1, H6N1 2000 3 H1N1 2001 2 H1N1 2002 9 H1N1, H9N9, H10N7 2003 5 H1N1, H3N2, H?N2 2004 12 H1N1, H1N2, H3N2, 2005 17 H1N1, H3N1, H3N2, H3N? learned through experience by the pioneers of Once the concept of biosecurity is understood, modern poultry production, is not necessarily under- structural biosecurity provides the physical facilities stood by large corporation management involved in and the management schemes to prevent disease today’s poultry industries. introduction (42). Physical facilities such as farm 23 / Control of Low Pathogenicity Avian Influenza 517

Table 23.2. Avian infl uenza virus hemaglutinin (HA) subtypes from ducks and detection of infl uenza in turkey fl ocks in Minnesota (1980–1983 and 1998–2000) (22, 26). 1980–1983 1998–2000 HA Duck isolates Turkey outbreaks Duck isolates Turkey outbreaks

H1 20 3 0 3 H2 00 90 H3 59 1 28 0 H4 96 4 43 0 H5 20 2 11 0 H6 66 6 24 1 H7 61 50 H8 30 00 H9 01160 H10 8 2 1 0 H11 26 0 11 0 H12 5 0 1 0

layout, walk-through showers, fences, gates, locks, tion, isolation, and traffi c control to be targeted decontamination sites, wild bird-proof houses, farm- appropriately. specifi c equipment, and feed-handling systems contribute to disease prevention. Management schemes Identify Sources of Avian Infl uenza Virus such as all-in/all-out practices prevent problems The reservoirs of infl uenza virus and the environ- associated with multiple-age farms and provide brief ments associated with them are widely recognized downtime for separation between current and fol- as sources of the virus as previously described. lowing fl ocks. However, once an outbreak has occurred, attention Operational biosecurity involves the programs must be directed toward sources of LPAI virus and protocols that guide day-to-day activities of within infected poultry populations. It is not possible people to prevent disease introduction and spread to categorize with certainty that a given fl ock is not (42). Because operational biosecurity depends on infected with LPAI virus. Consequently, every effort the day-to-day behavior of people, it is less depend- is directed toward avoiding direct and indirect expo- able than conceptual and structural biosecurity. It is sure to the most important known potential sources this level of biosecurity that receives the most atten- of virus: poultry manure, respiratory secretions, live tion in the face of a disease threat because it is the poultry, dead poultry, unwashed eggs (and reused most easily intensifi ed. Unfortunately, it is the con- egg packing materials), and equipment or people ceptual and structural biosecurity that is most impor- contaminated with manure or respiratory secretions tant and most effective in disease prevention, and (7, 11, 19, 27, 35, Carol Cardona, personal com- these levels of biosecurity are not as easily munication). In the United States and Canada, pet changed. birds and backyard poultry have not been associated Preventing disease exposure (biosecurity) requires with AI outbreaks in commercial poultry (40, Dennis an understanding of sources of contamination Senne, personal communication, Victoria Bowes, and their proximity to susceptible poultry; this com- personal communication), and the same conclusion prises a matrix of the source of the virus, how the was reached in the Netherlands following the HPAI virus moves, knowledge of poultry population outbreak in 2003 (15, 29). However, in Mexico, involved, cross-over points, and the exposure risk backyard or village poultry remained infected with level. The knowledge of the virus and host factors H5N2 LPAI virus after H5N2 HPAI virus was con- mentioned here allow the biosecurity tools of sanita- trolled in commercial poultry (54). Village poultry 518 Avian Influenza may involve multiple species and uncontrolled bird insemination crews and equipment transmitted LPAI movement, and if the poultry density is high enough, virus between fi ve turkey breeder farms in Califor- this system supports the existence of an LPAI virus nia (Fig. 23.1) while leaving some 300 other poultry reservoir. This may have happened and may be hap- fl ocks in the same area untouched (34). Some more pening with the H5N1 HPAI virus in parts of east recent examples are as follows: (1) in Minnesota, Asia. live haul crews and equipment were implicated (19); (2) in Virginia, a common rendering plant and hired Identify How the Virus Moves labor were involved (35); (3) in Italy, manure hauling In nature, the LPAI virus is moved from place to contributed to spread (11); and (4) in California, place by the movement of waterfowl and shorebirds. movement of spent hens and egg-handling materials In domestic poultry, the virus is moved from place were identifi ed (Carol Cardona, personal commu- to place by the activities of people. The existence of nication). The striking similarity between these the virus in either the waterfowl or market reservoir methods of spread is the amount of gross contamina- is not the problem; rather, it is movement of virus tion involved. Lack of aerosol transmission has been or infected birds into close proximity to susceptible noted as early as 1925 (31) and as recent as 2003 birds that causes transmission of AI. Depending on (52), but it was considered to have been possible in the type of poultry, the type of poultry operation, the Fraser Valley, British Columbia, HPAI outbreak and regional considerations (e.g., poultry density), in 2004 (40). Attempts to detect airborne live virus for any infl uenza outbreak it is critical to quickly outside the poultry houses in that outbreak were not identify how the virus will be moved about by the successful (40). activities of people. Recognizing the ways the virus Another reason that bird movement is so danger- is apt to be moved in a particular outbreak is the ous is that it intimately exposes birds to equipment most important part of the control strategy. Eighty and personnel previously used to move other birds. years ago, it was recognized that contaminated If the moving equipment is not almost perfectly poultry crates and railcars spread fowl plague cleaned and disinfected, it may infect a few birds, halfway across the United States (31). Twenty years which will then amplify the virus, eventually result- ago, it was documented that contaminated artifi cial ing in infection of the entire fl ock.

22 Miles 40 Miles

A 158 B 6 DAY 1

9 110 Miles C 22 Miles DAY 8 100 3 E 34 DAY 41 D DAY 24

Total Poultry Premises by County Infected Premises with no Symptoms ■ Suspected virus source Infected Premises with Symptoms DAY Interval, in days, between onset of symptoms in flock B and onset in remaining flocks Mileage

Figure 23.1. Spatial and temporal aspects of avian infl uenza (H5N3) on fi ve turkey breeder premises allied to one company and insemination crew in relationship to total poultry premises at risk in a six-county area (34). (Used by permission of International Symposia on Avian Infl uenza.) 23 / Control of Low Pathogenicity Avian Influenza 519

Identify Specifi c Avian Populations within a single company. Managers of these profi t Industry practices are specifi c for different types of centers may make decisions that contribute to disease poultry. Some types of poultry are moved from one transmission. For example, the contractual need for place to another, have access to the outdoors, have product by the marketing division may drive deci- contact with live poultry markets, or have contact sions by the processing division that may precipitate with wild birds. Recognition of these practices and dangerous movement of infected birds or contami- taking appropriate mitigation steps have an impact nated materials that consequently expose nonin- on control measures. fected birds in the production division to AI virus. The susceptibility of different poultry groups to Another example, the cost of routinely cleaning and AI outbreaks differs greatly from one group to disinfecting equipment may not be perceived by another and depends to a great extent on exposure managers to have economic benefi t if no immediate to natural or man-made sources of AI viruses, on the disease threat is recognized. host adaptation of the virus itself, on movement of birds, their products and their manure, and on the Commercial broiler industry extent of human activity during the production In the case of broilers, there is generally no move- period. For example, during the 2002 Virginia LPAI ment of these birds from the time they are placed outbreak, it was observed that turkeys in the growing after hatching until they are marketed about 7 weeks facilities, after being moved, were far more fre- later. They are often grown in all-in/all-out fl ocks of quently infected than those in brooder facilities, 30,000 to 50,000 birds; this usually allows for resi- having never been moved (35). Confi nement poultry dent labor (living on the farm) to accomplish the and nonconfi nement poultry likewise have different routine labor needs. The litter is generally removed characteristics and risks. once a year. A service person may visit periodically and vaccines may be applied by a mass method. Characterize Totally Confi ned Populations It is curious that there are approximately 20 times The commercial poultry industries reduce their risk more broilers raised in the United States (9 billion) of LPAI virus introduction by avoiding exposure to than layers, turkeys, and breeders combined, but the wild bird or live poultry market reservoirs; if AI there are few instances of large broiler outbreaks is introduced, their risk is related to the high popula- with LPAI. All large outbreaks of LPAI in the tion density and intraestablishment movement. In United States have largely, if not exclusively, general, the large integrated commercial industries involved commercial turkeys (e.g., Minnesota, Utah, are characterized by total confi nement and comprise Virginia) or commercial egg layers (e.g., Pennsyl- the broiler industry, the turkey industry, and the egg vania, Connecticut, California). layer industry. These industries generally have It is possible that the broiler industry has been exposure to neither the wild bird reservoir nor the much more diligent in providing education, prevent- man-made reservoirs. Further, they avoid contact ing the introduction of AI, and conducting surveil- with nonconfi ned poultry and other potential sources. lance than the turkey and layer industry. It is more Integration means that these industries are further likely that the structural biosecurity of this “com- characterized by total control over and physical and partment” is the most biosecure from an AI stand- functional separation of breeding operations, hatch- point. ery operations, commercial production of meat or eggs, and processing operations. Bird movement, Commercial turkey industry when it occurs, is intraestablishment and usually, Turkeys are generally moved from brooder facilities but not always, involves a whole fl ock moving from at 5 to 7 weeks of age to grower facilities on the one production facility to another or to market. same or a different farm where they stay until they As poultry production has evolved from farmer- are marketed between 12 and 22 weeks of age. This driven production-oriented decisions to industry- movement of birds exposes them to hauling equip- driven consumer-oriented decisions, certain ment and personnel who may previously have been pressures have arisen that can complicate disease in contact with other fl ocks. Turkey farms are more prevention efforts. The functional separation of likely to require hired labor than are broiler farms. operations causes the development of profi t centers Like broiler farms, turkey farms may be visited 520 Avian Influenza periodically by a service person and mass vaccina- swine-origin infl uenza in turkey breeders that happen tions may be administered. Litter from brooder houses often enough that vaccinating turkey breeders against is routinely cleaned out after every fl ock, but litter swine infl uenza is common. from grower fl ocks may be removed only annually. Modern small holdings Commercial egg layers Modern small holdings, including some backyard Commercial egg layer replacement pullets spend fl ocks, may duplicate the confi nement, segmenta- approximately 16 weeks in a pullet house before tion, and movement control of integrated compa- being moved to a layer facility, where they live for nies. Resident labor is often suffi cient to care for the another 70 to 90 weeks. Replacement pullets are birds. usually handled individually during the rearing period: once to be beak trimmed and again for Characterize Nonconfi ned Populations vaccine administration. Pullet facilities are often, In general, owners of nonconfi nement birds reduce but not always, single age, but recently constructed their risk of AI loss by their small size and low egg production facilities are generally multiple-age population density; their risk of exposure is great farms with 1 million hens or more with egg process- and is related to direct or indirect contact with one ing and feed manufacturing on the same premises. of the reservoirs of LPAI and interestablishment Scores of employees are necessary for the produc- movement. These populations may be characterized tion and processing functions on a modern egg layer by one or both of the following: exposure to a natural complex. Manure removal from pullet and layer or man-made reservoir and interestablishment trans- facilities may occur on a daily basis in the case of fer of ownership or movement of birds. They are houses with manure belts or as seldom as annually frequently kept in pens with wild bird access and on in high-rise facilities. Eggs produced at one of these farms with more than one generation and species. In farms might leave as unwashed “nest run” eggs, they contrast to totally confi ned commercial poultry, may leave as washed and sanitized eggs in new these nonconfi ned operations have exposure to res- materials, or they may leave as liquid egg product. ervoirs, bird movement to or from another establish- It is likely that weaknesses in both structural bio- ment, and multiple functions on one site. Frequently, security (e.g., moving birds from site to site using there is a population mixture of different species and shared equipment and multiple age farms) and oper- sources. They also usually have fewer birds and at ational biosecurity (e.g., use of hired labor and inad- lower density than totally confi ned poultry. equate sanitation of moving equipment), as well as The term backyard birds has no specifi c meaning greater longevity of the birds, may account for the in determining AI risk because they may consist of a greater prevalence of LPAI in turkeys and layers single age and species kept in confi nement with no than in broilers and breeders. contact with live poultry markets, or they may consist of multiage, multispecies reared outdoors and with Parent breeder fl ocks live poultry market contact. Thus, it is clear that Multiplier fl ocks (10,000 to 30,000 birds is typical) backyard fl ocks have to be characterized in order to for these major commercial industries are also main- make meaningful decisions about their potential contained in confi nement, usually under better biosecu- tribution to AI spread and control. Backyard fl ocks rity than and far removed from commercial fl ocks. in the United States probably have little resemblance Replacements are usually grown in a single age to backyard fl ocks in east Asia or Africa. setting and moved to a single-age breeder facility. Upland game and waterfowl that are raised for Resident labor is often adequate for their care. Mul- release; organic, range, and pastured poultry; back- tiple vaccines that require individual handling may yard, exhibition, or zoo birds; and game fowl (fi ght- be administered prior to egg production. The layer, ing chickens) may have exposure to wild birds. In turkey, and broiler breeders are not frequently addition, live birds may be transferred from one loca- involved with AI. Tight structural and operational tion to another (birds raised for release, exhibition biosecurity can be credited for this success. birds, zoo birds, and fi ghting cocks), although there An exception to the above observation about may be little or no movement of backyard birds. parent breeders involves perennial outbreaks of Other poultry may be grown for the live poultry 23 / Control of Low Pathogenicity Avian Influenza 521 market system, where the exposure comes from the Recently, an exposure risk index (ERI) has been transfer of contaminated materials, or, rarely, live proposed as a way to make this determination more birds from market to farm, where susceptible poultry objective (25). Briefl y, using available objective then multiply the infection and become a source for information about the proximity of susceptible additional contamination of the market. animals to disease reservoirs, an index is calculated that has been shown to correlate signifi cantly with Identify Cross-Over Between Populations poultry veterinarians’ opinions of disease exposure The new World Organization of Animal Health risk. By dividing the reservoir (expressed as micro- (Offi ce Internationale des Epizooties [OIE]) chapter bial load) by a proximity measurement (area), one on AI promotes the concept of compartmentaliza- obtains an exposure risk measurement that is tion for maintaining poultry trade in the event of a then expressed as a log. An ERI of 7 or greater notifi able AI outbreak. Although poultry establish- was correlated with intolerable or high risk accord- ments make great efforts to distance themselves ing to a group of poultry veterinarians, while an from others producing the same type or different ERI of 4 or less was considered moderate or low types of poultry, sometimes there are opportunities risk. Using this index, it is possible to estimate rela- for exposure due to cross-over. Cross-over occurs tive risk of disease exposure from disparate sources. where production-related activities of one establish- The ERI highlights the importance of the mass of ment utilize the same resources as other establish- the contaminant and its proximity to susceptible ments. For examples, a rendering truck may visit birds and facilitates communication to describe the broiler, layer, and turkey farms, contract labor importance of reducing contamination and increas- (crews) may provide services to many different ing the distance between susceptible poultry and companies, individuals may work on more than one sources of contamination. It is also possible to eval- crew, pullet-moving dollies may be used on multiple uate mitigation strategies. For example, the ERI farms, egg-moving materials often go to different indicates that pullet-moving equipment contami- farms, and feed trucks may provide feed to multiple nated with 1 kg of manure with 106 AI viruses per establishments. Eliminating these cross-over points gram still has an unacceptable ERI even after clean- demonstrates compartmentalization and is critical ing and disinfecting remove 99.99% (4 log10) of the for any establishment that is interested in exporting contamination (Table 23.3). This indicates that poultry meat or eggs. the cleaning and disinfecting procedure requires modifi cation. Identify Specifi c Exposure Risk Level Once exposure risk is estimated, investments in Determining the risk of disease exposure has been a fi xed and variable costs of biosecurity can be evalu- subjective exercise for poultry professionals. ated in light of possible economic impact of AI

Table 23.3. Exposure risk index (ERI) for various sources of inﬂ uenza viruses. Hazard Proximity to poultry house ERI 99.99% Mitigation

Estimated 1% available 40 tons infected turkeys 100 meters 7 3 10 tons contaminated manure 100 meters 7 3 1 kg (20 to 30) wild birds inside 9 5 Estimated 100% available 1 kg manure on pullet dollies inside 11 7 10 g poultry dust on clothes inside 9 5 1 g contamination on hands inside 8 4 ERIs of 7 or greater are correlated with “intolerable risk,” and ERIs of 4 or less are correlated with “moderate risk.” From Halvorson and Hueston (25). 522 Avian Influenza outbreaks. In the past the biosecurity costs were replacing operational biosecurity with structural weighed against risk of disease effects on perfor- biosecurity. mance: mortality, body weight, feed effi ciency, egg production, etc. As the poultry industries have PREVENTION AND CONTROL PROGRAMS evolved from meat and egg production–oriented All LPAI prevention and control programs have or companies to global consumer–oriented companies, should have a similar goal for poultry to be free of today an AI outbreak has the potential to have far infection. This requires preventing the introduction greater impact than just on poultry production. Food of AI virus, controlling an outbreak by preventing safety may be called into question, exports may be spread, and then eliminating or eradicating any at risk, public health may be questioned, and brand infection introduced into domestic poultry popula- name survival may be threatened. This increased tions. These programs, even though they may have impact means that increased spending for biosecu- different approaches in response to infected fl ocks, rity is justifi ed in the global economy. all have similar needs to provide education, to prevent the introduction of AI, to detect infection, Sanitation and a reliance on biosecurity to control an outbreak. Everything and everyone entering a poultry facility These components (education, prevention, surveil- must be free of contamination that could initiate an lance, and biosecurity) are essential for AI control AI infection. One way to maintain freedom from in all types of poultry, but they must be adapted to contamination is to prevent any contaminated mate- fi t the specifi c population and region. rials and people from accessing the facility. Where In the Minnesota program (19, 39), the industry, that is not possible, sanitation (cleaning and disin- state, and federal health professionals worked in fecting [C&D]) is utilized to reduce contamination partnership to detect and eliminate the infection of equipment, people, and clothing (13). Like pas- quickly and effi ciently. It is now widely recog- teurization, this process of sanitation reduces patho- nized that industry provides the infrastructure and gen load, it does not eliminate it. Consequently, the operational skills while government provides the greater the level of contamination, the better the organization and enforcement necessary for an sanitation must be, and the greater is the risk that integrated control program. Different relationships sanitation will fail. exist in each state, but, no matter what the rela- The sanitation tools for reducing infl uenza virus tionship, it is important to maintain trust between contamination are physical removal and inactiva- industry and government personnel. This is espe- tion. Physical removal involves removal of contam- cially important with the public health concerns inated material, dry cleaning, and wet cleaning. about AI. Washing with detergent is an inactivation process. Most disinfectants are effective against infl uenza Education: The Most Important Component virus—the selection is based on the environment in None of us is able to write policies, protocols, and which the disinfectant is used. Heat alone is also programs to address all the possible ways that AI effective against the AI virus and it facilitates disin- virus might enter and move about in a population of fectant activity as well; that is, disinfectants gener- domestic birds. Farmers and employees directly ally do not work well under cold conditions. working with the poultry are in a unique position to Under some conditions, such as in extreme cold, contribute to disease prevention and control pro- C&D might be impossible. Some equipment may be grams. By relying on education, it is possible to diffi cult or impossible to C&D. In addition, C&D is involve all poultry growers, employees, and their a part of operational biosecurity and depends on families in the development of farm-based preven- satisfactory human performance to be effective. For tion programs that address the specifi c farm, produc- these reasons, some establishments provide each tion system, region, and species of poultry involved. farm with all the necessary equipment, precluding Education about AI virus allows people to under- the need to move equipment from farm-to-farm. stand where the virus resides and how it moves Shifting from the need to C&D equipment to pro- about, and equips them to elucidate prevention strat- viding farm-specifi c equipment is an example of egies that work in their situation. 23 / Control of Low Pathogenicity Avian Influenza 523

If people are knowledgeable about the virus and Wild birds—examples of specifi c preventive steps how it moves and understand the matrix of virus Several critical prevention steps should include that source, specifi c host population, cross-over points (1) people should not hunt, trap, or fi sh or have other and exposure risk, and broad intervention strategies, contact with waterfowl or their environment the they can identify specifi c biosecurity steps necessary same day they care for poultry; (2) clothing and to prevent AI virus introduction to farms. These boots worn while engaged in such activities should broad strategies include understanding the reservoirs not be worn on poultry farms until they have been and controlling movement of live and dead birds, laundered; (3) equipment and vehicles used in such manure, and egg-handling materials, equipment, and activities should not be brought on the farm; (4) people. people should never travel directly from these environments into poultry houses; and (5) people should Preventing the Introduction of Avian Infl uenza never use pond water for watering poultry. Virus In the United States, outbreaks of LPAI in turkeys Live poultry markets—examples of specifi c typically have come from wild bird sources, whereas preventive steps outbreaks in chickens have come from live poultry Several critical prevention steps should include that market systems. (1) people should not have any contact with live poultry markets or any other place where live poultry Sources Associated with Accidental may be kept, sold, exhibited, or fought for at least Introduction 24 hours before they care for poultry; (2) no clothing Apparently healthy wild or domestic waterfowl and worn while in such contact should be brought on the poultry, particularly associated with live poultry farm until it has been laundered; and (3) vehicles markets or range production, may be infected with and crates or batteries that have contact with live LPAI virus. The virus is found in the droppings of poultry markets or other live poultry should never infected waterfowl and each bird may excrete 1010 enter the farm. mean embryo infectious doses (EID50) of virus per day (55). In gallinaceous poultry the LPAI virus is Sources Associated with Illegal Behavior found in both the respiratory secretions at up to 107–8 There may be illegal behavior that might introduce 5–7 EID50 of virus per gram and in feces at up to 10 AI viruses onto a poultry farm. Farming is a business

EID50 of virus per gram. To put this in perspective, involving trust; historically, contractual deals involv- Tumpey et al. (51) found that turkeys could be ing tens of thousands of dollars were once done with 1 infected with less than 10 EID50 of H7N2 LPAI only a handshake. Written contracts are the now the virus from the 2002 Virginia outbreak. Cold environ- norm, but the culture of trust is very strong in agri- ments will allow the virus to survive for weeks at cultural enterprises. This culture of trust is an obsta- 4º C or for months in a frozen state, but at 21º C, AI cle to preventing potential illegal activity that could virus is usually inactivated in 7 days (48). Continu- introduce disease agents such as infl uenza virus. ous excretion of virus in cold weather will result in Theft of birds from a commercial poultry farm for an accumulation of even higher levels of virus in the personal use or for sale at a live poultry market, environment. Thus, not only are waterfowl or poultry smuggling birds or bird products, contact with fi ght- a source of virus, but also their environment can be ing chickens, or intentional introduction of virus are an important source of infection. To prevent the all ways that people who engage in illegal activities introduction of AI, any conceivable contact between could introduce AI virus into a fl ock. Security the high-risk contamination areas and commercial systems need to address these sources of introduc- poultry must be avoided. Prevention programs must tion, and the fi rst thing needed is locked doors to all address the risk associated with the birds and also the poultry buildings. associated with the environment. Consequently, it is recommended that people who have contact with Surveillance wild waterfowl environments or live bird market The early detection of LPAI is the key to controlling environments not have contact with other poultry. its spread. Often the fi rst fl ocks to be infected go 524 Avian Influenza through a silent infection or become ill from second- a veterinary diagnostic laboratory. Positive AGID or ary disease agents so that the diagnosis of AI is ELISA sera and positive RRT-PCR (43), antigen- missed (8, 16). Clinical signs and lesions may lead detection, or virus isolation samples are submitted to an incomplete diagnosis of pasteurellosis or coli- to the national AI reference laboratory for confi rma- bacillosis. These fl ocks, as well as incubating and tion and additional characterization. It is worth convalescent fl ocks, may be excreting virus while noting that no surveillance should be undertaken they appear healthy; thus, there is no such thing as until it has been established how any positive fi nd- “known noninfected” fl ock. For LPAI, active sur- ings will be reported and what the response will veillance is of critical importance to detect early be. inapparent infections so control programs can be initiated. The same can be said for the detection of Reporting viruses that are biologically LPAI but HPAI based If AI virus or infections are detected with the active on sequencing of the hemagglutinin cleavage site. or passive surveillance program, there must be a In general, monitoring programs have relied on reporting scheme set up in advance to alert appropri- serological surveillance for evidence of LPAI infec- ate stakeholders of the detection of AI. Prompt tion, but that is changing with the availability of reporting is necessary to achieve industry-wide antigen detection systems and real-time reverse tran- control. The stakeholders include the farmer, hatch- scriptase–polymerase chain reaction (RRT-PCR) eries, processing plants, feed mills, other poultry tests for infl uenza. owners and employees, support and service person- The National Poultry Improvement Plan (NPIP) nel, poultry organizations, and regulatory offi cials. is an organization in the United States that coordi- Industry stakeholders are the primary responders in nates serological monitoring of breeder birds as well any LPAI control program, particularly in the fi rst as commercial birds for AI. Satisfactory compliance 24 to 96 hours, and the reporting schemes must with NPIP requirements leads to recognition of AI include them. status of fl ocks, hatcheries, and processing plants. Positive AGID, ELISA, RRT-PCR, antigen- Individual states and industries also have moni- detection, and virus isolation results indicate a toring programs designed to detect the introduction suspect fl ock and should be reported to the proces- of AI. These programs are designed to fi t the produc- sor, grower, and people directly involved, who in tion type, poultry density, perceived exposure, and turn notify employees, customers, suppliers, and regional needs of the industry. In range-reared birds neighbors. When the results are confi rmed at the in a high-risk area, weekly surveillance may be jus- National Veterinary Services Laboratories (NVSL), tifi ed, while in confi ned birds, market testing may the whole industry is notifi ed. Reporting outbreaks be adequate, and in broilers, periodic testing is prob- to industry personnel who are in direct or indirect ably suffi cient. In all cases, marketing pressures may contact with poultry is necessary so that people who result in more intensive surveillance efforts. For are most likely to be involved in inadvertent trans- example, a model surveillance program in a high mission and are best positioned to institute control risk area could include collection of 20 blood measures can respond appropriately. samples from every fl ock at the processing plant and Historically, in the Minnesota Cooperative Avian conducting agar gel immunodiffusion (AGID) or Infl uenza Control Program, press releases have not enzyme-linked immunosorbent assay (ELISA) tests been part of the reporting system. Today, with for detection of antibodies to type A infl uenza (23). greater interest and attention by the public, the media Such active serological monitoring is not designed are more likely to be involved. One danger from to detect the index case of LPAI in an area, and for media involvement is the negative impact of public- this reason passive surveillance of diagnostic cases ity on growers, markets, or companies with infected is important. A critical source of monitoring samples birds that might reduce their incentive to report sick is a sick fl ock, particularly one exhibiting depres- birds. In response to this, media representatives sion, respiratory signs, or a drop in egg production. should be sensitized to this potential harm to control Such fl ocks must be routinely checked for AI by efforts. This is especially true in reporting unverifi ed submission of sick and dead birds or submission of AI outbreaks, which can fuel rumors and cause unin- cloacal swabs, tracheal swabs, and blood samples to tended economic harm. 23 / Control of Low Pathogenicity Avian Influenza 525

Response however, it has been observed that seropositive Immediately (within the fi rst few hours) after detec- fl ocks are not associated with risk of transmission to tion of AI virus in an establishment, area, or com- other fl ocks (21, 24, 30). Ziegler et al. (56) demon- partment, a quick epidemiological assessment is strated the recurrence of LPAI H7N2 with oviduct needed to identify what fl ocks are most likely to trophism in fl ocks of layers, but this appears to be a have been exposed. This initial assessment, tracing rare event. By contrast, the danger of seronegative backward and forward, should concentrate on the AI-incubating fl ocks cannot be overemphasized. previous 14 days and the most likely sources of virus Flocks incubating AI virus may be infectious for up movement: live birds, dead birds, manure, egg-han- to 14 days before detection, and evidence of AI virus dling materials, equipment, and people who have in fl ocks that do not seroconvert has been docu- been in bird contact. mented (24).

Do No Harm Specifi c Control Measures The fi rst consideration in responding to an AI out- Once an outbreak occurs, certain things have been break is to avoid inadvertently spreading the infec- identifi ed that greatly contribute to spread: move- tion. Surveillance needs to be intensifi ed, but in ment of contaminated equipment or people (process- some past AI outbreaks control efforts, such as diag- ing trucks, egg-handling materials, loaders or loading nostic sampling and depopulation activities, have crews), movement of dead birds, partial fl ock mar- contributed to the transmission of AI in the Pennsyl- keting, and marketing an actively infected fl ock vania 1983–1984 (30) and 1997 outbreaks (27) and (19). The following management steps are designed the British Columbia 2004 outbreak (18, 40). Do not to keep AI from escaping affected premises (inclu- increase human farm-to-farm traffi c in response to a sion biosecurity) and from entering nonaffected problem. Because fl ocks can have undiagnosed or premises (exclusion biosecurity) (49). silent AI virus infection and can excrete the virus Infected birds and their manure, and people and for up to 14 days prior to the onset of illness, it is equipment in contact with them, are the major risks not safe or possible to say for certain that any fl ock for AI virus transmission. All methods for stopping was not exposed or is not infected. Initially in an the spread of LPAI are based on controlling the outbreak, all epidemiologically linked fl ocks (within movement of birds, egg-handling materials, manure, the same establishment or compartment) must be people, and equipment from affected premises, considered either infected or potentially infected which prevents contact with susceptible birds. These until demonstrated as not infected, and movement of methods, however, must be appropriately adapted materials from such fl ocks must be briefl y inter- to the population affected and must be regionally rupted to allow time for assessment and planning. specifi c.

Don’t Rush Things—promote Virus Infected Birds Inactivation Flocks that have been infected (or that may be The second consideration is that AI virus is gener- infected) are a signifi cant threat. Once a fl ock has ally inactivated in 1 week at temperatures of 21º C. been infected, it is considered infected for life. If movement of birds, manure, people, and equip- Infected fl ocks should be left in situ in the house for ment is curtailed, application of heat and passage of at least 2 weeks until the clinical signs (and virus time favor virus inactivation. excretion) subside. Approved RRT-PCR tests (43) and antigen-detection tests available today make it Focus on Seronegative Flocks possible to monitor fl ocks for detectable virus prior The third consideration is that seropositive fl ocks are to movement. High-risk factors should be controlled not a signifi cant risk for transmission of AI virus as follow: (30). As birds seroconvert, virus shedding declines, usually to an undetectable level, but recovered fl ocks 1. Dead bird disposal should be isolated. On farm should be isolated to prevent contact with suscepti- disposal by composting, burial, or incineration ble fl ocks. Once a fl ock is infected, it should be reduces risk of transmission. Composting dead considered a potential source of virus for life (48); birds is an economical and environmentally 526 Avian Influenza

friendly procedure that has been shown to inac- near the poultry house to become contaminated with tivate AI virus within 10 days (41). Off-site dis- manure, and do not allow service, feed, or delivery posal can be safe, but collection sites must be vehicles onto areas grossly contaminated with maintained properly. Be aware that rendering manure. trucks, barrels, and dumpsters can spread the disease. Control traffi c to and from bird disposal People areas. People who come in contact with birds or their 2. Range production should be eliminated. However, manure should not move from farm to farm. Anyone if range rearing cannot be eliminated, isolate can mechanically transmit AI virus, and people who range birds from confi ned birds and service per- have direct contact with birds or their manure have sonnel should not travel between these two types been the most frequent cause of AI virus transmis- of production. sion. Prevent or minimize visitors to the farm. If 3. Birds taken to the diagnostic laboratory should visitors are allowed, use a log book in each house to be double-bagged to reduce risk of vehicle record visits. If infection occurs, this log will help contamination. track down other potentially exposed fl ocks. Spe- 4. Be aware that offal, feathers, dead-on-arrivals, cifi c measures to follow are listed next. and condemned birds from AI-infected fl ocks are all potential sources of the virus. Growers 5. Send eggs to processing or the hatchery only on Allow no unnecessary or unauthorized visitors into dedicated, washed, and disinfected plastic or new the fl ock. Do not allow other growers to visit. Make paper fl ats and dedicated cases, pallets, or no unnecessary visits to other farms. Conduct a racks. review of policy with all employees and include (1) no other poultry on the farm, (2) no other poultry at Eggs home, and (3) no family members of farm workers Eggs per se are not ordinarily a signifi cant risk for can work in a poultry meat–processing plant, egg- LPAI virus transmission, but their surface could be processing plant, or hatchery or assist in load-out. contaminated with manure and AI virus before washing, so egg-moving materials that traverse farm Visitors to processing to farm require close attention. Eggs Visitors should be discouraged except for essential that are being moved from one farm to another (side- personnel such as repair persons. All visitors should loaded eggs) are a risk and such movement should be provided with boots and coveralls. Clothing for be curtailed during an outbreak. Egg processing visitors or service persons may be kept in the entry- plants and hatcheries must have biosecurity proto- way without laundering. Inspect everyone who cols to manage fl ow so that nonsanitized materials comes to the farm for cleanliness and determine if cannot be inadvertently moved to farms with sus- they have had recent bird contact. Do not allow truck ceptible birds. The risk of hatching eggs being a drivers to enter the building. Require part-time help source of hatchery disseminated AI virus is low, but and crews to wear freshly laundered clothing or the potential must be acknowledged and managed. clothing supplied on the farm each day. Do not allow Paper chick boxes should be discarded. Plastic chick persons employed at other poultry operations on the boxes and delivery trucks should be carefully washed premises. Require visitors to wash their hands with with detergent and disinfectant to avoid bringing soap before entering. virus to the hatchery. Service persons Manure When possible, conduct fl ock service calls by tele- Contaminated manure should not be moved until phone. In the event of an outbreak, it is not possible suffi cient time has elapsed to promote inactivation to safely visit more than one fl ock per day. If you of the AI virus. If possible, manure should be com- must visit more than one fl ock per day, disinfect posted in the poultry house to inactivate the virus. your vehicle, shower and wear clean boots, cover- If moved off site, manure should only be transported alls, and hats at each site. If there are several farms in a covered vehicle. Do not allow the traffi c area in the company, establish zones to prevent one 23 / Control of Low Pathogenicity Avian Influenza 527 person from traveling to all farms. Establish a pattern poultry market reservoir that introduces AI to com- for necessary traffi c by supervisors. Consider each mercial poultry, so also the simple existence of fl ock you have visited infected and each fl ock you infected poultry fl ocks is not as great a threat for AI plan to visit free from infection. Bring nothing to a transmission as the movement of infected or suspect fl ock and take nothing away. After outerwear has birds or any materials associated with them. Many been used, it should be left on each farm or placed issues of human traffi c are common to most popula- in a garbage bag to avoid contaminating your vehicle. tions such as people involved in dead bird disposal, Wash clothing, rubber boots, coveralls and gloves manure movement, fl ock service, utilities installa- to be used at another farm with detergent in hot tion and repair, pest control, maintenance of equip- water. ment, repair of buildings and equipment, and delivery of supplies. Vehicles and Equipment Vehicles and equipment that come in direct contact Commercial broilers with birds or their manure should not be moved from All-in/all-out practices result in no birds being farm to farm. If vehicles and equipment must be present after a fl ock is marketed. It is rare in com- moved, specifi c measures should be used as mercial broiler production for part of a fl ock to be follows: marketed and the balance left to grow for additional time. Even if live haul equipment, clean out equip- 1. Establish protocols to prevent contamination of ment, and live haul crews are unsanitary (which is personal and service vehicles. unlikely), if there are no chickens remaining on 2. Apply heat (32º to 35º C) for as long as possible the farm, AI virus infection is unlikely to occur. In (up to a week). addition, resident workers reduce the likelihood of 3. Wash with detergent and disinfect moving and introduction. load-out equipment such as loaders, trailers, tarps, panels, and screens. Parent breeders 4. Wash with detergent and disinfect vehicles used To reduce risks, specifi c weaknesses associated with in loading and moving birds after unloading. this sector must be recognized and mitigations Cabs of these vehicles must be cleaned. implemented. High-level biosecurity must be prac- 5. Wash and disinfect farm clean-out equipment ticed to reduce risk of activities such as vaccinating such as tractors, trailers, pumps, and sprayers that individual replacement birds, moving replacements are taken from farm to farm. between farms, transporting eggs, spiking broiler 6. Make sure that service persons’ vehicles are not breeder fl ocks with extra males partway through the contaminated by litter or birds. They should be egg production period, moving or culling broody cleaned and disinfected at least daily or after turkey hens, inseminating turkey breeder hens being on a farm where AI is suspected. weekly, and maintaining turkey stud farms that 7. Do not allow shavings trucks or chick trucks to provide semen to multiple hen farms. enter the poultry house. 8. If possible, feed suppliers should set aside a truck Turkeys and commercial egg layers to be used only for deliveries to infected farms. These industries, accounting for most of the LPAI Do not pick up feed from an affected or suspect in U.S. commercial poultry, are both characterized affected farm. by the need to move birds from one site to another, frequently to multiple-age farms, and in the case of Preventing the Spread of AI in Different layers, movement of eggs means constant traffi c and Populations possible exposure to materials from other farms. Once an outbreak occurs, the control of AI must be However, movement of eggs is not that different adapted to the specifi c avian population that is from breeder farms. Hired labor is common on both involved, because each population will have its own commercial turkey and egg layer farms. characteristics as outlined in the biosecurity section. An example of a practice that increases the vul- Just as it is not the presence of AI virus, but the nerability of the turkey industry to AI is when birds movement of the virus from the wild bird or live are removed from the fl ock to make additional fl oor 528 Avian Influenza space available for the remainder. This is called an attempt to codify methods to reduce risk of inter- partial fl ock removal and is dangerous because the country transfer of poultry and poultry products, but presence of live haul equipment and personnel that whether they will help in maintaining fair trade is has had previous contact with other fl ocks is in not clear. contact with remaining susceptible birds and thus Depopulation, by either destruction or controlled increases the risk of exposure to pathogens. marketing, is the usual step to end infection on a An example of a practice that might increase risk farm. Depopulation during acute disease may create for the layer industry is called back fi lling and occurs a bigger risk of disease spread (e.g., generation of when hens are added to a fl ock to bring the house to infectious dust, hauling infected carcasses, and capacity at the time of recycling or forced molting. failure to control movement of contaminated crews) Usually the source of these hens is the same farm, than allowing the fl ock to remain under quarantine but this activity exposes the existing fl ock to person- unless special biosecurity and procedural measures nel and equipment that may have been used to move are utilized (30). It is not the existence of the AI pullets or spent hens on other farms. Because eggs virus but rather the movement of virus that causes usually move from a farm to a processing facility or virus spread during an outbreak. A fl ock that has hatchery, they are not associated with AI virus trans- been infected should not be moved until virus shed- mission, but egg-handling materials, because they ding subsides. Otherwise, the heavy contamination may be inadvertently moved from affected to unaf- of people and equipment used to load and move fected farms, require close attention. them and dispersal of virus on feathers and dust generated by the activity greatly increase the risk of Nonconfi ned poultry exposing other fl ocks and farms (18, 27, 30, 39, 40). These populations, if involved in an LPAI outbreak, Once it is accepted that infected fl ocks actively must be quickly evaluated for exposure to AI virus shedding virus should not be moved, a discussion by interestablishment movement. If unconfi ned can proceed on how to dispose of birds after virus poultry have the population density and multidirec- is no longer present. tional movement of birds necessary to maintain AI virus infection, this is the most diffi cult situation to Destruction control. In the United States, the live poultry market- One approach has been to depopulate by euthanizing ing system has been able to maintain H7N2 LPAI and disposing of infected birds. In principle, the host virus from 1994 to 2006. is removed, virus production stops, and the source Examples of practices that increase the risk of is eliminated. If these steps are taken after virus interestablishment AI transfer in nonconfi ned poultry shedding has subsided such as when the fl ock is include crates that move in and out of the live poultry seropositive, success is easier to achieve. However, market system, “topping off” of birds for the live this raises an important question: Why destroy the poultry market (partial fl ock removal), movement of fl ocks if they are no longer shedding LPAI virus? game birds for release at hunting preserves, move- One answer is to remove a threat to export markets ment of birds to and from exhibitions, exchanges of even though LPAI viruses are not found in meat or birds between collections, multiple species and ages eggs. Mass culling of fl ocks, especially when it of poultry in the village production system, and con- involves healthy convalescent fl ocks or healthy gregation of birds and people from multiple sources potentially exposed fl ocks, often leads to objections at cock fi ghts. by the public and serious erosion of public support (10, 32). In addition, if the government authorities What To Do with Flocks That Have Been do not pay compensation for destroyed fl ocks, this Infected with LPAI economically penalizes the farmer and company and How to dispose of or utilize fl ocks that have been will discourage future cooperation. For destruction infected with LPAI virus is a subject of disagree- of LPAI virus–infected fl ocks to be successful, ade- ment among poultry health professionals, regulatory quate compensation is essential. veterinarians, and corporate management. The threat Furthermore, destruction programs create the of export markets being closed as a result of an LPAI need to dispose of tons of potentially contaminated outbreak is very real. The new OIE guidelines are carcasses. On-farm disposal of carcasses is generally 23 / Control of Low Pathogenicity Avian Influenza 529 favored because it is economical and it eliminates Both approaches have been successful; however, the movement of potentially hazardous material and the economics favor controlled marketing. In 25 results in reduction of AI virus transmission. For years of LPAI in Minnesota, the total costs were less negative environmental reasons, burial and incinera- than $25 million, one sixth the cost of the Virginia tion on site are less favored (36), but composting of H7N2 LPAI outbreak ($150 million), while involv- carcasses can be an economical and environment- ing over fi ve times as many fl ocks (1100 fl ocks in ally friendly manner of disposal. Off-farm disposal Minnesota and about 200 in Virginia). includes taking the birds to central rendering, burning, or burial at landfi ll sites. If the carcasses Flock Scheduling must be moved, rendering has the economic advan- Whatever approach is used (destruction or controlled tage, but renderers have been reluctant to take such marketing), the fl ock schedule must be adjusted to materials for fear of customer rejection of stigma- make sure there is no live AI virus on the farm tized product despite rendering being effective at before another fl ock is placed. Whether the poultry killing AI virus. Central burning is expensive, is not house is totally cleaned, washed and disinfected or suited to large depopulation, efforts and creates real heat treated for 2 weeks, no susceptible poultry can environmental concerns (36). That leaves landfi lls be moved onto the farm until all procedures have as a satisfactory disposal option. No matter what been completed. This ordinarily means that there off-site disposal option is selected, moving the car- must be a delay in placement of the next fl ock on casses in sealed conveyances is essential. the farm to avoid placing susceptible birds into a potentially contaminated environment. Hatching Controlled marketing and placement schedules are very tight in commer- A second approach has been orderly or controlled cial production and altering them is often very dif- marketing of clinically healthy, virus-negative, serofi cult. Corporate management pressures may be positive fl ocks. This approach, termed a responsible diffi cult to resist. response (37), is based on a set of procedures designed to avoid increasing the economic hardship Cleaning and Disinfection caused by LPAI virus infection. It involves the vol- The AI virus is labile. It is important to recognize untary isolation of infected fl ocks by the owner to that AI outbreaks are not often associated with prevent transmission to other fl ocks. Often doing moving a susceptible fl ock into a facility that previ- nothing to move the virus is the single most impor- ously held an AI virus–positive fl ock. When AI tant thing to reduce the spread of disease. Rigorous virus infections are associated with moving suscep- measures to prevent the contamination and control tible poultry into a facility previously housing the movement of people and equipment are required infected fl ocks, it has been associated with failure to in order to stop the spread of this disease to suscep- clean the facility and not the failure of the cleaning tible fl ocks. Controlled marketing of fl ocks is sched- procedure. Once infected fl ocks have been removed uled after they have recovered from infection. from the premises, attention is directed at reducing Generally, live AI virus cannot be detected 2 weeks and eliminating the viral load that is present. A after clinical signs peak. Today with RRT-PCR (43) simple procedure that can greatly reduce viral load or antigen-detection tests on daily mortality, fl ocks is the application of heat. Warming the poultry can be evaluated for the presence of virus in birds house and its contents to 32º to 35º C for a week just hours before moving them to market. greatly reduces viral load and in turn reduces the The principle involved is that an informed indus- risks associated with removal and movement of try develops its own isolation and movement control manure. procedures necessary to stop the spread of LPAI Once the application of heat has reduced the virus. Steps to further reduce risk include routing amount of live AI virus in manure, the removal, dry processing trucks away from poultry farms and mar- cleaning, washing with detergent, and disinfection keting convalescent fl ocks at the end of the week to will almost certainly be effective. Manure can be allow more time for virus inactivation, more time composted for added safety. for crews to be away from other poultry, and more In cold climates, it is especially important to time for cleaning and disinfecting equipment. direct C&D attention to the area immediately outside 530 Avian Influenza the poultry house doors so that virus is not tracked against AI-related morbidity, mortality, condemna- into the building after clean up. tion, and resulting economic losses; to reduce viral shedding if fl ocks get infected; to reduce AI virus VACCINATION: THE SECOND LINE OF transmission between fl ocks; to increase fl ock resis- DEFENSE tance to AI virus infection; and, in the case of Vaccination alone will not control an AI outbreak domestic waterfowl, to reduce environmental con- (54), but inactivated infl uenza vaccine is an effective tamination with AI virus (45). Controlled vaccina- tool in the prevention and control of LPAI (20, 21, tion should be used, if necessary, as a part of an 45, 46). Vaccination for AI has been reviewed thor- overall strategy to eliminate LPAI and reduce the oughly (21, 45–49) Because immunity is hemag- risk of HPAI emergence from an uncontrolled H5 or glutinin subtype specifi c and birds are susceptible to H7 LPAI outbreak. (10, 12, 20, 21). all 16 hemagglutinin subtypes, preventive vaccination prior to an outbreak is not usually practiced with Mechanics of Vaccination the exception of vaccinating turkey breeders against H1N1 and H3N2 swine infl uenza. Once a subtype is Available Vaccines identifi ed in poultry and biosecurity practices appear There are only two types of licensed vaccines in the to be inadequate, however, controlled vaccination United States. There are inactivated whole virus oil- can be an effective tool to reduce the susceptibility emulsion vaccines and fowlpox-vectored vaccines of poultry populations. Reducing the number of sus- with an H5HA gene insert. Both types of vaccine ceptible fl ocks has been shown to contribute to the must be administered individually, requiring birds containment of an AI outbreak (44). to be handled, and both types of vaccine are very In some countries, fi nancial constraints preclude effective (45). wholesale slaughter and burial to control HPAI; in The vaccine strain must have the same hemag- some countries, export markets are not an issue and a glutinin as the fi eld strain and, in any particular slow systematic program of vaccination and controlled outbreak, studies should be done to demonstrate marketing might be used as an adjunct to eradicate effectiveness. Standardized manufacturing and HPAI; and in some HPAI outbreaks, the stamping-out quality control are necessary to ensure consistent attempts alone may be unsuccessful. Although the vaccine performance (safety, potency, and effi cacy). rationale for using controlled vaccination might be Policies and procedures are needed for proper similar for both HPAI and LPAI, government eradica- storage, distribution, handling, and administration tion programs for HPAI accompanied by indemnity (45). payment means the government determines the rules for how the eradication program is conducted. Where To Use the Vaccine Cost benefi t and risk analyses contribute to selection Purpose of birds to vaccinate. Swayne (45) has suggested the The purpose of vaccination is to aid in a coordinated following algorithm: high-risk situations (outbreak control program to prevent the spread of LPAI virus, zone), rare captive birds, valuable genetic stock, to reduce disease and disease-related economic long-lived poultry (commercial egg or hatching egg effects, and to eliminate the infection from the producers), and, last, meat production poultry. Eco- poultry populations in an area. Vaccination is the nomic considerations are the cost of the vaccine second line of defense against LPAI (3–5, 20, 21). ($0.10 to $0.15 per bird including administration), Leaving convalescent birds in place and controlled the value of the bird being vaccinated, and the eco- immunization of fl ocks at risk with an inactivated nomic implications of the outbreak as well as using vaccine reduces the susceptibility of the poultry vaccine. The individual grower may wish to vacci- population, while removal of fl ocks and reschedul- nate to protect his fl ocks, but industry and governing (e.g., delayed replacement) reduces the bird ment may wish to utilize vaccine as an aid to density in an area. eliminate the disease. Ring vaccination is a term to describe the practice Goals of Vaccination of vaccinating susceptible animals in all the premises The main goals of a program incorporating con- surrounding an infected herd or fl ock. This proce- trolled vaccination against LPAI are to protect dure is not known to have ever been used for the 23 / Control of Low Pathogenicity Avian Influenza 531 control of AI. The reason is clear: AI is not known to use a test to detect antibodies against the nonstruc- transmit from premises to premises by direct contact tural protein that will be present in AI virus–infected or airborne spread, but is transmitted by human activ- birds but not in vaccinated birds (50). Alternatively, ity often over many miles (see Fig. 23.1). What has a pox-vectored AI vaccine can be used and the AGID been used in aiding AI control is what could be called test will be negative in vaccinated noninfected birds. blanket vaccination, which describes the total vac- Both vaccinated and convalescent fl ocks can be cination of all the susceptible animals in an area at treated in the same way—isolated until marketed risk. Blanket vaccination describes the procedure (4,21). A plan is needed for determining when vac- that was successfully used in Utah, Italy, and Con- cination is no longer required or desirable. necticut (1, 10, 12, 21). Neither ring vaccination nor blanket vaccination has been used in Minnesota. In Justifi cation the early years of vaccine use, Minnesota turkey Vaccination as part of an offi cial eradication effort farmers on premises with a history of AI would is justifi ed when that plan incorporates controlled attempt preemptive or preventative vaccination. This marketing of vaccinated and convalescent fl ocks usually met with failure because a different subtype before quarantine is released (4) or when the poultry of AI infected the turkeys than what they had been industry is trying to eliminate LPAI infection and vaccinated against. After that experience, Minnesota likewise isolates vaccinated and convalescent fl ocks turkey producers used the vaccine more judiciously, until they are marketed (21). Vaccination against vaccinating fl ocks at risk (epidemiologically linked) LPAI has had demonstrated success in Utah (21), once an outbreak subtype was identifi ed. Connecticut (1), Italy (10), and against HPAI in Hong Kong (17). Administering the Vaccine Transporting people and vaccination equipment Concerns About the Use of Inactivated AI from farm to farm is a risk for transmission of AI, Vaccines but blood-sampling crews, bird-moving crews, and A common objection to the use of inactivated depopulation crews are also a risk. This risk must be vaccine is that if a vaccinated fl ock is exposed to managed. fi eld virus, birds may be infected and shed virus. The basis of these objections is experimental studies Evaluation showing failure of such vaccines to completely A surveillance program is necessary to determine block infection and shedding when high doses of whether AI virus is circulating in vaccinated fl ocks. challenge virus are used (21). Laboratory results Routine serosurveillance to detect convalescent have shown that vaccination eliminates (9) or greatly fl ocks is complicated by inactivated vaccines. Anti- reduces shedding of virus in experimentally chal- body against nucleoprotein and matrix proteins is lenged birds (28). If a vaccinated fl ock gets infected induced by circulation of LPAI viruses, like the anti- it will excrete approximately 99% to 99.99% less body induced by inactivated vaccines, and is detected virus than a nonvaccinated infected fl ock. by the commonly used AGID and the commercial Field use of vaccine has not increased the risk of ELISA tests. Surveillance must be conducted on undetected infection; in fact, fi eld experience has vaccinated fl ocks to determine whether the fi eld indicated that vaccination greatly enhances a control virus is circulating and whether the control strategy program. In Utah during 1995, no new cases of is working (45). LPAI in turkeys were detected 6 weeks after the Nonvaccinated sentinel fl ockmates can and should initiation of a widespread vaccination of over 200 be left in the vaccinated fl ocks and serologically fl ocks with a killed H7N3 AI vaccine and the disease monitored periodically to detect evidence of AI was subsequently eliminated (20, 21). Similar results infection until the vaccinated fl ocks are marketed. have been observed in Connecticut and Italy (1, 10). Another approach is to use the “differentiating If a nonvaccinated fl ock is exposed to fi eld virus, infected from vaccinated animals” (DIVA) strategy 100 to 10,000 times more virus is produced. (12). One way to do this is to vaccinate with a Some have suggested that vaccinated fl ocks are a vaccine containing a different neuraminidase than risk for transmitting AI virus to other fl ocks. Epide- the fi eld virus so a neuraminidase inhibition test or miological observations have shown that serologi- an N-specifi c ELISA can be used. Another way is to cally positive birds are not associated with LPAI 532 Avian Influenza virus transmission (30). Recent experimental studies probably because AI outbreaks usually occur in have demonstrated that AI vaccine prevents or immunologically naïve poultry fl ocks. If a large reduces transmission (47, 53). Nevertheless, vacci- population of poultry were immunized (by either nated birds can never be assumed to be free of AI natural infection or by vaccination), it is reasonable virus and carry some, but a reduced, risk for trans- to assume that a variant could emerge. Recent mission. Cardona et al. (14) demonstrated that H6N2 research, however, showed protection from a single LPAI fi eld vaccinated hens, when challenged in the recombinant vaccine against diverse H5 challenge laboratory, were able to transmit virus to contact viruses from three continents isolated over a 38-year hens. period (47). It is not clear what would be the effect of continuous vaccination in an area. This potential Incentives and Disincentives for emergence of variants is one reason that reliance With no indemnity from government, vaccine as a on vaccination alone is not a good strategy for LPAI safety net is critical (5). Without such a program, control. However, it is also a reason not to allow certain parts of the industry are more prone to disas- LPAI outbreaks to spread unchecked. trous effects of LPAI than others. If there is industry The history of vaccine use in Minnesota illustrates pressure to voluntarily destroy an LPAI seropositive that, as the turkey industry became more aware of fl ock, a producer may delay reporting or may be its limitation, vaccine use declined. The amount of motivated to market an actively infected fl ock. vaccine used annually from 1978 to 2002 is shown If LPAI strikes egg-laying birds, it can be disas- in Table 23.4. In the fi rst years after vaccine became trous, but if the same birds are infected before the onset of egg production, there is likely to be little, if any, clinical effect (48). Lack of a vaccine pro- Table 23.4. Avian infl uenza vaccine usage in vides the egg producer with an incentive to expose turkeys in Minnesota from 1979 to 2002. replacement pullets or replacement breeders to AI Year Doses prior to the onset of egg production (20). A meat bird grower may also be motivated to expose meat 1979 3,816,000 type growing birds. While intentional exposure may 1980 5,394,000 reduce fi nancial loss for the producer, it does not 1981 1,820,000 contribute to disease control and will delay elimina- 1982 1,382,000 tion by effi ciently transmitting the LPAI virus. It 1983 1,187,000 may also increase the chances of a fi eld H5 or H7 1984 113,000 LPAI mutating and becoming an HPAI virus. 1985 320,000 The needs of the various poultry industries may 1986 764,000 be different, but they all benefi t from elimination of 1987 515,000 a disease threat in an area. If the overall goal of 1988 183,000 elimination of LPAI is recognized, then each indus- 1989 2,117,000 try can pursue the goal using the most effective and 1990 155,000 applicable measures available. Long-lived birds 1991 918,000 such as breeders, commercial layers, exhibition 1992 295,000 birds, and game fowl are most suitable candidates 1993 385,000 for vaccination, and the protection induced by killed or pox-vectored AI vaccine is clear. 1994 313,000 1995 2,266,000 Vaccination Limitations 1996 442,000 Vaccination alone has never eliminated LPAI. Con- 1997 185,000 tinuous vaccination for LPAI is not a sound strategy 1998 39,000 for control. Antigenic drift directly attributable to 1999 120,000 vaccination has not been documented in poultry 2000 40,000 (although it may have occurred in Mexico and 2001 50,000 Central America and may be occurring in Asia), 2002 90,000 23 / Control of Low Pathogenicity Avian Influenza 533 available, up to 10% of the market turkeys were fl ocks deemed to be at risk by industry veterinarians vaccinated until growers learned that preemptive (where regulatory veterinarians are informed of all vaccination was not effective. In recent years, vaccine use), monitoring sentinel birds left in the vaccine usage has declined to 0.2% of turkeys pro- vaccinated fl ocks or other appropriate monitoring duced and involves swine infl uenza vaccine used in methods, isolating or quarantining convalescent and some turkey breeder fl ocks. vaccinated fl ocks, and controlled marketing of convalescent and vaccinated fl ocks. CONCLUSION We now recognize that science-based LPAI While the global epidemiology of AI is complex, at control programs with totally different approaches the local level, AI prevention and control are usually can have the same outcome. Combining the best simple but rarely easy. In the natural environment, features of existing programs has the potential to AI virus is moved by wild bird activity, whereas in improve the existing LPAI control strategies, to agricultural systems, it is primarily spread by human reduce the objections that have been raised about activity. In totally confi ned poultry, the only sig- them, and to reduce disease control costs. nifi cant risk of AI virus introduction is movement of virus into close proximity of poultry by the activities REFERENCES of people. People move AI virus by moving poultry 1. Adriatico, N. 2005. Controlling AI by vaccination: and manure and things contaminated by poultry and the Connecticut experience. In: Proceedings of the manure. With this basic information, AI prevention North Central Avian Disease Conference, Saint and control programs are designed around the char- Paul, Minnesota, March 2005, pp. 25–28. acteristics of the virus and host in conjunction with 2. Bahl, A.K., A., Langston, R.A. Van Deusen, B.S. Pomeroy, J. Newman, D. Karunakaran, and D. local poultry population density and customs. Before Halvorson. 1979. Prevention and control of avian any program is complete, it must address the biology, infl uenza in turkeys. In: Proceedings of the 83rd ecology, and epidemiology of the LPAI viruses; the Annual Meeting of the United States Animal poultry population(s) at risk; how the virus moves Health Association. U.S. Animal Health Associa- in the specifi c population; risk of cross-over from tion, Richmond, VA, pp. 355–363. other poultry groups; how poultry are exposed; and 3. Beard, C.W. 1982. Immunization approaches to sanitation principles important for LPAI virus. avian infl uenza. In: R.W. Bankowski (ed.). Pro- Biosecurity is essential to the control of LPAI. Its ceedings of the First International Symposium on application is the core of all successful LPAI pre- Avian Infl uenza, April 1981, Beltsville, Maryland. vention and control programs. Education, prevent- Symposium on Avian Infl uenza, US Animal Health ing introductions, surveillance, reporting, and a Association: Richmond, VA, pp. 172–177. 4. Beard, C.W. 1987. To vaccinate or not to vacci- responsible response provide the framework for all nate. In: B.C. Easterday and C.W. Beard (eds.). AI control programs. Proceedings of the Second International Sympo- Sometimes biosecurity is not suffi cient to stop the sium on Avian Infl uenza, Georgia Center for spread of LPAI. In general, infl uenza virus is only Continuing Education, the University of Georgia, maintained in a dense population of susceptible Athens, Georgia, USA, September 3–5, 1986. hosts. When there is a large population of suscep- Symposium on Avian Infl uenza, US Animal tible poultry in an area, use of an inactivated AI Health Association: Richmond, VA, pp. 258– vaccine can contribute to AI control by reducing the 263. susceptibility of the population. 5. Beard, C.W., 1992. The role of vaccines and vac- Use of licensed vaccines have reduced a fl ock’s cination. In: B.C. Easterday (ed.). Proceedings of susceptibility to AI infection, have reduced the the Third International Symposium on Avian Infl u- enza, May 27–29, 1992, Madison, Wisconsin. quantity of virus shed post challenge, have reduced Symposium on Avian Infl uenza, US Animal Health transmission post challenge, and have markedly Association: Richmond, VA, pp. 293–305. reduced disease losses. Controlled vaccination 6. Beard, C.W., M. Brugh, and D.C. Johnson. 1984. against LPAI, including H5 and H7, should be avail- Laboratory studies with the Pennsylvania infl uenza able as part of a science-based infl uenza control viruses (H5N2). In: R.W. Bankowski (ed.). Pro- strategy that includes tight biosecurity, monitoring ceedings of the First International Symposium on all fl ocks at risk, using controlled vaccination for Avian Infl uenza, April 1981, Beltsville, Maryland. 534 Avian Influenza

Symposium on Avian Infl uenza, US Animal Health 18. Etsell, G. 2005. Political impact of avian infl uenza. Association: Richmond, VA, pp. 462–473. North Central Avian Disease Conference, Saint 7. Bowes, V. 2004. An outbreak of HPAI in British Paul, Minnesota, March 2005, pp. 14–24. Columbia, Canada: or how to learn about AI 19. Halvorson, D.A. 1987. Avian infl uenza—a Minne- the hard way. In: Proceedings of the Avian Infl u- sota cooperative control program. In: B.C. Easter- enza Diagnostic Workshop. National Poultry day and C.W. Beard (eds.). Proceedings of the Improvement Plan. Athens, GA, November 16– Second International Symposium on Avian Infl u- 18. enza, Georgia Center for Continuing Education, 8. Bowes, V.A., S.J. Ritchie, S. Byrne, K. Sojonky, the University of Georgia, Athens, Georgia, USA, J.J. Bidulka, and J.H. Robinson. 2004. Virus char- September 3–5, 1986. Symposium on Avian Infl u- acterization, clinical presentation, and pathology enza, US Animal Health Association: Richmond, associated with H7N3 avian infl uenza in British VA, pp. 327–336. Columbia broiler breeder chickens in 2004. Avian 20. Halvorson, D.A. Strengths and weaknesses of vac- Diseases 48(4):928–934. cines as a control tool. 1997. In: D.E. Swayne and 9. Brugh, M., C.W. Beard, and H.D. Stone. 1979. R.D. Slemons (eds.). Proceedings of the Fourth Immunization of chickens and turkeys against International Symposium on Avian Infl uenza, May avian infl uenza with monovalent and polyvalent 29–31, 1997, Athens, Georgia. Symposium on oil emulsion vaccines. American Journal of Vet- Avian Infl uenza, US Animal Health Association: erinary Research 40(2):165–169. Richmond, VA, pp. 223–227. 10. Capua I., and S. Marangon. 2003. Vaccination 21. Halvorson, D.A. 2002. The control of H5 or H7 policy applied for the control of avian infl uenza in mildly pathogenic avian infl uenza—a role for inac- Italy. Developmental Biology (Basel) 114:213– tivated vaccine. Avian Pathology 31(1):5–12. 219. 22. Halvorson, D.A., C.J. Kelleher, and D.A. Senne. 11. Capua, I., S. Marangon, M. dalla Pozza, C. Terre- 1985. Epizootiology of avian infl uenza: effect of gino, and G. Cattoli. 2003. Avian infl uenza in Italy season on incidence in sentinel ducks and domestic 1997–2001. Avian Diseases 47(3 Suppl.):839– turkeys in Minnesota. Applied and Environmental 843. Microbiology 49(4): 914–919. 12. Capua, I., F. Marangon, M. Dalla Pozza, and U. 23. Halvorson, D.A., C.J. Kelleher, B.S. Pomeroy, V. Santucci, 2000. Vaccination for avian infl uenza in Sivanandan, A.S. Abraham, J.A. Newman, D. Italy. Veterinary Record 147:751. Karunakaran, P.E. Poss, D.A. Senne, and J.E. 13. Cardona, C.J. 2008. Farm and regional biosecurity Pearson. 1987. Surveillance procedures for avian practices. In: D.E. Swayne, ed. Avian Infl uenza. infl uenza. In: B.C. Easterday and C.W. Beard Blackwell Publishing: Ames, IA, pp. 353–367. (eds.). Proceedings of the Second International 14. Cardona, C.J., B. Charlton, and P.R. Woolcock. Symposium on Avian Infl uenza, Georgia Center 2006. Persistence of immunity in commercial egg- for Continuing Education, the University of laying hens following vaccination with a killed Georgia, Athens, Georgia, USA, September 3–5, H6N2 avian infl uenza vaccine. Avian Diseases 1986. Symposium on Avian Infl uenza, US Animal 50(3):374–379. Health Association: Richmond, VA, pp. 155–162. 15. de Wit, J.J., J.H. van Eck, R.P. Crooijmans, and A. 24. Halvorson, D.A., S. Kodihalli, E. Laudert, J.A. Pijpers. 2004. A serological survey for pathogens Newman, B.S. Pomeroy, D. Shaw, and V. Sivana- in old fancy chicken breeds in central and eastern ndan. 1992. Infl uenza in turkeys in the U.S.A. part of the Netherlands. Tijdschr Diergeneeskd (1987–1991). In: B.C. Easterday (ed.). Proceed- 129(10):324–327. ings of the Third International Symposium on 16. Elbers, A.R., T.H. Fabri, T.S. de Vries, J.J. de Wit, Avian Infl uenza, May 27–29, 1992, Madison, Wis- A. Pijpers, and G. Koch. 2004. The highly patho- consin. Symposium on Avian Infl uenza, US genic avian infl uenza A (H7N7) virus epidemic in Animal Health Association: Richmond, VA, pp. the Netherlands in 2003—lessons learned from the 33–42. fi rst fi ve outbreaks. Avian Diseases 48(3):691– 25. Halvorson, D.A., and W.D. Hueston. 2006. The 705. development of an exposure risk index as a rational 17. Ellis, T.M., C.Y. Leung, M.K. Chow, L.A. Bissett, guide for biosecurity programs. Avian Diseases W. Wong, Y. Guan, and J.S. Malik Peiris. 2004. 50(4):516–519. Vaccination of chickens against H5N1 avian infl u- 26. Hanson, B.A., D.E. Stallknecht, D.E. Swayne, enza in the face of an outbreak interrupts virus L.A. Lewis, and D.A. Senne. 2003. Avian infl u- transmission. Avian Pathology 33(4):405–412. enza viruses in Minnesota ducks during 1998– 23 / Control of Low Pathogenicity Avian Influenza 535

2000. Proceedings of the Fifth International mercial poultry farms in Virginia. Journal of the Symposium on Avian Infl uenza. Avian Diseases American Veterinary Medical Association 226(5): 47(3 Suppl.):867–871. 767–772. 27. Henzler, D.J., D.C. Kradel, S. Davison, A.F. 36. Mixson, M.A. 1992. Stability/lability of avian Ziegler, D. Singletary, P. DeBok, A.E. Castro, H. infl uenza viruses in the depopulation process. In: Lu, R. Eckroade, D. Swayne, W. Lagoda, B. B.C. Easterday (ed.). Proceedings of the Third Schmucker, and A. Nesselrodt. 2003. Epidemiol- International Symposium on Avian Infl uenza, May ogy, production losses, and control measures asso- 27–29, 1992, Madison, Wisconsin. Symposium on ciated with an outbreak of avian infl uenza subtype Avian Infl uenza, US Animal Health Association: H7N2 in Pennsylvania (1996–98). Avian Diseases Richmond, VA, pp. 155–158. 47(3 Suppl.):1022–1036. 37. Poss, P.E., and D.A. Halvorson. 1987. The nature 28. Karunakaran, D., J.A. Newman, D.A. Halvorson, of avian infl uenza in turkeys in Minnesota. In: B.C. and A. Abraham. 1987. Evaluation of inactivated Easterday and C.W. Beard (eds.). Proceedings of infl uenza vaccines in market turkeys. Avian Dis- the Second International Symposium on Avian eases 31(3):498–503. Infl uenza, Georgia Center for Continuing Educa- 29. Koch, G. Poultry Foreign Animal Disease Aware- tion, the University of Georgia, Athens, Georgia, ness Training Course. USDA-APHIS-VS National USA, September 3–5, 1986. Symposium on Avian Veterinary Services Laboratories: Ames, IA, Infl uenza, US Animal Health Association: Rich- March 30–April 1, 2004. mond, VA, pp. 112–117. 30. Kradel, D.C. 1992. Avian infl uenza—are recov- 38. Poss, P.E., D.A. Halvorson, and D. Karunakaran. ered seropositive fl ocks a risk? In: B.C. Easterday 1982. Economic impact of avian infl uenza in (ed.). Proceedings of the Third International Sym- domestic fowl in the United States. In: R.W. posium on Avian Infl uenza, May 27–29, 1992, Bankowski (ed.). Proceedings of the First Interna- Madison, Wisconsin. Symposium on Avian Infl u- tional Symposium on Avian Infl uenza, April 1981, enza, US Animal Health Association: Richmond, Beltsville, Maryland. Symposium on Avian Infl u- VA, pp. 43–49. enza, US Animal Health Association: Richmond, 31. Krohn, L.D. 1925. A study on the recent outbreak VA, pp. 100–106. of a fowl disease in New York City. Journal of the 39. Poss, P.E., K.A. Friendshuh, and L.T. Ausherman. American Veterinary Medical Association 20(2): 1987. The control of avian infl uenza. In: B.C. East- 146–170. erday and C.W. Beard (eds.). Proceedings of the 32. Landman, W.J., and C.C. Schrier. 2004. Avian Second International Symposium on Avian Infl u- infl uenza—eradication from commercial poultry is enza, Georgia Center for Continuing Education, still not in sight. Tijdschr Diergeneeskd 129(23): the University of Georgia, Athens, Georgia, USA, 782–796. September 3–5, 1986. Symposium on Avian Infl u- 33. Lu, H., A.E. Castro, K. Pennick, J. Liu, Q. Yang, enza, US Animal Health Association: Richmond, P. Dunn, D. Weinstock, and D. Henzler. 2003. VA, pp. 318–326. Survival of avian infl uenza virus H7N2 in SPF 40. Power, C. 2005. The source and spread of the avian chickens and their environments. Avian Diseases infl uenza virus in the Lower Fraser Valley of 47(3 Suppl.):1015–1021. British Columbia during an outbreak in the winter 34. McCapes, R.H., R.A. Bankowski, and G.B.E. of 2004—an interim report. Available at http:// West. 1987. Avian infl uenza in California—the www.inspection.gc.ca/english/anima/heasan/ nature of the clinical disease 1964–1985. In: B.C. disemala/avfl u/2004rep/epie.shtml. Canadian Food Easterday and C.W. Beard (eds.). Proceedings of Inspection Agency, Animal Products, Animal the Second International Symposium on Avian Health and Production Division. Accessed March Infl uenza, Georgia Center for Continuing Educa- 16, 2007. tion, the University of Georgia, Athens, Georgia, 41. Senne, D.A., B. Panigrahy, and R.L. Morgan. USA, September 3–5, 1986. Symposium on Avian 1994. Effect of composting poultry carcasses Infl uenza, US Animal Health Association: Rich- on survival of exotic avian viruses: highly patho- mond, VA, pp. 118–132. genic avian infl uenza virus and adenovirus of egg 35. McQuiston, J.H., L.P. Garber, B.A. Porter-Spald- drop syndrome-76. Avian Diseases 38(4):733– ing, J.W. Hahn, F.W. Pierson, S.H. Wainwright, 737. D.A. Senne, T.J. Brignole, B.L. Akey, and T.J. 42. Shane, S.M. 1997. Prevention of diseases. In: Holt. 2005. Risk factors for spread of low patho- Handbook on Poultry Diseases. American Soybean genicity H7N2 avian infl uenza virus among com- Association: St. Louis, MO, pp. 20–23. 536 Avian Influenza

43. Spackman, E., D.A. Senne, T.J. Myers, L.L. 50. Tumpey, T.M., R. Alvarez, D.E. Swayne, and D.L. Buluga, L.P. Garber, M.L. Perdue, K. Lohman, Suarez. 2005. A diagnostic aid for differentiating L.T. Daum, and D.L. Suarez. 2002. Development infected from vaccinated poultry based on antibod- of a real-time reverse transcriptase PCR assay for ies to the nonstructural (NS1) protein of infl uenza type A avian H5 and H7 subtypes. Journal of Cli- A virus. Journal of Clinical Microbiology 43(2): nical Microbiology 40(9):3256–3260. 679–683. 44. Stegeman, A., A. Bouma, A.R. Elbers, M.C. de 51. Tumpey, T.M., D.R. Kapczynski, and D.E. Jong, G. Nodelijk, F. de Klerk, G. Koch, and M. Swayne. 2004. Comparative susceptibility of van Boven. 2004. Avian infl uenza A virus (H7N7) chickens and turkeys to avian infl uenza A H7N2 epidemic in the Netherlands in 2003: course of the virus infection and protective effi cacy of a com- epidemic and effectiveness of control measures. mercial avian infl uenza H7N2 virus vaccine. Avian Journal of Infectious Diseases 190(12):2088– Diseases 48(1):167–176. 2095. 52. Van der Goot, J.A., G. Koch, M.C. de Jong, and 45. Swayne, D.E. 2006. Principles for vaccine protec- M. van Boven. 2003. Transmission dynamics of tion in chickens and domestic waterfowl against low and high pathogenicity A/chicken/Pennsylva- avian infl uenza: emphasis on Asian H5N1 high nia/83 avian infl uenza viruses. Avian Diseases pathogenicity avian infl uenza. Annals of the New 47(3 Suppl.):939–941. York Academy of Sciences 1081:174–181. 53. Van der Goot, J.A., G. Koch, M.C. de Jong, and 46. Swayne, D.E. 2008. Vaccines, vaccination and M. van Boven. 2005. Quantifi cation of the effect immunity. In: D.E. Swayne (ed.). Avian Infl uenza. of vaccination on transmission of avian infl uenza Blackwell Publishing, Ames, IA, pp. 407–451. (H7N7) in chickens. Proceedings of the National 47. Swayne, D.E., M. Garcia, J.R. Beck, N. Kinney, Academy of Sciences U S A 102(50):18141– and D.L. Suarez. 2000. Protection against diverse 18146. highly pathogenic H5 avian infl uenza viruses 54. Villarreal-Chavez, C., and E. Rivera-Cruz. 2003. in chickens immunized with a recombinant fowl- An update on avian infl uenza in Mexico. Avian pox vaccine containing an H5 avian infl uenza hem- Diseases 47(3 Suppl.):1002–1005. agglutinin gene insert. Vaccine 18:1088–1095. 55. Webster, R.G., M. Yakhno, V.S. Hinshaw, W.J. 48. Swayne, D.E., and D.A. Halvorson. 2003. Avian Bean, and K.G. Murti. 1978. Intestinal infl uenza: infl uenza. In: Y.M. Saif (ed.). Diseases of Poultry, replication and characterization of infl uenza viruses 11th ed. Iowa State University Press, Ames, IA, in ducks. Virology 84:268–278. pp. 135–160. 56. Ziegler, A.F., S. Davison, H. Aclund, and R.J. Eck- 49. Swayne, D.E., and D.A. Halvorson. 2008. Avian roade. 1999. Characteristics of H7N2 (nonpatho- infl uenza. In: Y.M. Saif (ed.). Diseases of Poultry, genic) avian infl uenza virus infections in 12th ed. Iowa State University Press, Ames, IA, in commercial layers in Pennsylvania, 1997–1998. press. Avian Diseases 42:142–149. 24 The Economics of Avian Influenza

Anni McLeod

INTRODUCTION BENEFITS AND COSTS OF CONTROLLING The epidemic of H5N1 highly pathogenic avian HIGH PATHOGENICITY AVIAN infl uenza (HPAI) that has spread across Asia, INFLUENZA Europe, and Africa since 2003 should not have been Economic justifi cation for controlling HPAI comes a surprise. Ecological conditions in today’s poultry from three sources. The case for low pathogenicity sectors are conducive to the emergence and spread avian infl uenza (LPAI) depends on the extent to of disease. Nevertheless, panic and economic losses which it is associated with continuation of HPAI. have been a continuing theme even where outbreaks Three types of benefi t justify HPAI control. have not occurred, or have been reported and rapidly stamped out, or have been caused by strains of HPAI Net Benefi ts of Avoiding a Human Pandemic other than H5N1. Losses to date have been large and The potential benefi ts from trying to prevent human estimates of potential loss are enormous. Equally infl uenza are so great that it has hardly been neces- important, this long emergency has caused the world sary to argue the case. Economic estimates of poten- to think seriously about the way that poultry should tial impact are very large, and this has resulted in be kept in the future. considerable international funding for avian and HPAI is a particular concern because it is a zoo- human pandemic control, as discussed in the next notic disease, the possible source of the next human section on fear of human infl uenza. infl uenza pandemic and a transboundary animal disease affecting all poultry. These reasons combine Net Benefi ts of Minimizing Human Disease to merit classifying freedom from HPAI as an inter- Contracted Directly From Birds national public good. The international response to Human cases and deaths from HPAI, although the wave of outbreaks reported since 2003 has been tragic, have so far been small in number and would in the order of US$2 billion. International and not have justifi ed huge international expenditure on national organizations have deployed considerable disease control. There were 1.2 million deaths refi nancial and human resources to deal with the corded for malaria in the annual statistics of 2002 immediate emergency and reduce risk for the future. and 2003 (29), while HPAI had caused 204 known In order to use these resources effectively, it is deaths at the time of writing. important to understand the economic and social factors that affect the success and impact of mea- Net Benefi ts From Improved Poultry sures used for control. This chapter addresses the Productivity Through Avoiding Disease in Birds economic imperatives faced by decision makers It is widely agreed that control of the disease at who must deal with avian infl uenza (AI) as a poultry its source in domestic poultry will be the most effec- disease, while remaining aware of the humanitarian tive way to prevent the occurrence of a human pan- and economic threat of a human pandemic. demic, and this chapter concentrates mainly on the

Table 24.1. Phases of disease and socioeconomic issues. Socioeconomic issues Preoutbreak Outbreak(s) Rehabilitation Long-term prevention

Market shocks Culling/compensation Movement control effects Vaccination costs Restocking costs Restructuring investment Long-term market access Financing animal health

economic impacts of disease and control methods in It also requires investment to revive animal health the poultry sector. Control should be done as cost systems that have suffered from neglect. effectively as possible and with a minimum of dis- Estimates of net benefi ts from avoiding disease in ruption to human lives and economies, although birds need to balance the impact from disease against crises do not create the best conditions for cost the impacts of control processes and assess differ- effectiveness. ential impact by sector and along market chains. A typical pattern of socioeconomic effects begin- Ideally, they will take into account all of the ning before an outbreak and progressing toward following. long-term control measures is shown in Table 24.1. The length and intensity of each phase are infl u- 1. Net impact of market shocks. Shocks occur when enced by the structure of the poultry sector and the demand and prices are disrupted by consumer response capacity of the animal health system. fears of disease or import bans of trading part- Market shock is the fi rst economic effect and may ners. The control process can also cause market occur even without an outbreak, created by con- disruption by restricting movement and sales or sumer fears. If an outbreak occurs, each element of exaggerating consumer fears through ill-judged the disease control process has associated costs and communication and may have impacts far beyond livelihoods effects, beginning with reporting of the area of infection. disease, stamping-out by culling and movement 2. Net impact on poultry productivity. Productivity control, providing compensation for birds culled, gains from controlling disease must be offset and later perhaps the introduction of vaccination. against the losses caused by the control process. The diverse character of poultry keeping and poultry These effects are greatest within areas where out- keepers offers huge challenges in designing control breaks occur, affecting producers and those programs that maximize the benefi ts from contain- immediately connected to them through market ing disease while at the same time balancing the chains. There may be wider effects if depopula- needs of small- and large-scale operators. As disease tion is extensive. is brought under control, rehabilitation of the poul- 3. Costs of dealing with diseased birds. These try sector begins. This is a straightforward process include treatment (if any) and disposal of car- if an outbreak has been quickly stamped out, but casses. more complicated if it is taking place under condi- 4. Direct costs of prevention and control processes. tions of recurring outbreaks. Where there are These include all of the human resource, capital, complex poultry market chains and continuing and consumables needed to carry out surveil- disease, there is pressure for governments to intro- lance, culling and disposal, movement control, duce long-term measures that will restructure the and vaccination. sector in a more biosecure way. However, this 5. Costs of rehabilitation. Restoring the operation carries the risk of excluding smallholders from of the poultry sector after an outbreak incurs a poultry keeping, with associated loss of livelihoods. restocking cost above the normal costs for main- 24 / The Economics of Avian Influenza 539

taining production cycles. In addition, it usually Neither human health effects nor loss of biodiver- requires investment in more biosecure manage- sity was considered. The costs included were those ment by farmers, traders, and market managers, of planning, surveillance, farm biosecurity, market as part of the effort toward preventing recurrence biosecurity, general public biosecurity, and vaccina- of disease. tion. Comprehensive restructuring of the sector has 6. Impacts of restructuring. Beyond the immediate been under discussion for some time in Vietnam, but impacts of dealing with disease, there may be it was not included in the calculations shown in changes in the structure of the poultry sector Table 24.2 because insuffi cient information was resulting from heightened animal health and food available at the time to evaluate the potential impacts safety regulations or restrictions in the places of restructuring. where production and processing may take place. The results cannot be considered conclusive and These measures require investment and will are currently being revised, but they do illustrate result in improved productivity for some but three interesting points. First, the benefi ts as shown reduced market access for others. They may also, here are very stable. By the time these estimates although this has not yet been evaluated, result were made, Vietnam had been dealing with HPAI in a loss of animal genetic resource. for more than a year, had passed the market shock phase, and was experiencing small numbers of out- If the control strategy is well designed and imple- breaks that were quickly controlled. Second, the cost mented, the losses from control should be consider- of farm biosecurity, while based on quite conserva- ably less than those that would have occurred from tive estimates, was nevertheless very high. This cost an uncontrolled disease outbreak, but the impacts on is assumed to be borne almost entirely by the private different stakeholders may be uneven. Compliance sector. Finally, the recurrent costs even without con- with disease control regulations will depend on the sidering farm biosecurity were not insignifi cant. benefi t that each stakeholder group perceives from Approximately US$4 million a year was estimated them. For example, providing compensation does to be necessary for improved surveillance, into the not reduce the production loss from culled birds, but indefi nite future, and it was assumed that vaccina- shares the loss between producers and others in tion would continue at decreasing levels for 7 society, providing an incentive for producers to years. cooperate with culling teams. One of the diffi culties of making a comprehensive A complete benefi t cost-benefi t or cost-effective- global estimate arises from the unreliability of the ness analysis for global control has not yet been data on loss of birds from disease. Estimates to date attempted. At the start of the present H5N1 HPAI (from Food and Agriculture Organization of the epidemic, it would have been highly speculative, United Nations [FAO] searches) suggest that and an ex-post evaluation cannot be completed approximately 250 million poultry had died or been while the disease is still spreading. Preliminary esti- culled in H5N1 HPAI outbreaks between the begin- mates have been made for some countries and ning of 2004 and October 2007. This fi gure is prob- regions at different stages of disease, some giving a ably an underestimate, but the reported country detailed snapshot for a particular country and time fi gures suffer from unreliability of base data, the and others talking vaguely of potential costs running diffi culty of accurate recording under crisis situa- into billions, but none gives a complete picture. tions, and the limited scope of surveys. Estimates One example of a snapshot assessment of animal based on proxy data such as household surveys or health control measures was made for Vietnam at agricultural census fi gures are complicated by the the end of 2005 when the country was introducing fact that the disease behaves differently in each pro- mass vaccination (Table 24.2). At that stage, approx- duction system. An uncontrolled outbreak of H5N1 imately 43 million birds had died or been culled, HPAI in a shed of chickens reared on litter will there had been investments in veterinary infrastruc- usually result in 95% to 100% mortality. In caged ture, and the government was reviewing plans for poultry, the mortality rate will initially be lower, but the future. The assessment considered only the ben- if left unchecked most of the fl ock will die. In scav- efi ts to be gained by avoiding production losses and enging fl ocks, the mortality may be low because avoiding the cost of stamping-out further outbreaks. many birds will not be exposed to the virus (51). 540 Avian Influenza

Table 24.2. Beneﬁ ts and costs of continued HPAI control in the Vietnamese poultry sector based on preliminary assessment in 2005, after 1 year of control measures. Year 2006 2007 2008 2009–2010 2011–2012 2013–2015 Total

Beneﬁ t 31.3 31.3 31.3 31.3 31.3 31.3 312.6 Investment cost 196.7 191.8 72.2 71.5 603.7 Recurrent cost 126.48 126.53 121.90 121.86 121.84 117.89 1216.0 Investment without 22.5 17.6 0.7 40.8 smallholder biosecurity Recurrent without 16.48 16.53 11.90 11.86 11.84 7.89 116.0 smallholder biosecurity NPV at 10% including −1044.5 smallholder biosecurity NPV at 10% without 80.7 smallholder biosecurity Figures in US$1 million. Source: McLeod and Brandenburg, 2005.

It is unclear what proportion of birds have died of Nigeria, Thailand, Turkey, and Vietnam have suf- disease and what proportion have been culled. This fered heavy losses compared to their poultry popula- distinction is unimportant when calculating total tion, from a combination of death and culling. In economic loss but becomes important when esti- other countries, the effects have been smaller and mates are made of costs to different stakeholders. As relatively local. discussed later, birds that die of disease may not be The remainder of the chapter discusses in more eligible for compensation or may be compensated at detail the main economic effects identifi ed in this a lower rate. In Vietnam by April 2004, around 43 section. It covers the potential impacts of AI in million poultry had died or been culled in the control humans that may be avoided by control of HPAI in zone around infected farms with the majority being poultry, the contribution of poultry sector diversity culled. Further poultry were culled voluntarily by to HPAI impact, the effects of market shocks, food farmers due to loss of access to markets (51). In security and livelihoods impacts, the costs and pro- Indonesia, by contrast, the levels of culling have ductivity losses associated with outbreak control, the been quite low and death from disease has been restocking process, and the socioeconomic effects of proportionally higher. A survey carried out in 2005 restructuring. (8) found that 16.2 million birds had died or been culled, although the study largely ignored backyard FEAR OF HUMAN INFLUENZA producers. In Cambodia and Laos, there were rela- It is likely that the next serious discontinuity to tively low levels of reported death and culling but world development will originate from either a very limited testing was done in smallholder fl ocks humans infl uenza pandemic or a transformational away from the immediate outbreak areas, and so it world war (52). HPAI has the possibility to trigger was diffi cult to establish the extent of disease. Small the next human fl u pandemic and this is a major outbreaks in remote areas tend to go unreported, and contributing factor to the concern about AI. In social records are particularly poor in backyard fl ocks. and humanitarian terms, human pandemics are dev- Estimates from Egypt at the end of 2006 suggested astating—witness the impact of the relatively minor that between 13 and 17 million birds had been culled global SARS outbreaks in 2003, which killed less (48). Although precise numbers are diffi cult to than 800 people (9) but seriously disrupted the econ- gauge, the magnitude of impact seems clear. China, omies of Southeast Asian countries and Canada (19, Indonesia, Egypt, the West Bank and Gaza Strip, 12), and the lives of their citizens. The human fl u 24 / The Economics of Avian Influenza 541 pandemics in 1918–1919, 1957, and 1968 may have Even without a human fl u pandemic, the eco- killed 100 million, 2 million, and 1 million people, nomic costs of HPAI have been large and its control respectively. at source is essential. Various contributors to cost Pandemic effects depend not only on the numbers are discussed in further sections of this chapter. The of people killed but also on the demographic distri- H5 and H7 LPAI viruses, which normally would not bution of illness and death. A high proportion of rate much international attention, are receiving infection in economically productive age ranges, as greater vigilance and stricter control measures than in the case of HIV/AIDS, has the potential to cause might otherwise be the case, out of concern that they long-term damage to economies. Should a human fl u may mutate to HPAI viruses. pandemic occur, it is uncertain which age groups would be worst affected. With many uncertainties, THE GLOBALIZED POULTRY SECTOR precise estimates of economic impact for a new pan- Poultry are perhaps the most globalized of all live- demic are impossible. Recent calculations suggest stock. They are kept and their products are con- between 1.4 and 142 million human deaths and sumed in almost every country, by almost every losses between US$330 billion and US$4.4 trillion culture, and by every income group where meat is (36). One of the long-term impacts of a pandemic eaten, and the inputs and outputs of poultry systems could be to push large numbers of households below are traded across the world. Poultry production and the poverty line (6), and the low level of investment trade have shown steady growth (Table 24.3), and in public health in the poorest countries (45) is a projections suggest that demand will continue to cause for concern. rise. At the same time, the sector is highly diverse, The economic effects of a pandemic are likely to with production systems ranging from industrial, start with disruptions to businesses and economies specialized, and highly biosecure units to backyard and will place unusually high demands on some fl ocks with scavenging birds. services (through stockpiling essential items) and abruptly lower the demand for others (entertain- Production Systems ment, restaurants, hotels). National and corporate FAO and World Organization of Animal Health plans for operation in times of pandemic aim to (Offi ce Internationale des Epizooties [OIE]) (22, 16) allow government and business to continue in the defi ne four types of poultry production system clas- event that employees may be ill, caring for others, sifi ed as sectors 1 to 4. Sector 1, industrial poultry or unable to travel to work and to ensure the avail- with high biosecurity, is the system from which the ability of the most essential supplies (3). Consider- majority of internationally traded poultry is derived. able resources have been devoted to preparing for a Sector 2 includes large-scale commercial producers pandemic. It is tenuous to attribute all of this prepa- with good biosecurity and the farmers under contract ration to H5N1 HPAI virus. If a human fl u pandemic to large companies, who raise birds from day-old occurs, it could originate from some other source. chicks (DOCs) and have feed supplied by the con- Equally, terrorist attacks might create conditions tractors. Contract farming represents an opportunity where travel is impossible and work disrupted. for new market entrants, requiring technical skill but Much of the expenditure on preparedness for disas- a lower level of investment than independent farming ter, however, would not have been made or planned because the contractor supplies many of the inputs. without the present threat of a human pandemic During the HPAI outbreaks of 2004–2005, contract originating from HPAI. farmers in Thailand, Vietnam, and Indonesia were

Table 24.3. Production and international trade of poultry meat 1991–2006 in million tons. Year 1991 1993 1995 1997 1999 2001 2002 2003 2005 2006

Production 37.8 47.3 54.5 59.8 65.2 70.1 74.3 76.5 81.9 80.5 Trade 2.1 2.7 4.9 5.9 6.9 7.8 7.8 8.2 8.1 7.8 Source: Morgan, 2006, from FAO data. 542 Avian Influenza

Figure 24.1. Poultry system continuum in 2006 with country examples. (Adapted from World Bank et al., 2006.)

buffered from fi nancial loss by their contractors (30, tribute to cash fl ow and from minimal investment; 50). Sector 3 consists of commercial units of a small they can produce a return of up to 600% (43). They to medium scale, where poultry are confi ned and fed contribute directly to household nutrition and to but biosecurity investment is low. This is a highly social capital, because they are exchanged as gifts diverse sector. In developed countries, some of the and eaten on social occasions (35, 48) and their meat high-value niche market production such as organic is often preferred to that of commercial broilers. and free range might be considered to fall into this They play a part in farm and household ecology by group, as might specialist producers of rare breeds eating snails and insects (4, 25) and are used in who keep them in free-range systems. In developing social and religious ritual (a function where chickens countries sector 3 consists chiefl y of small-scale are considered irreplaceable in parts of Southeast commercial units with limited investment in facili- Asia). A number of endangered breeds are kept ties, rapid turnover, and a growing market. Their within this system. Millions of smallholders keep numbers are not great, typically around 10% of the sector 4 fl ocks but most are not recorded in formal poultry population in a country where the poultry registration systems. From available data and esti- sector is growing, but they represent a transition mates, they appear to represent between 10% and route out of poverty and a way of meeting a growing 99% of birds and producers in different countries. demand for poultry meat. Sector 3 includes large Sectors 1 and 2 predominate in industrialized fl ocks of herded ducks in delta areas of Vietnam, nations, while developing nations, even those with China, Thailand, and Bangladesh that graze on the strong commercial sectors, still have predominantly crop residues and snails of paddy rice systems (4, small fl ocks. Figure 24.1 shows the different situa- 10). Until the rise of HPAI, they represented a secure tions that countries face. Those with the highest form of income that was tightly embedded in the diversity of systems have the greatest challenge in farming systems of these specialized ecological controlling disease in a way that is both effi cient and regions. As “silent carriers” of AI, ducks are the equitable. center of a debate about the future of certain poultry- keeping systems. A very specialist group that may Market Chains be included in sector 3 are the fi ghting cocks, banned Feed, vaccines, eggs, DOCs, poultry meat, and in most countries but still popular and highly valu- feathers are traded through international market able and representing a unique part of the gene pool. chains, so that an outbreak of a poultry disease in Sector 4 includes the backyard, scavenging system, one country can have economic impacts in several where birds may be housed at night or not at all, and others. The main international market chains are small urban fl ocks kept in houses in the towns and concentrated and integrated, while domestic market cities of developing countries. Sector 4 fl ocks are chains in developing countries involve many par- small and productivity levels are low, but they con- ticipants and a variety of contractual relationships. 24 / The Economics of Avian Influenza 543

Live bird markets form an important part of this both cases, DOCs from a single source lead into system, and sell a wealth of species and products, various products and market channels. In Thailand, brought in by numerous traders and producers. three types of farms raise the meat birds: those owned The length and international reach (in the case of by the company that produces the chicks, farms con- sector 1) and complexity (for sectors 2 and 3) of tracted to the breeding company, and independent poultry market chains make it important to consider farmers, often on a small scale. In Egypt, much of the the whole chain when identifying risks and assessing production of eggs and spent hens lies within inte- beneﬁ ts and costs. Figure 24.2 shows the intercon- grated systems (those owned by a single company), nectedness of the formal and informal sectors for which are an important source of products for infor- broiler chickens in Thailand and layers in Egypt. In mal markets supplying domestic consumers.

THAILAND Breeding company MEAT <20%

Day Old Chicks Individual farmer Farm owned by Contract farmer breeding company

Finished chickens Small scale Slaughterhouse owned by public and breeding company illegal slaughtering 45% 55%

Export Processing Wholesaler company

Supermarket. May be owned by Local retailer - wet breeding company market

EGYPT Parent stock producers LAYERS

Hatcheries Day Old Chicks, Day Old Chicks fertileeggs Commercial layers

Spent hens Table eggs

Live bird markets Informal markets Formal markets & Export Upper Nile large cities food processors

Small scale Export producers Domestic Consumers

Figure 24.2. Poultry meat market chains in Thailand, 2003, and layer market chains in Egypt, 2006. Boxes in gray show integrated systems. (Adapted from Costales et al., 2006, and Rushton, 2006.) 544 Avian Influenza

Market chains not only have a functional form, as HPAI—notably, the United States and Brazil, which shown here, but they also exist in geographical between them supply almost 70% of the world space. Where human and poultry populations are export trade. Brazil was able to expand poultry dense, different market chains tend to be physically meat exports but suffered from lower international close. Live bird markets and small slaughter points prices for DOCs. Estimates made by FAO suggest are important interaction points for different chains. that an extensive outbreak of HPAI in the European The same participants provide feed, veterinary ser- Union would cause international price rises in vices, and transport to more than one chain and poultry and substitute meats and a fall in poultry create physical contacts between them. meat prices in Europe (39). Fortunately, this has not happened. MARKET SHOCKS International market chains are not restricted to formal systems but fl ow informally across the International Markets borders of neighboring countries in the Mekong Outbreaks of HPAI have occurred in the context of Delta, Southern Asia, Africa, and the Middle East. an already volatile international poultry market, This means that both disease and market shocks adding a new source of volatility. Effects have been have the potential to cross borders. Nepal suffered a substantial in terms of shifts in prices, volumes, and fall in local demand when India fi rst had HPAI, and location, driven by consumption drops. a price drop for live birds to 52% of the former level Restrictions on exports from Asian countries was reported informally from Mauritania after an affected by HPAI outbreaks in 2004 and the fi rst half outbreak in Nigeria. of 2005 contributed to a nearly 20% increase in international poultry prices over the period. Con- Domestic Market Shocks sumers switched to other protein sources, and export HPAI has caused shocks to domestic markets in of live birds and chilled meat from major Asian most countries that have suffered outbreaks and producers, particularly Thailand and China, was some that have not. No domestic market shock has banned. At the same time as international prices been recorded to date associated with LPAI. Typi- rose, domestic prices fell in the infected countries cally, demand for poultry products falls when an because of reduced domestic demand and the release outbreak fi rst occurs, with a resulting fall in price. of products intended for export onto their domestic This seems to be exaggerated by dramatic announce- markets (39). Asian poultry populations fell because ments of outbreaks by the media or governments of culling, and between 2003 and 2004 there was an coupled with limited information on appropriate 8% fall in the volume of global poultry trade. Global risk-avoiding behavior, although the extent to which trade bounced back in 2005 and rose again in 2006, communication promotes or may mitigate market although to a lower level than predicted before shocks has not been fully analyzed. There is usually HPAI. panic selling of birds, especially those suspected of The location of exports has moved toward South being sick, and there may be panic culling by bird America. In 2003, Asia had 21% of the world export owners. Sales together with offi cial and unoffi cial market, Europe had 16%, and South America had culling lead to a fall in the poultry population. It may 27%. By 2005 the fi gures had shifted to 13%, 10%, take weeks before restocking is permitted, and even and 40%, respectively (FAO fi gures). Thailand lost when it is allowed, establishing the sources of supply approximately 50% of its export trade in 2004, and may be delayed. If consumer confi dence is restored, the Netherlands, which suffered an outbreak of market prices rise again, sometimes to above preout- H7N7 AI in 2003, lost about 20% of its export break levels. market and saw the bankruptcy of its largest pro- Two examples of market shock are shown in ducer (26). LPAI has also played a part in market Tables 24.4 and 24.5, for meat and egg prices in shifts; for example, in August 2006 an H7 LPAI Egypt during the outbreak in early 2006 and egg strain was detected at a poultry farm in the Nether- prices in Cambodia during an outbreak early in lands, resulting in import bans from Taiwan and 2004. Cambodian chicken meat prices followed the Hong Kong. The other major players on the interna- same pattern as eggs, but duck meat prices were not tional market have suffered less impact from affected. 24 / The Economics of Avian Influenza 545

Table 24.4. Prices (Egyptian Pounds) of fall in prices. Although prices swung sharply upward chicks, broilers, and table eggs in Egypt to above their previous level when markets returned through an HPAI outbreak. to normal, poultry populations and hence sales were Broiler Broiler/kg Table egg/ lower than before (13). In Indonesia’s outbreaks of Month/year chick live carton 2004–2005, there were reports (8) of a fall of 45% to 60% in the demand for DOCs and feed inputs Oct 05 1.5 5.9 3 during the outbreak and a reduction of just over a Nov 05 1 4.6 3 third in the employment in the poultry industry. In Dec 05 0.5 4.7 3 France, there were job losses among casual workers Jan 06 0.4 3.7 3 when an HPAI outbreak in 2006 resulted in trade losses and market restrictions, although the majority Feb 06 0.25 2.9 2 of them would have been able to obtain social secu- Mar 06 0.25 3 3 rity, a way of sharing risk across society (42). Apr 06 0.8 4.85 4 From a consumer perspective, this is a complex May 06 1.5 6 6 situation to analyze. Consumer decisions to stop Jun 06 2 8.4 10 eating poultry have a profound effect on markets. In Jul 06 3 7.4 12 their turn, consumers may experience changes in Source: Shalaby, 2006. diet and effects on the household food budget as prices of poultry and prices and availability of substitute proteins shift. It is not clear to what extent Table 24.5. Market prices (Cambodian Riels) different factors affect consumer perception of risk. of eggs in Cambodia through an HPAI A study in Vietnam suggested that older and younger, outbreak. rural and urban, populations had different risk per- Month/year Chicken eggs Duck eggs ceptions (24). In the European Union, countries that suffered a similar level of risk showed different con- Mar 03–Feb 04 193 275 sumer behavior (for example, the drop in demand in Mar 04 135 223 Italy was much greater than in the United Kingdom). Apr 04 135 207 In Thailand, which has seen three waves of out- May 04 220 303 breaks since 2003, progressive communication and Jun 04 240 320 quality control combined with a falling human case- Jul 04 245 330 load gradually minimized market shocks. The fi rst Aug–Sep 04 245 338 wave was accompanied by a 37% fall in demand on the domestic market (14), but various efforts by the Source: VSF, 2004. private sector to establish and communicate about food safety, and perhaps the recognition by consum- Producers, traders, retailers, and those employed ers that no cases had been associated with eating in the poultry industry are affected by loss of sales well-cooked meat, meant that by the third wave the and market restrictions as well as fall in prices. In domestic market shock was minimal. Cambodia, market retailers realized slightly lower Substitution effects have been seen for alternative margins during the 6 months during and immedi- proteins. In Cambodia, when the prices of chicken ately after the HPAI outbreak of 2004 than they had meat and chicken and duck eggs fell during an HPAI in the corresponding months of the previous years, outbreak in 2004, the local prices of pork, beef, and with individual impacts ranging from no change to fi sh all rose and stayed slightly higher than normal a 64% decrease (56). In Turkey, egg production even after poultry prices had increased (56). In from the formal sector appears to have dropped by Vietnam, the price of pork rose from 15,000 VND/ about 15% during the 2006 outbreak, and a stock of kg (approximately US$1) to VND 24,000 in Novem- 1 billion eggs (10% of annual production) was even- ber/December 2005 at the lowest point in the poultry tually liquefi ed (25). In Vietnam, traders and retail- market (1). Other diseases also affect the prices of ers were affected by heavy restrictions on the substitute proteins, such as foot and mouth disease movement of live birds that reduced sales and by a (FMD) and bovine spongiform encephalopathy— 546 Avian Influenza both contributed to low world prices for beef between seem to be quite brief. More serious effects on 2001 and 2004 (41). markets may result from longer-term disease pre- Direct food security impacts (reduced energy vention measures, and this is discussed later in the intake, protein, or micronutrients) do not seem to be chapter. a major effect of poultry market shocks. Indirect effects through loss of livelihoods are more of a FOOD AND LIVELIHOODS INSECURITY concern than direct effects, because they reduce the Livelihood insecurity and, in some cases, food inse- possibility to purchase alternative proteins and may curity may be caused by a market shock, by disease, continue for several weeks during an outbreak and or by disease prevention and control measures, in into the rehabilitation period. A market shock may particular, those associated with depopulation and result in these impacts being felt over a wide area. market restrictions. Losses of birds and markets and Poultry and eggs can be sold at short notice for a reduced prices all have effects on producers, while rapid source of cash in small quantities, to buy food consumers may suffer from lack of food products or and other daily household needs, and poor house- raised prices. holds are as likely to sell poultry products as they An AI outbreak would have to be extremely pro- are to eat them, particularly in urban and peri-urban longed and extensive and, occurring in a closed areas (31). The questions of food security and liveli- market economy, to cause a national food security hoods are examined in more detail in the next problem. It is extremely unlikely that it would section. directly cause national food insecurity (that is, by It has been suggested (54) that different types of reducing the food available to eat) unless the country markets might be expected to adjust in different was already on the brink of a food crisis or a small ways to market shocks. In a closed economy, the island state that found it diffi cult to restock. relative strength of fall in demand and fall in supply However, household food security may be affected determines the fi nal impact, because there is no in the immediate area of an outbreak or in a wider opportunity to compensate for a fall in the poultry area impacted by market closure. Using conserva- population by importing. A net importing country tive estimates, 78 million people in Africa and 280 can use imports to buffer shifts in domestic supply million people in Asia are in food insecure house- and demand. For an exporting country, a ban on holds that keep poultry (31). In such households, exports may be damaging to the whole sector and loss of poultry through HPAI may create a food not only those fi rms involved in exports. A country security problem, mostly as an indirect effect result- where no outbreaks have occurred may still suffer ing from loss of livelihood (21). Poultry products demand shifts. While this analytical framework has generally constitute less than 1% of daily calorifi c not been fully tested, the impacts suggested can be intake in Africa and about 3% in Asia, between 5% seen to some degree in countries mentioned in this and 15% of protein consumption, and 20% to 50% chapter. In Cambodia, a largely closed economy that of meat consumption. They appear to be most experienced a limited outbreak, there was a classic important to the diets of the poor in the poorer coun- pattern of demand fall followed by price fall fol- tries of central Asia and the Middle East. The source lowed by limited depopulation and price rise when of livestock products consumed by poor households demand was restored. In Egypt, which had a ban on varies by area and income group. In the more remote poultry imports, the same pattern was experienced areas in Vietnam, for example, the proportion of but the import ban was later relaxed. In Thailand, a poultry consumed and used within the household is major exporter, the fall in demand for poultry prod- 91%, while it is only 9% in areas with good access ucts occurred at the same time as a ban on Thailand’s to markets (53). Regardless of location, in very poor exports and resulted in products produced for export households, people are more likely to sell livestock competing with those on the domestic market. products than to eat them. Brazil, Nepal, and Mauritania all suffered from Because of this variability in consumption habits, some degree of market disruption without experi- losses of poultry through disease or culling, or encing HPAI outbreaks. increases in the price of poultry products from In the cases where market shocks have been market shocks, have impacts that vary by location. studied in detail, the impacts, although dramatic, Smallholders in Turkey affected by HPAI outbreaks 24 / The Economics of Avian Influenza 547 mentioned that they had to buy eggs instead of pro- in the south, as a result of biosecurity measures ducing them to eat (25), but they regarded this as an imposed on markets, there was a concentration from inconvenience rather than a crisis. In studies of 134 wholesale markets selling fresh eggs to 75, and HPAI impacts conducted in fi ve Southeast Asian from 1300 small shops and 250 markets selling countries in 2005 (8, 14, 16, 30), direct loss of food chicken to 6 poultry “selling points” and 1 shop did not emerge as a concern to any of the farmers selling frozen poultry meat (1). questioned, but many reported income losses. Small- Thailand is estimated to have lost 29% of its total holder poultry development schemes have resulted chicken population in the fi rst wave outbreak, in increased consumption, improved nutrition, and including some 18 million native chickens, and increased income (15). Income from poultry is often more than 20% of the duck population (14). It was managed by women, and income controlled by particularly diffi cult for the duck farmers to return women often goes directly into child nutrition or to production as biosecurity regulations were intro- education. duced requiring them to register and invest in The studies of HPAI impact in Southeast Asia improved housing. Cambodia (56) and Laos (55) showed that impacts differed by sector. The total experienced outbreaks at about the same time but value of dead and culled birds was greatest for pro- with much more localized effects. In Cambodia, the ducers in sectors 1 and 2. Producers in sector 3, with main impacts seem to have come from the market smaller fl ocks, experienced a lower absolute value shocks previously described. However, the affected of loss, but it could be a large proportion of their households experienced a severe loss of assets: total asset and they were likely to be in debt if they between July 2003 and July 2004, the mean number lost a fl ock. Backyard poultry keepers in sector 4 of birds fell by 44% and the number of households lost the smallest number and value of birds, but they owning 0 to 10 birds rose from 5% to 25%. In two were the most likely to be excluded from offi cial provinces of Turkey, farmers ranked loss of poultry compensation schemes because birds died before eggs and meat for their direct consumption the most culling teams arrived or were not properly regis- serious impact of culling and loss of income from tered. Data for Vietnam suggest a pattern of effects poultry within the top three impacts (25). Loss of for a country that has been badly affected by HPAI companion animals and the stress of the culling and subsequently imposed strict control measures. operation were also mentioned by more than half of Fifty-eight of 64 provinces were affected in 2004– the farmers interviewed. In China, once a farm is 2005 (47). A survey of smallholders showed the depopulated, it is not supposed to restock for 6 average losses per farm affected by HPAI to be months (51), creating an income and food security between US$70 and US$108 and the loss per bird gap for poor families. at around US$2.70 including slaughter, disposal, Livelihood losses have also been experienced by and some down-time (13). Based on a poverty line others in the market chain: traders, DOC suppliers, of US$1 per person per day and a family size of fi ve, and those who work in the livestock industry. Unless this is equivalent to 2 to 3 weeks’ income for a very death or culling is suffi ciently widespread to cause poor family. Four months after an outbreak in 2005, serious depopulation, losses for those other than a survey found that 27% of backyard farmers and poultry producers tend to be an effect of market 19% of small commercial poultry keepers had not shocks or movement controls rather than direct restocked and were considering leaving the poultry effects of poultry deaths. sector (30). Sector 3 farmers who continued to keep birds fed them the minimum rations necessary to IMPACTS OF OUTBREAK STAMPING-OUT sustain them (1), and this resulted in a considerable MEASURES loss of feed sales. Poultry sales were banned in many Stamping-out measures for HPAI outbreaks are the markets of the Red River and Mekong Deltas (areas standard measures employed against any notifi able of the country where poultry production is most livestock disease, namely movement control, culling dense) from December 2005. Some traders remained with compensation, disinfection, and disposal of in the markets to deter others from occupying their carcasses. If this is successful in containing disease, space, while others traded in other commodities with no further measures are needed, but if disease mixed success. In Ho Chi Minh City, the largest city becomes widespread, vaccination may be used. 548 Avian Influenza

Table 24.6. Timeline for an HPAI outbreak in Turkey. Outbreak event Date Behavior

National television broadcast January 2006 Three children died of HPAI in eastern Turkey, causing instant panic countrywide Birds start dying in own ﬂ ock February 2006 Some panic sales or village Veterinarian called Same day Farmer reports to Mukhtar, who calls veterinarian Veterinarian collects samples Same day Lab test result 5 days later Announcement via Mosque loudspeakers Culling team arrives 1 day later Some not willing to hand over birds; people told what compensation they would get Compensation 1 month later Compensation paid to those who registered their name and tax number Source: Geerlings, 2006a.

Where LPAI is notifi able, similar measures would of animal health workers; messages to farmers via also be employed. television, radio, posters, and leafl ets; and commu- Table 24.6 shows the timeline for an outbreak in nity involvement. It is quite widely agreed that com- a Turkish community in 2006. It shows the progres- munication needs to be 10% or more of the total sion from the fi rst announcement of disease in the budget for control of a notifi able animal disease and country, through reporting of a suspected outbreak, involves messaging, negotiation at all levels, and laboratory confi rmation, culling, and compensation. advocacy backed up by a sound disease control strat- The potential impact of each element of stamping- egy that is seen to deliver results (57). The greatest out are discussed in the sections that follow. investment to date has been in broadcast or printed messages, usually slanted toward protection of Reporting and Confi rmation people. The cost of mass communication depends Timing is critical in outbreak control as every delay on the concentration of poultry keepers and the increases the chance that disease will spread and all methods by which messages must be broadcast. For of the associated control costs will increase, although instance, mass media campaigns in Vietnam have an outbreak in an isolated rural fl ock may die out cost US$1 million a year (33), but where communi- without veterinary intervention while one in a cation can be done through industry groups, private dynamic marketing system will spread rapidly. The veterinarians, and the Internet, the cost should be situation shown in Table 24.6 is commendable, with less. Table 24.6 shows the importance of religious the veterinary services notifi ed on the same day that channels and other community institutions where a the fi rst suspect case was noticed and the test result communication infrastructure already exists. available fi ve days later. This is symptomatic of a country that had recently experienced a human death Compulsory Disease Reporting, Legislative and where good laboratory facilities existed. Back-Up, Enforcement Capacity, and Ingredients of a good reporting system include the Incentives to Report (33) following. A report on the Australian system of fi nancing disease control (2) highlighted the need for both Farmer Awareness of Disease Reporting “carrot” (compensation) and “stick” (penalties for Pathways (33) nonreporting including withholding of compensa- Efforts have been made in various countries to tion) approaches to improve reporting. Finding an improve reporting, using a combination of funding effective balance of carrot and stick is not easy. It to veterinary services for surveillance; engagement often requires legislation change and improved 24 / The Economics of Avian Influenza 549 enforcement capacity, which may not be costly but staff, and these were expanded during 2006 in may be extremely time consuming. Compensation, response to HPAI. The cost of setting up a network which is discussed in more detail later, requires in one district and training staff was estimated at good management procedures and can also be around US$3000 with an additional US$1200 a year expensive. There may also be a need to invest in to run it (data from project fi nancing estimates of the infrastructure to improve the ability to spot suspect World Bank Avian Infl uenza Emergency Recovery cases. In Hong Kong, improvement in biosecurity Project). A review of classic swine fever control measures in live bird markets, including reporting of (40) estimated that the cost of contracting CAHWs suspect cases, has required long negotiation with to do surveillance work supervised by district vet- market stakeholders, provision of better stalls and erinary staff would be between US$3000 and hygiene facilities, and severe penalties for those who US$4600 per district per year, excluding any addi- do not comply with regulations. tional costs for training or setting up a communications network. In Indonesia, a pilot system has been An Effective Animal Health Information set up for participatory surveillance and reporting at System (33) district level, but it is too soon to assess what the An animal health information system may be con- costs will be for expanding and maintaining it. sidered to be effective if disease information going into it is timely and accurate and decisions made on Competent Laboratory Staff in Properly the basis of the information improve disease control. Equipped Laboratories (33) For a country engaged in international trade, an Laboratory testing for precise identifi cation of HPAI effective information system will improve trade virus strains has been beyond the capability of many prospects by inspiring confi dence in trading part- affected countries, leaving them with the option to ners. Not only the hardware and computer software send samples to approved laboratories for testing or, but also the human aspects need to be considered. if the need for testing was likely to be prolonged, to In Vietnam, the animal health information system is upgrade laboratory facilities and train staff in new being upgraded as part of the effort to control HPAI, tests. The cost of building or upgrading a laboratory at an estimated cost of US$340,000 to upgrade lab- ranges from $500,000 to $50 million, but cannot all oratory and province level systems for two thirds be attributed to HPAI control since the laboratory of the provinces (33). The human element requires can also be used to test for other diseases. The costs competent fi eld-based personnel and good links of virus isolation or real-time reverse transcriptase– between farmers, private animal health staff, and polymerase chain reaction (RRT-PCR) tests are government veterinary staff. The information needs approximately $10 to $20 per sample (51). In Thai- of each stakeholder in the system and their some- land, where detailed house-to-house surveys are times complex relationships with each other need to conducted as part of an active surveillance program, be understood and catered to (17). Regardless of the laboratory diagnostic testing associated with legislation, reporting of initial suspicion is more each survey is reported to cost approximately $1 likely to occur between individuals who know and million (33). In Hong Kong, surveillance and moni- trust each other and who have at least a basic under- toring costs are approximately $US0.12 per bird standing of the problem. In sectors 1 and 2 and for sold (51). most farmers in developed countries, the most trusted contact is likely to be the farmer’s regular Movement Control private veterinarian. For sectors 3 and 4 in develop- In the Turkish outbreak described in Table 24.6, ing countries, the link between farmers and the state there was panic selling of birds before control mea- veterinary service is through para-vets and commu- sures were put into place. This is a common occur- nity leaders (16). rence and birds may continue to be moved after a In Vietnam, efforts have been made over several movement ban has been imposed. OIE recommends years to strengthen the community animal health that movement control should be put into place at worker (CAHW) networks. Pilot initiatives have the earliest suspicion (58). Table 24.6 does not used regular meetings and training to promote links mention movement controls, but this may have been between CAHWs, farmers, and district veterinary because the farmers interviewed were smallholders 550 Avian Influenza consuming poultry products at home or selling only changed to pig production, while others reduced into local markets. Control measures may include their agricultural activities. Sector 1 producers have the requirement for movement permits, closure of been the most adaptable to movement restrictions. live animal markets, and even quarantining of They have attempted to sell packaged meat to super- animals on farm in infected areas. Costs associated markets and have opened up their own selling points with movement control include the costs of impos- for poultry products (1). ing it and the cost of lost sales and animals kept beyond their normal production cycle. In a country Culling and Compensation with predominantly sector 1, few units are involved, Culling of birds normally extends to those that are it is easy to monitor them, and the penalties for infected and in close contact, but “close contact” noncompliance can be made very severe. The main may cover as much as a 3-km ring around the fi rst costs involved are those of communication, inspec- infected premises. Table 24.6 shows a typical situation of vehicles, and issuing of movement permits. tion where the culling is carried out by a government In countries with many sector 3 and 4 premises, it team announced shortly in advance. In this case, is almost impossible to impose control as birds are although farmers were given information that there moved by foot, bicycle, taxi, and van and carried in would be compensation, some were still reluctant to plastic bags and small crates. The veterinary service present birds. Women and children in particular often does not have the power to stop and search; were very distressed. this must be done by the police. No comprehensive Economic impacts of culling include the estimates of movement control costs for HPAI were following. available at the time of writing. In Vietnam, the cost to establish and run movement control units for an Costs of Carrying Out the Culling, Disposal outbreak of classic swine fever has been estimated and Disinfection at between US$330 and US$3000 per province (A. Culling and disposal are normally carried out by the McLeod, data). government, and the cost should always be borne by Costs of lost sales follow a similar pattern to those the government. Costs of disinfection should also be from a market shock, but when markets are closed borne by the government. Because disinfection takes or animal movements are restricted, there may be place after the farm has been free of poultry for a additional costs of maintaining market space against period, it may be carried out by the farmer who competitors or feeding animals beyond their normal should then be recompensed for time and materials. production cycle. In Hong Kong in 2001, all live Costs depend on the production system and the poultry markets were closed for 1 month. As Hong process used, with some economies of scale and Kong had no dedicated slaughter plants for chick- experience. With HPAI there is a need for protective ens, the outlets for poultry were lost. The govern- clothing to prevent people from being infected. In ment culled market weight birds on farms and made Vietnam, it was estimated to cost about US$0.25 per a payment to farmers for the birds (51). In Vietnam, bird to destroy and dispose of 200 chickens per farm, the cities of Ho Chi Minh City and Hanoi closed live and the process of assembling teams has become bird markets within the city and restricted entry of very streamlined. In Nigeria, teams were organized birds from other provinces. Birds could only be sold on an ad hoc basis and the costs are estimated at from approved slaughterhouses. Before the AI out- about US$1 per bird if the team culled 1000 birds breaks, sector 4 farmers typically sold birds and within a day (33). eggs at the local market or to neighbors or assemblers at the farm gate. Birds sold to assemblers were Loss of Production taken to wholesalers or consumers (1). After the AI Approximately 250 million birds are estimated to outbreaks, the birds could no longer be taken beyond have died or been culled because of H5N1 HPAI. their communes and local markets. Sector 3 farmers An average market value for an adult chicken just had a larger range of marketing channels, some of before an outbreak might be around $5, giving a which are now closed (1). They could previously very rough estimate of US$1.25 billion, but this does have supplied supermarkets, but this is no longer not take into account the full production value lost possible owing to new regulations. Some farmers before restocking or the much higher value of parent 24 / The Economics of Avian Influenza 551 stock, geese, turkeys, and fi ghting cocks. The inten- million birds (46). In November 2005, the compen- tion of culling is that, while more birds may die in sation rate was raised to just under US$1 per bird the short term, less will do so in the longer term. If culled, representing about 50% of market value. Had compensation is provided for culled birds, the loss this been used from the commencement of the out- is shared between the government and producers; break, it would have resulted in a compensation cost otherwise, it is entirely borne by producers. Losses of around US$40 million. A variety of rates have to others in the market chain are not compensated been set and paid for compensation in HPAI out- under offi cial schemes for control of any animal breaks (58), with fl at rates from $1 to $10 for a disease in any country. Psychological distress for chicken and more for geese and turkeys, or percent- farmers and cullers is often a side effect. Farmers ages of market value ranging from 20% to over are never paid in full and often not at all for the loss 100%. of income while they wait to restock. Where the time The compensation scheme needs of sectors 1 and to restocking is long because of government regula- 2 differ from those of sectors 3 and 4 (58), and this tions, as in China, or through the seasonality of has implications for costs and cost sharing. For outbreaks, as in Turkey, the effect of “down-time” example, in sectors 1 and 2 the emphasis is on may be quite severe. An unusual positive effect was precise valuation of birds, meaning that the costs of experienced in 1998 after poultry farms in Hong a valuation process need to be considered. Contract Kong had been depopulated and cleaned in 1997, farmers may need to be compensated although they when farmers reported that the fi rst batches of may not legally be the owners of birds, because they poultry grew much faster than those before the out- have faced production costs. In sectors 3 and 4, break (51). while the value paid for birds should not be too low, Compensation is a transfer payment between the speed of payment is more important and it may be government and farmers. The value of dead birds acceptable to use a blanket valuation on broad cat- does not change if compensation is provided for egories of birds provided that compensation is paid them; the loss is simply borne by a different stake- quickly. There is need to disperse money widely, holder. However, a well-designed compensation sometimes in cash, and this carries administrative scheme should improve compliance with culling overheads. Sectors 1 and 2 are most interested in requirements and may encourage disease reporting, rapid disease control to restore markets and may be ultimately reducing the spread of disease and overall willing to endure severe culling to achieve this. loss. No estimates have yet been made of the impact Sector 3 is concerned about markets but may not see of compensation on disease reporting or spread in the need for severe culling, and debt may become the countries affected by H5N1 HPAI. Many of widespread unless compensation takes place quickly. them had little experience with compensation There can also be diffi culties with restocking, which schemes for animal disease. Moreover, those that is discussed later. With sector 4, one of the biggest have experienced widespread or persistent problems challenges is to include farmers in offi cial culling (such as Vietnam, China, Indonesia, Egypt, and schemes so that they can be compensated, because Nigeria) are mainly countries that fall into the central compensation has rarely been paid for dead birds. area of Figure 24.1, with high diversity in their Systems of registration and disbursement need to be poultry systems, while the countries with most expe- planned with consideration for country and local rience of compensation are those with a predomi- conditions; there are no one-size-fi ts-all solutions. nance of sector 1. Cost estimates to date report the In terms of funding, countries where sectors 1 and amounts paid to farmers but not the administrative 2 are strong tend to be those with cost sharing, using overheads of schemes. In Thailand as of March public and private sector payments into a joint 2004, farmers had been compensated for about 61 animal health fund from which payments can be million heads of poultry (16), resulting in the made for compensation as well as other animal payment of US$46.5 million. During 2004 and 2005 health expenses. These funds are associated with in Vietnam, compensation rates differed between strict regulations on biosecurity. In theory, producer provinces and the effective cost share of the govern- groups in sector 3 might also contribute to such ment was only about 20% to 30% of the market funds but there are no examples of good practice on value, with a cost of US$18.5 million for 41.3 which to draw. For sector 4, compensation will 552 Avian Influenza always need to be funded by the government, and nation together with other control measures, and this for reasons of effi ciency ideally from central or ear- presumably had a positive effect on tourism. How- marked funds. ever, Thailand and Malaysia, two countries that Culling in a reduced area with ring vaccination in were concerned about impacts on tourism, chose not a wider area is a possibility, provided that the vac- to vaccinate. cination is administered safely and effectively. In Vaccination that reduces culling and poultry the areas of China with median poultry density, deaths may also be a valid strategy for preserving using ring vaccination in a 5-km zone with limited biodiversity if endangered species kept in sector 4 culling to stamp out an HPAI outbreak, instead of fl ocks, or valuable genetic stock in grandparent culling in a 3-km zone, has the potential to save the fl ocks can be vaccinated and excluded from destruction of poultry valued at approximately culling. $84,000 for meat birds of moderate value, from an Vaccination does not remove the impacts of investment of about $14,000 (51). Ring vaccination market shocks or movement control, and in some costs may be shared between the government and cases, farmers may be better off having their fl ocks farmers or borne fully by the government. culled and receiving rapid compensation than having them vaccinated and being unable to sell them. Preventive Vaccination Countries that have experienced widespread and Will Vaccination Have an Adverse Effect repeated outbreaks of HPAI have developed long- on International Trade? term control strategies that minimize culling. For a country with predominantly sector 1 produc- Vietnam, Indonesia, China, and Hong Kong have ers, the preferred option is to try to stamp out without introduced wide-scale vaccination against H5N1 vaccination, but as the Netherlands and Canada have with varying levels of coverage and success. Thai- discovered, the costs can be very high. Thailand was land has modifi ed its approach to stamping-out and in the top fi ve exporters of poultry meat before surveillance but does not use vaccination (2). HPAI. It chose not to use vaccination, but to attempt A simple framework (38) can be used to assess eradication by other measures. Until now eradica- whether it is appropriate to include vaccination in an tion has not been achieved and there has been a shift AI control strategy. toward processed products for export. France and the Netherlands have vaccinated parts of their Is Vaccination Technically Viable? poultry stock as a preventive measure (33). Existing vaccines against H5N1 HPAI give good protection in chickens and ducks, although there is Is There an Ensured Source of Funding for a continual need to be vigilant for emerging new Continued Vaccination? strains and to improve the formulation of vaccines. An advantage of preventive vaccination over emergency activities is that many of the associated costs Does Vaccination Benefi t Livelihoods of can be planned in advance. However, a vaccination Vulnerable People? program once begun may need to continue for 3 There is likely to be a positive effect of vaccination years or longer. Investment may be needed in staff if it minimizes disease spread and minimizes depop- training for fi eld operations, improvements to labo- ulation, particularly in situations where adequate ratory facilities and establishment, or upgrading of compensation is not available. For maximum impact, cold chains. Recurrent costs must cover mass cam- it needs to be accompanied by clear signals that paigns or the costs of making vaccine continuously restocking is welcomed, reopening of markets, and available to sector 1 and 2 farmers. They also need assurance to consumers that vaccinated birds are to cover the cost of serosurveys to monitor effective- safe to eat. There will also be a positive effect on ness. Reported costs of vaccination tend to empha- livelihoods if vaccination minimizes the disruption size the campaigns and ignore the costs of of other sectors linked to livestock, such as leisure monitoring and quality control. Recent estimates and tourism, but this effect has not been estimated suggest that campaign costs of equipment, vaccine, for HPAI. In Vietnam, the cases of human deaths vaccinators and communication constitute 65% to have dropped since the introduction of mass vacci- 85% of the total for a 3-year program, with the 24 / The Economics of Avian Influenza 553 remainder spent on upgrading of facilities and mon- include the fi nancing and management of restock- itoring (A. McLeod, J. Hinrichs, and A. Riviere Cin- ing, and the potential to invest in biosecurity namond, unpublished data). To provide a high level improvements before restocking takes place. Small- of protection in backyard chickens with existing holders fi nd it harder than large commercial farmers vaccines may require four to six campaigns a year, to restock and to adopt good biosecurity measures which is clearly impractical with campaigns, even (20). in densely populated Asian systems. In a well- planned program, wide-scale vaccination will give Restocking way to targeted campaigns in high-risk areas. While Two considerations in restocking are fi nance and the this is likely to yield better economic returns, to be source of the birds. For sector 1 through 3 farmers, effective it may require a vaccine stockpile, with fi nance is either internally generated or provided by associated maintenance costs. credit, and birds are sourced from commercial producers of DOCs. After a severe outbreak, if breeder Can Vaccination be Delivered in a Cost- farms have been culled it takes time to reestablish a Effective Manner that Takes Account of production cycle. Normally cycles are staggered so the Production Systems in which It is that there is a regular fl ow of chicks. When breeder Applied? farms are destocked, the production of eggs and In sectors 1 and 2 and large sector 3 farms, the most DOCs needs to be restarted in a staggered fashion cost-effective system is for the farmer to make and this means that it takes time to build up to full arrangements and pay for the vaccine to be admin- fl ow. In Canada, contingency planning for HPAI istered by the farm’s usual veterinarian and farm stamping-out had to take into account that there workers. Even medium-sized farmers will take the would be delays in providing DOCs to some farmers, initiative to do this if vaccine of reasonable quality and this might justify higher compensation rates for is available (50). In smaller sector 3 chicken farms, farmers experiencing longer down-time (7). sector 4 chicken fl ocks, and free-ranging duck For sector 4 fl ocks, birds for restocking are tradi- systems, vaccine will need to be funded by the gov- tionally sourced locally. Farmers may split their ernment and probably delivered through offi cial fl ocks and keep part of the fl ock with a relative to campaigns, employing animal health workers when avoid risk, or obtain birds for restocking from within they are available. If birds can be housed before the their own village. If widespread culling takes place vaccination team arrives, the time taken and cost are and all of their traditional sources are culled out, considerably reduced. there may be a delay in fi nding birds of indigenous The cost per dose of vaccine delivered in Vietnam breeds with which to replace them. In Indonesia, at US$0.05 to US$0.06 cents differs signifi cantly native chickens are found in all provinces and were from those estimated for Nigeria at between US$0.14 estimated to be kept by 30 million households in and US$0.38 (33). In Vietnam, the vaccination is 2005. In Vietnam, over 90% of the poultry-keeping done on a contract basis with teams travelling by households fall into sector 4 (8 to 9 million), and a motorcycle and paid per bird vaccinated. In Nigeria, study in 2005 found that 60% of village and back- there are a limited number of trained vaccinators yard chicken farms sourced their chicks within the available and villages are a long distance apart. village. In Thailand, about 36% of the chicken population is indigenous (15). POST OUTBREAK REHABILITATION Sector 3 farmers are likely to rely on credit to Even during contingency planning for outbreaks, maintain the poultry production cycle and will expe- there is value in thinking beyond outbreak control rience fi nancial problems particularly if birds die to the rehabilitation process because it may affect close to the end of the cycle when much has been the decisions made during outbreak stamping-out. invested in feed. In Vietnam in 2004–2005, the gov- For example, providing compensation in kind rather ernment encouraged banks to extend credit periods than cash or linking it to restocking would signal an so that farmers could restart production. In Indone- intention to promote restocking and would also sia, many small- and medium-scale farmers had mean that payment of compensation is delayed. Eco- problems in paying or rescheduling credit (32). nomic considerations during the rehabilitation phase Some became contract farmers in order to obtain the 554 Avian Influenza inputs to restart production. In Lombok Island of In sector 3, improving biosecurity in duck systems Indonesia, contracted broiler growers were forced to might require a complete change of management destock for a month during the 2003–2004 outbreaks system, if free-ranging ducks herded in rice fi elds when contracts were suspended but were paid for are required to be enclosed or indoor housed. Not destocked birds at the contract price set by the con- only would this entail investment and increased tractors prior to the outbreak (50). recurrent costs, but also it could result in loss of crop In Turkey, after HPAI in 2006, many people yields or the need to introduce pesticides. Continued whose birds were culled did incomplete restocking vaccination might be preferred to making such for several reasons: fear of contracting HPAI, or that drastic changes. Sector 3 chicken systems are likely the government would cull their birds again; because to need improved night houses with netting (under they did not know that restocking was permitted; or $200 in Vietnam for a small unit), more regular because they normally partly destock during the cleaning, disposable or washable footwear, and winter and prefer to restock after the wild bird exclusion of visitors. There will undoubtedly be migratory season (25). economies of scale and FAO and others are working on estimates of minimum viable sizes for biosecure Increasing Biosecurity units. In developed countries, niche markets such as When the poultry sector is being rehabilitated after organic and free-range systems may benefi t from a serious outbreak, farmers and governments are specialist approaches based on knowledge of sea- likely to think about upgrading their biosecurity. sonal risk from wild birds, because the main concern Ideally they would do it in advance as a preventive in these systems is not the cost of extra biosecurity measure, but this seldom happens, except in sector measures but rather the potential loss of premium 1, where continuous improvements to systems are markets if birds can no longer be kept outside. In necessary to keep pace with international food safety sector 3, improved biosecurity is most likely to be requirements. Biosecurity and hygiene improve- achieved if it is linked to progressive registration ments may be needed in feed supplies, on farms, in and inspection of farms. In Hong Kong, there has transport systems, at live bird markets, at slaughter- been progressive tightening of biosecurity require- houses, and at meat retailers. ments for farms, including wild bird proofi ng, con- On farms, different approaches are needed for struction of disinfectant baths to clean equipment, poultry going into long market chains rather than and transportation and other measures. In a recent local sale. Sectors 1 and 2 see investments in bios- offer to farmers wanting to leave the industry, a sum ecurity as part of normal business practice, and the of about $US19,300 for a farm of 10,000 to 20,000 potential loss of markets is viewed as a much greater caged meat birds was provided to compensate for concern than the cost of upgrading biosecurity. Gov- the improvements made by farmers (51). ernment-run farms do not face the same commercial In sector 4, rural and urban fl ocks may need dif- imperatives; in Vietnam, it has been necessary to ferent approaches. Rural fl ocks may need commu- make major biosecurity improvements to 12 govern- nity as well as individual household measures, ment breeding farms with costs per farm of $10,300 because investing in confi nement of individual to $75,500 for construction, $12,750 to $41,875 for fl ocks is not likely to be economically viable. equipment, and about $1500 for training (51). In In all cases, there is a need to fi ne-tune the advice Lombok in Indonesia, a contract farmer needing to and training given to farmers and their advisors with upgrade biosecurity to meet contractor requirements training. One survey (50) found that adoption of might spend $3000 in 2002 fi gures for a 2500-head biosecurity measures by farmers could be affected broiler house meeting the requirements of the con- by industry structure, whether farmers were contracting fi rm (50). To be a contract farmer to one tracted to agribusiness fi rms or operating indepen- integrator in Vietnam, a poultry keeper must agree dently, type of products, and the stage of development to construct a coop on the farm; build a good road of the poultry industry. For instance, a contracted so that trucks can access the farm year-round; grower might adopt biosecurity measures when provide the birds with clean water and light; and required to by a contractor, while an independent raise chickens, ducks, and pigs apart from the family farmer might choose to diversify away from poultry residence (1). rather than make investments in biosecurity. 24 / The Economics of Avian Influenza 555

Governments face a dilemma about whether they cities is an example of this and so is banning poultry should ban live bird markets or insist on better keeping in cities. There was a shift of poultry pro- hygiene. These markets provide a livelihood to duction away from Bangkok between 1992 and many people, and freshly killed meat from previ- 2000, with densities reducing in the 50 km away ously inspected birds is preferred by many consum- from the city, encouraged by tax incentives (27). A ers. There is potential to make a fi nancially viable consequence of moving slaughtering facilities away investment in upgrading markets as demonstrated by from cities is likely to be a larger number of super- the experience of the Marikina market in the Philip- market outlets in cities. The impact of re-siting can pines. By engaging stakeholders, exploring various be negative (less accessible to poor consumers and possible sources of fi nance, and introducing changes small traders) or positive (creating employment in progressively (23), the Marikina market went from rural areas, moving smells and water contamination a state of “total chaos” to one with a mission to away from human residence). “provide consumers and vendors best quality of Poultry production zones are included in the long- service focusing on cleanliness, security, discipline term planning of Malaysia and Vietnam. Zoning and orderliness” (5). The cost of relocating and limits poultry production to specifi ed areas. Infra- rebuilding the market was US$1.3 million. Opera- structure can be concentrated in these areas so that tional costs of providing improved services by 2006 production becomes more cost-effi cient, and were US$303,000 a year. but the market was gener- hygienic premises can be built to house, slaughter, ating a total of US$466,000 from stall rentals, penal- and process birds. The cost of these developments, ties, certifi cates, and inspections of which US$32,600 however, will include loss of livelihoods for came from chicken sellers (5). Although stall rental small-scale producers who are unable to meet the is US$39 a month, stalls are never vacant and a con ditions needed to participate and potential envi- renter can make up to US$300 a month income ronmental externalities of concentrating livestock (5). production and processing in small geographical spaces. FAO and the World Bank are working with LONGER-TERM MEASURES FOR HPAI the Vietnam government to fi nd ways to minimize PREVENTION AND CONTROL externalities from restructuring. Several wholesale Prevention measures for AI that are robust in the markets have already been closed in major centers long-term combine improved response to outbreaks in Vietnam. Some of the people who formerly with reducing risk that they will occur. Benefi ts worked in these establishments have found alterna- should include reduced market shocks and increased tive employment, while others are waiting for market stability, increased consumer confi dence, poultry sales to resume (1). risk sharing, and hence a greater buy-in to animal health. However, sustained investment is needed Reduced Complexity and some of the changes likely to be introduced have One consequence of increased attention to biosecu- the potential to exclude people from making a live- rity may be separation of more and less formal lihood from the poultry sector. The most likely mea- chains, a reduction in complexity from the situation sures include biosecurity upgrading, which has illustrated by Figure 24.2. As some chains become already been discussed, restructuring of market more regulated, with higher food safety standards, chains, and investments in the animal health system their contact with other chains tends to reduce. In to provide support to poultry production. Vietnam, the formal and informal chains have seen some separation particularly in terms of the sources Restructuring of birds to high value retailers. Gains from increased When a market chain is restructured, changes may trade have largely accrued to sectors 1 and 2 (1). occur in several features of the chain, as follows. Increasingly, it is being recognized and defi ned in international guidelines that disease freedom may Changed Location apply not only to a country but also to a zone or a Parts of the chain may be re-sited away from areas compartment. Thailand has been exploring the pos- with high densities of humans and other livestock. sibility of defi ning disease-free and vaccination-free Moving wet markets and slaughterhouses outside of compartments for export of poultry (34), which 556 Avian Influenza might allow targeted vaccination in smallholder ble market chains tended to push the risk toward fl ocks outside the compartments. If the compartment small-scale producers at the end of the chain. It is is successfully established, then an isolated outbreak not impossible for small-scale producers to be outside of it should not affect its trading status. It included in formal market chains, but they may need has thus far proved diffi cult for Thailand to arrive at to upgrade from sector 3 to 2 in terms of their bio- a technically and fi nancially viable design that is security and to be operating from a secure fi nancial acceptable to importers (38). basis to cope with delayed payments. They will also face fi erce price competition with larger producers Increased Concentration and Changed unless they can offer a differentiated product such Composition as a traditional or organic bird. One possibility for The commercial poultry sector is already concen- smallholders would be to increase the strength of trated in terms of ownership and numbers of prem- their producer associations to negotiate with buyers. ises and there is a tendency toward further There has been some success with this in Latin concentration in countries whose poultry sectors are America with horticulture products, but no obvious modernizing. Twenty-eight large-scale companies examples to follow in poultry production. own approximately 80% of commercial production Some of the countries tempted by restructuring in Thailand (44). Five fi rms dominate the broiler will be those where domestic demand for meat is industry in Indonesia, with over 95% of production growing. There is still a strong demand for fresh or under contract (18). Poultry production and process- chilled rather than frozen meat, and this offers hope ing lend themselves to economies of scale, and the for domestic production. The tendency is to assume transactions costs of sustaining and certifying bio- that this demand will best be met by fewer, larger, security are lower for fewer, larger units. Fewer effi cient units that may also form the basis for break- large production units tend to be matched by fewer ing into export markets. However, in a country larger traders. where demand is growing, sectors 3 and 4 supply There is a trend toward mechanization in devel- consumers who cannot afford to access supermar- oped and emerging economies. In developing coun- kets, and sector 3 has the fl exibility to expand and tries, the low price of labor currently favors the use contract quickly to meet shifting demand. An abrupt of people rather than of machines, but the need change in the composition of the sector is likely to to implement ever more rigorous quality manage- harm consumers as much as small producers and ment systems for food safety may tip the balance. If may create a demand gap that must be fi lled by this happens, the potential for restructured poultry imports from neighbors with uncertain AI status. chains to offer employment to displaced smallholder farmers will diminish rapidly. Cessation of backyard Investment in Animal Health Systems poultry production in Vietnam alone could lead to Managing avian diseases, including HPAI, in a income foregone in the order of US$550 million a poultry sector that continues to grow will take year, equivalent to 2.5 million “full-time” jobs at the support from animal health systems in surveillance, minimum rural wage rate (43). In Thailand, 3% of rapid response to suspected cases, and preventive the poultry population were ducks (28), but biosecu- measures and border controls. This calls for both rity measures and breeding restrictions are making investment and some rethinking of the systems. it increasingly diffi cult for the extensive systems to HPAI has highlighted the particular diffi culty of survive. managing infectious diseases in decentralized fi nancing and decision making systems. The con- More Formal Relationships tainment and eradication of transboundary diseases Relationships in poultry market systems include require standardization and guidelines that cut across integration, where one fi rm owns several parts of the international, national, and administrative bound- chain; written contractual arrangements; verbal, but aries (16). It would be absurd to suggest wholesale still fi rm, contracts; and more casual arrangements. reorganization because there are strong arguments The need for higher biosecurity along a market chain to maintain decentralization for functions other than tends to push relationships toward formal contracts epidemic disease control, but it is possible to learn with lower transactions costs. It has also in vegeta- from the examples of good practice in managing the 24 / The Economics of Avian Influenza 557 funding of animal health control and major decision in the economic and social fabric of the countries in making on legislation and enforcement. Even in a which they exist. When these systems are examined, decentralized system, funds for animal health can be it becomes obvious that the control strategy for an pooled at a central point and their use authorized by individual country, while it follows certain general a trusted group of decision makers. As the commer- principles must be tailored to the mix of systems in cial sector grows, it becomes possible to develop a the country and their stage of development as well fund with contributions from central government, as to local fi nancial and human resources. decentralized government, and private industry that There is a strong case for continued international is managed according to strict guidelines agreed on fi nancing of HPAI control but also for fi ne-tuning of by representatives of all of the contributors. recommendations to make them more cost-effective It is more problematic to provide funding to within particular situations. In order to support this support sector 4 and the least biosecure parts of process, work needs to continue in learning more sector 3, whose members are not in a position to about the benefi ts and costs of control processes. contribute to a national fund. It may be in the inter- Areas of particular interest for continued investiga- est of the commercial sector to subsidize them. It is tion include the following. also in the public interest to support the formation of local producer groups through which training and Economics of Surveillance Systems services can be delivered, centered on animal health Emerging diseases, particularly those of a zoonotic paraprofessionals. There has already been consider- nature, are a continuing threat and their economic able work in learning what supports and what impact increases sharply with delays in fi rst response. hampers the ability of paraprofessionals to support A clearer understanding is needed of the incentives communities, and this needs to continue even if it is to reporting, including more effective use of expensive. It will be a particular challenge to direct compensation in developing country smallholder energy toward service for poultry, because small production systems and deeper engagement at com- poultry fl ocks receive the least animal health inputs munity level in decisions about disease control. of any livestock. Despite strong efforts to legitimize Regional cooperation in surveillance must also be the position of paraprofessionals, their relationship improved given the extent of offi cial and unoffi cial with the government is still irregular. One way to cross-border trade in poultry. build their capability is to give them regular work, with formal contracts, in surveillance, quality The Future of Smallholder Poultry Production control, and vaccination. Finally, with the increasing Sector 3 has been promoted as a pathway out of tendency toward emerging zoonotic diseases, it may poverty, but it is not clear under what conditions it be possible to forge closer links between animal and can continue to play this role. If biosecurity require- human health paraprofessionals, particularly for sur- ments continue to be raised, what will be the poten- veillance. tial for small-scale production with the biosecurity levels of sector 2? Sector 4 is likely to remain CONCLUSIONS untouched for much longer because it plays a differ- The chapter has briefl y highlighted the main issues ent economic role. However, it is not yet clear in the economics of AI control, particularly of HPAI. whether improved biosecurity would be possible for AI has not initiated the changes taking place in the this sector, what would be required to implement it, global poultry sector, because trends toward concen- and the cost and management implications. tration and reorganization were already under way, but it has brought them to the attention of people all Investment Needs for Animal Health Systems over the world and accelerated change. With the likelihood of continued changes to poultry This review has described the chronology of sector structure and continued threats from emerg- social and economic issues that must be addressed ing diseases, there is a need to examine the way that at different stages of dealing with disease. It has animal health systems are designed and funded. drawn attention to the different concerns of four Those in developing countries were poorly prepared types of poultry system, defi ned by their biosecurity and underfunded to respond to HPAI. Those in and level of commercialization, that all play a part developed countries continue to negotiate with 558 Avian Influenza industry over risk management measures and miti- 9. Centers for Disease Control and Prevention. 2005. gation of market shocks and to refi ne their fi nancing Frequently Asked Questions About SARS. Avail- and decision-making mechanisms. able at http://www.cdc.gov/ncidod/sars/faq.htm. AI is a wake-up call. If we review the economic Centers for Disease Control and Prevention: lessons, we may be better prepared to deal with other Atlanta, GA. Accessed May 3, 2006. 10. CIVAS (Center for Indonesian Veterinary Ana- livestock diseases that will surely emerge in the lytical Studies). 2006. A Review of Free Range future. Duck Farming Systems in Indonesia and Assess- ment of Their Implication in the Spreading of the REFERENCES Highly Pathogenic (H5N1) Strain of Avian Infl u- 1. Agrifood. 2006. Poultry Sector Rehabilitation enza Virus (HPAI). A Report for FAO. Food and Project—Phase I. The Impact of Avian Infl uenza Agriculture Organization: Rome, Italy. on Poultry Sector Restructuring and Its Socio-eco- 11. Costales, A., P. Gerber, and H. Steinfeld. 2006. nomic Effects. Prepared for FAO by Agrifood Underneath the Livestock Revolution. Livestock Consulting International April 2006. Food and Report 2006:15–27. Food and Agriculture Organi- Agriculture Organization: Rome, Italy. zation: Rome, Italy. 2. APEC 2006. SOM III, Danang. HTF APEC Capac- 12. Darby, P. 2003. Economic Impact of SARS, ity Building Seminar on Avian Infl uenza. http:// Special Briefi ng May 2003. Conference Board of www.apecorg/content/apec/documents_reports/ Canada. Available at http://www.dfait-maeci.gc. health_task_force/2006.html#cbs. ca/mexico-city/economic/may/sarsbriefmay03. 3. APEC 2006. SOM III, Danang. HTF Symposium pdf. Accessed July 2006. on Functioning Economies in Times of Pandemic. 13. Delquigny, T., M. Edan, N. D. Hoan, P. Kien, and http:/www.apecorg/content/apec/documents_ P. Gautier. 2004. Evolution et Impact de l’èpidémie reports/health_task_force/2006.html#sfetp. d’Infl uenza Aviaire et Description de la fi lière 4. AVSF 2006. Review of Free-Range Duck Farming Avicole au Vietnam. Rapport Final. Vétérinaires Systems in Northern Vietnam and Assessment of Sans Frontières: Hanoi, Vietnam. Their Implication in the Spreading of the Highly 14. DLD (Department of Livestock Development, Pathogenic (H5N1) Strain of Avian Infl uenza Ministry of Agriculture and Co-operatives). 2005. (HPAI). Report for FAO, March 2006. Food and Socio-economic Impact Assessment for the Avian Agriculture Organization: Rome, Italy. Infl uenza Crisis: Gaps and Links Between Poultry 5. Benigno, C.C. 2005. Benefi ts and Costs of Reduc- Production and Poverty in Smallholders. Food and ing Risks in Wet Markets: Marikina City Wet Agriculture Organization: Rome, Italy. Market Case Study. Presentation to FAO-APHCA 15. Dolberg, F. 2003. Review of Household Poultry and OIE Regional Avian Infl uenza Economic Production. PPLPI Working Paper No. 6. Food and Assessment Workshop, September 26–29, 2005, Agriculture Organization, Rome. Available at Bali, Indonesia. http://www.fao.org/ag/againfo/projects/en/pplpi/ 6. Bloom, E., V. de Wit, and M.J. Carangal-San Jose. docarc/wp6.pdf. 2005. Potential Economic Impact of an Avian 16. Dolberg, F., E. Guerne-Bleich, and A. McLeod. Flu Pandemic on Asia. ERD Policy Brief Series 2005. Post-Avian Infl uenza Rehabilitation in Cam- No. 42. Asian Development Bank: Manila, The bodia, Laos, Vietnam and Indonesia: Summary of Philippines. Project Results and Outcomes (TCP/RAS/3010). 7. Burden, R. 2006. Considerations Regarding “Insur- Food and Agriculture Organization: Rome, Italy. ance”: Some initial thoughts and observations. 17. Dung D.H, N.M. Taylor, and A. McLeod. 2006. Presentation made July 2006 at Food and Agricul- Improving veterinary surveillance in Viet Nam—a ture Organization: Rome, Italy. knowledge management approach. In: Proceedings 8. CASERED (Indonesian Centre of Agricultural of the 11th International Symposium on Veterinary Socio-Economic Research and Development). Epidemiology and Economics, 2006. Available at 2004. Socio-Economic Impact Assessment of the www.sciquest.org.nz. Avian Infl uenza Crisis in Poultry Production 18. Fabiosa, J.F., H.H. Jensen, and D. Yan. 2004. Systems in Indonesia, with Particular Focus on Output Supply and Input Demand System of Com- Independent Smallholders. Final Report for FAO’s mercial and Backyard Poultry Producers in Indo- TCP/RAS/3010 “Emergency Regional Support for nesia. Working Paper 04-WP 363, May 2004, Post Avian Infl uenza Rehabilitation”. Food and Center for Agricultural and Rural Development: Agriculture Organization: Rome, Italy. Iowa State University, Ames, IA. 24 / The Economics of Avian Influenza 559

19. Fan, E.X. 2003. SARS: Economic Impacts and Livestock, Ministry of Agriculture and Coopera- Implications. ERD Policy Brief No. 15. Available tives: Bangkok. at http://www.adb.org/Documents/EDRC/Policy_ 29. Global Health Facts. Available at http://www.glo- Briefs/PB015.pdf. balhealthfacts.org/topic.jsp?i=25&srt=2#table. 20. Food and Agriculture Organization. 2005. Social Accessed November 1, 2006. and Economic Impacts of Avian Infl uenza Control. 30. GSO. 2005. Impacts of HPAI in Vietnam. Unpub- In: Proceedings of a workshop held in Bangkok, lished report from Vietnam Government Statistics December 8–9, 2004. Food and Agriculture Orga- Offi ce. Food and Agriculture Organization: Rome, nization: Rome, Italy. Italy. 21. Food and Agriculture Organization. 2006. Com- 31. Hancock, J. 2006. Exploring Impacts of Avian mittee on World Food Security, Thirty-Second Infl uenza on Food Security. Internal working paper Session, Rome, 30 October–4 November 2006, prepared for the FAO ECTAD Socio-Economic Assessment of the World Food Security Situation. Working Group, November 2006. Food and Agri- Available at ftp://ftp.fao.org/docrep/fao/meeting/ culture Organization: Rome, Italy. 011/j8096e.pdf. 32. Hartono, D. 2004. Economic impact of AI on price 22. Food and Agriculture Organization/Offi ce Interna- and supply of poultry product. Paper presented at tionale des Epizooties. 2005. A Global Strategy for National Workshop on Post Avian Infl uenza the Progressive Control of Highly Pathogenic Recovery, October 4–5, 2004, Jakarta, Indonesia. Avian Infl uenza (HPAI). Available at www.fao. 33. Hinrichs, J., L. Sims, and A. McLeod. 2006. Some org/docs/eims/upload//210745/Glo_pro_HPAI_ Direct Costs of Control for Avian Infl uenza. In: oct05_en.pdf. Proceedings of the 11th International Symposium 23. Food and Agriculture Organization/Offi ce Interna- on Veterinary Epidemiology and Economics, 2006. tionale des Epizooties/World Health Organization. Available at: www.sciquest.org.nz. 2005. Consultation on Avian Infl uenza and Human 34. Marchot, P., and L. Ratananakorn. 2005. Draft Health: Risk Reduction Measures in Producing, Compartmentalisation Model. Presentation made Marketing and Living with Animals in Asia, July at the DLD/EU Workshop on Poultry Farming 3–5, 2005, Kuala Lumpur, Malaysia. Compartmentalisation Project in Thailand, August 24. Fournier, T. 2004–2005. Perceptions d’un Risque 15, 2005, Siam City Hotel, Bangkok, Thailand. Sanitaire par les Consommateurs. Mémoire 35. Mathias, E. 2006. Gender and Socio-Economic de DESS Sciences Sociales Appliquées à Issues in Avian Infl uenza Control. Concept paper L’alimentation. Available at http://www.malica- prepared for the FAO Gender and Development asia.org/publications/publications.html. Service. Food and Agriculture Organization: 25. Geerlings, E. 2006a. Rapid Assessment of HPAI Rome, Italy. Socio-economic Impacts in Turkey. Report pro- 36. McKibbin, W.J., and A.A. Sidorenko. 2006. Global duced for the FAO ECTAD Socio-economic Macroeconomic Consequences of Pandemic Infl u- Working Group, November 2006. Food and Agri- enza. Lowy Institute of Economic Policy, February culture Organization: Rome, Italy. 2006, Sydney, Australia. 26. Geerlings, E. 2006b. Literature Review of the Dutch 37. McLeod, A., and A. Brandenburg. 2005. Estimates Poultry Sector and Impacts of AI. Internal Report, of Benefi ts and Costs in the Poultry Sector of Con- Food and Agriculture Organization: Rome, Italy. tinued HPAI Control Made for the Ministry of 27. Gerber, P., T. Wassenaar, P. Chilonda, H. Menzi, Agriculture and Rural Development. Summary and H. Steinfeld. 2004. Geographical Shifts of the presented to Joint Donor committee, November Livestock Production: Land Use and Environmen- 2005, Hanoi, Vietnam. tal Impact Implications. Paper given at the confer- 38. McLeod, A., and J. Rushton. 2008. Economics of ence “Structural Change in the Livestock animal vaccination. OIE Scientifi c and Technical Sector—Social, Health, and Environmental Impli- Revue. cations for Policy Making,” Bangkok, Thailand, 39. McLeod, A., N. Morgan, A. Prakash, and J. Hin- January 27–29. richs. 2005. Economic and Social Impacts of Avian 28. Gilbert, M., and J. Slingenbergh. 2004. Highly Infl uenza. Paper provided to donor conference in Pathogenic Avian Infl uenza in Thailand: An Anal- Geneva. Available at: http://www.fao.org/ag/ ysis of the Distribution of Outbreaks in the 2nd againfo/subjects/en/health/diseases-cards/confer- Wave, Identifi cation of Risk Factors, and Pros- ence/documents/Economic-social-impact.pdf pects for Real-Time Monitoring. Food and Agri- 40. McLeod, A., N. Taylor, N.T. Thuy, and L.T.K. culture Organization, Rome and Department of Lan. 2003. Control of Classical Swine Fever in the 560 Avian Influenza

Red River Delta of Vietnam. A Stakeholder Anal- Producers after the HPAI Outbreak, Egypt. Food ysis and Assessment of Potential Benefi ts, Costs and Agriculture Organization: Rome, Italy. and Risks of Improved Disease Control in Three 50. Simmons, P. 2005. Perspectives on the 2003 and Provinces, Phase 3 Report to Strengthening of Vet- 2004 Avian Infl uenza Outbreak in Bali and erinary Services in Vietnam. Department of Animal Lombok. Paper Contributed to the 49th Annual Health: Hanoi, Vietnam. Conference of the Australian Agricultural & 41. Morgan, N. 2006. Meating the Market: Outlook Resource Economics Society, Coffs Harbor, New and Issues. Presentation made to the International South Wales, Australia, February 2005. Poultry Council at the Seminar on Global Trends 51. Sims, L. 2006. Assessment of Long-Term Costs in Meat Production and the Impact of Animal Dis- for Control of Highly Pathogenic Avian Infl uenza eases, VIV Europe 2006, May 16–18, 2006, in Indonesia, Thailand, Viet Nam, Cambodia, Laos Utrecht, the Netherlands. and Hong Kong PDR China. Draft working paper. 42. Nave, S. 2006. Some Costs of the Avian Infl uenza Food and Agriculture Organization: Rome, Italy. Outbreak in France. Internal report. Food and 52. Smil, V. 2005. The next 50 years: fatal disconti- Agriculture Organization: Rome, Italy. nuities. Population and Development Review 43. Otte, J. 2006. The Hen Which Lays the Golden 21(2):201–236. Eggs: Why Backyard Poultry Are So Popular. 53. Tung, D.X. 2005. Smallholder Poultry Production PPLPI features February 2006. Food and Agricul- in Viet Nam. Marketing Characteristics and Strat- ture Organization: Rome, Italy. egies. Network for Smallholder Poultry Develop- 44. Poapongsakorn, N. 2004. Dynamics of Southeast ment. Available at http://www.poultry.kvl.dk/ Asian Livestock Markets and Their Sanitary and upload/poultry/ workshops/w25/papers/tung.pdf. Technical Standards. Paper presented to the FAO 54. Upton, M. 2006. A framework for identifying Expert Consultation on “Dynamics of Sanitary and market and trade impacts of HPAI and its control. Technical Requirements in Domestic Livestock Paper presented at a symposium on Market and Markets: Assisting the Poor to Cope.” Food and Trade Dimensions of Avian Infl uenza Prevention Agriculture Organization: Rome, Italy. and Control, held in conjunction with the 21st 45. Preker, A., J.C. Langenbrunner, and S. Emi. 2004. Session of the FAO Intergovernmental Group on Defi cit fi nancing of health care for the poor. In: Meat and Dairy Products. Food and Agriculture Preker A (ed.). Health Financing for Poor People: Organization: Rome, Italy, November 13–16, 2006. Resource Mobilization and Risk Sharing. World 55. Vannasouk, T. 2004. Review of Livestock Market- Bank: Washington, DC. ing in Lao PDR. Report for FAO’s TCP/RAS/3010. 46. Riviere-Cinnamond, A. 2005. Compensation and Food and Agriculture Organization: Rome, Italy. Related Financial Support Policy Strategy for 56. VSF (Veterinaires Sans Frontieres). 2004. Review Avian Infl uenza. Emergency Recovery and Reha- of the Poultry Production and Assessment of the bilitation of the Poultry Sector in Viet Nam. Food Socio-Economic Impact of the Highly Pathogenic and Agriculture Organization: Rome, Italy. Avian Infl uenza Epidemic in Cambodia. Final 47. Rushton J., R. Viscarra, E. Guerne Bleich, and A. Report for FAO’s TCP/RAS/3010. Food and Agri- McLeod. 2005. Impact of Avian Infl uenza Out- culture Organization: Rome, Italy. breaks in the Poultry Sectors of Five South East 57. WCCD (World Congress on Communication for Asian Countries (Cambodia, Indonesia, Lao PDR, Development). 2006. Special Event on Reframing Thailand, Viet Nam): Outbreak Costs, Responses the AI Communication Discourse. Rome, Italy, and Potential Long-Term Control. Report for October 2006. Available at http://www.devcomm. FAO’s TCP/RAS/3010. Food and Agriculture org/worldbank/public.asp?idmacro=4&idmicro=2 Organization: Rome, Italy. 6&txt=12#a. 48. Rushton, J. 2006. Compensation for HPAI in Egypt. 58. World Bank, Food and Agriculture Organization, Report produced for the FAO ECTAD Socio- IFPRI, Offi ce Internationale des Epizooties. 2006. Economics Working Group, November 2006. Enhancing Control of Highly Pathogenic Avian Food and Agriculture Organization: Rome, Infl eunza in Developing Countries through Com- Italy. pensation: Issues and Good Practice. World Bank: 49. Shalaby, M.A. 2006. Rapid Assessment of Highly Washington, DC. Available at http://www.fao.org/ Pathogenic Avian Infl uenza Producers for Reim- docs/eims/upload//217132/gui_hpai_compensa- bursement of Backyards and Industrial Poultry tion.pdf. 25 Global Strategy for Highly Pathogenic Avian Influenza: Progressive Control and Eradication, and Postoutbreak Recovery

Juan Lubroth, Subhash Morzaria, and Alejandro B. Thiermann

INTRODUCTION The global prevention, control, and eradication The global strategy for highly pathogenic avian strategies for HPAI broadly parallel those for other infl uenza (HPAI) is more than a strategy: it encom- similar highly infectious diseases of livestock (and passes broad principles for the prevention, detection, some zoonoses), which are transboundary in nature. control or eradication, requirements for standards, Transboundary animal diseases (TADs) are defi ned best practices advice for different conditions and as those that can easily spread to other countries, farming systems, the importance of epidemiological causing epidemics, jeopardizing food security, and intelligence, communication and enabling policy inhibiting trade in livestock and livestock products. and legislation, the need for robust partnership with Many TADs are zoonotic and cause disease and a wide range of stakeholders, and consideration for death in humans. They often affect several countries the application of immediate- and longer-term mea- or regions simultaneously, and cause signifi cant eco- sures through a programmatic approach for tackling nomic impact to warrant regional and international this highly contagious disease in domestic poultry. cooperation for management and control. Because The strategy’s success is best determined at the local of these reasons, the control of many TADs is con- level by a number of impacts such as reducing pro- sidered as international public good. Historically, duction losses, maintaining robust trade in poultry most outbreaks of HPAI have been generally cir- and poultry products, improving livelihoods of poor cumscribed, and control and eradication has been poultry producers, improving food safety and secu- successfully based on elimination of infected and rity, preserving the genetic diversity of domestic exposed birds and application of strict biosecurity poultry and wild bird populations and, in the case of measures. HPAI is unlike many other TADs where zoonotic avian infl uenza viruses, minimizing risk of vaccination has been the mainstay for long-term human infection. This strategy is not a blueprint or success of control programs. a prescription for control and eradication. It provides When the Food and Agriculture Organization broad approaches for dealing with HPAI and main- (FAO) of the United Nations published the Recom- tains fl exibility to allow for inclusion of new meth- mendations on the Prevention, Control and Eradica- odologies and approaches and response to different tion of Highly Pathogenic Avian Infl uenza (HPAI) and varied global farming systems. in Asia, only 10 countries and administrative regions

Avian Influenza Edited by David E. Swayne 561 © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7 562 Avian Influenza in Asia had reported infections in poultry with the sive Control of Highly Pathogenic Avian Infl uenza, H5N1 HPAI virus (10). Since the mid to late 2005, which was produced in response to the recommen- the disease has spread more widely with outbreaks dation made during the Second FAO/OIE Regional reported in Eurasia, Middle East, Caucasus, and Meeting on Avian Infl uenza Control in Asia held in Africa in poultry and wild birds. The international February 2005 (11, 12). In this document, the term and regional organizations, the multilateral and progressive control is used to underline a series of bilateral donors, fi nancial institutions, and infected sequential steps to be undertaken to reduce inci- and at-risk countries have collaborated closely to dence and spread and eradicate the disease nation- respond to the H5N1 HPAI emergency. While work ally and regionally. Key to the principles at continues to address the current HPAI crisis at the progressive control is to tackle the disease at source, national level, greater efforts are required to which is domestic poultry. In addition, the OIE and strengthen regional integration and international FAO have published Ensuring Good Governance to coordination to backstop the national efforts and Address Emerging and Re-Emerging Animal Disease secure longer-term success for control and eradica- Threats, which identifi es short-, medium-, and long- tion. In this regard, several important meetings have term actions for the better preparedness to fi ght these taken place, notably the FAO/World Organization diseases through strengthened and rationalized vet- of Animal Health (Offi ce Internationale des Epizo- erinary services (29). oties [OIE]) Regio-nal Meetings on Avian Infl uenza This chapter is very much based on the Global Control in Asia (Bangkok, February 2004, and Ho Strategy for the Progressive Control of Highly Chi Minh City, February 2005), the OIE/FAO Inter- Pathogenic Avian Infl uenza and discusses an inte- national Scientifi c Conference on Avian Infl uenza grated approach to include H5N1 HPAI control at (Paris, April 2005), the World Bank/European Com- national, regional, and international levels. It focuses mission and Government of the People’s Republic on a number of issues related to policy and regula- of China donor pledging conference (Beijing, tory measures, technical appropriateness, restructur- January 2006), and the FAO/OIE International Con- ing of poultry industry, and capacity building. The ference on Wild Birds and Avian Infl uenza (Rome, needs for strong veterinary services, well-equipped May 2006) among others, to ensure understanding laboratories, adequate manpower, regional coopera- by decision makers and the scientifi c community of tion and networks on diagnosis and surveillance, and the strategies, research needs, and fi nancial require- epidemiological information are highlighted. ments to undertake in order to achieve the required The long-term vision of the strategy is to mini- control measures. These meetings have lead to mize the global threat and risk of HPAI for domes- greater and common understanding of the global tic poultry, through progressive control and importance of HPAI and greater collaboration and eradication of HPAI, particularly that caused by increased funding to support regional and interna- H5N1 virus, from domestic poultry, and to avoid tional efforts to control the disease. Many countries transmission to humans. Achieving this goal will have made exceptional progress in controlling the diminish a threat of a human pandemic from this disease in Southeast Asia; however, there are still virus, stabilize poultry production, enhance a robust several countries in Asia and Africa where the regional and international trade in poultry and disease has become endemic and continues to spread. poultry products, increase human and food safety, Cases of avian infl uenza in humans continue to rise and improve the livelihoods of the rural poor (3, 13). with over 50 cases reported in last 12 months. The The role of wild birds in disease introduction is of potential for H5N1 to infect different species was concern, but this concern strays from where inter- confi rmed, with infections reported in approximately vention is plausible at the farm, household, and nine mammalian and 88 avian species through market levels. March 2006 (24), raising additional concerns about Broadly speaking, poultry is defi ned in this chapter the potential effects of the disease in humans and as domesticated birds used for the production of endangered species. meat and eggs for consumption, for the production FAO and OIE, in collaboration with the World of other commercial products, for restocking sup- Health Organization (WHO), recently developed a plies of game, or for breeding these types of birds. document entitled Global Strategy for the Progres- Wild birds are those species that are free-living and 25 / Global Strategy for Highly Pathogenic Avian Influenza 563 play an important role in natural ecosystems and or at least to their exclusion from large livestock habitats. With the exception of subsistence hunting, producing zones, is a prerequisite to meet the future these wild species are usually not represented pre- global demand for safe and wholesome livestock dominantly in the human food. Waterfowl, on the products. other hand, can encompass both wild birds or those The emergence of H5N1 HPAI in 1997 is the Anatidae raised for human advantage. most recent example of transboundary zoonotic disease. Its rapid spread in Southeast Asia in 2003– BACKGROUND 2004 clearly underlined the need to strengthen Transboundary animal diseases, including those that capacity in disease surveillance, improve transpar- are zoonotic, continue to threaten economic and ency in reporting, and enhance regional collabora- social well-being of the increasing globalizing world tion and cooperation for a rapid response. Many (13). With a large and growing volume of regional Southeast Asian countries found themselves unable and international trade in livestock and livestock to control the disease that spread rapidly over a wide products and the rapid movement of large numbers geographical area in a relatively short period of of people across continents through air travel, several time, mainly because of the failure of disease event emerging infectious zoonotic diseases can spread reporting from the local level through the interna- widely and quickly over large geographical regions. tional stage. The delay by several months or even These diseases would have a wide-ranging impact longer will be proven to be one of the most costly on the livelihoods of farmers, regional and interna- exercises to date. The rapid spread of HPAI across tional trade, food safety, public health, international Southeast Asia, which caused high mortality in its travel, business, and tourism. Thus, emerging trans- previously unexposed, highly susceptible commer- boundary zoonotic diseases, such as H5N1 and cial and smallholder poultry populations, came as a H7N7 HPAI viruses, pose a serious and continual shock as it was realized that prevailing disease infor- threat to global economy and public health (3). mation systems and the veterinary capacity to deal While the economic losses from TADs, such as with the outbreaks were far from adequate to handle foot and mouth disease (FMD) and classic swine the scope of the emergency. Retrospective analysis fever (CSF) in Europe, have been well documented, from unpublished fi eld missions indicates that the it is the newly emerging zoonotic diseases that are disease was arguably already present and had been causing increasing worldwide concern. The bovine spreading undetected in parts of Southeast Asia spongiform encephalopathy crisis in Europe pro- since early 2003. Thus, H5N1 HPAI, once present vides an example of an emerging zoonotic disease only in the People’s Republic of China and Hong moving into new areas by means of trade fl ows of Kong, became a major problem for all of Southeast cattle or bone meal (15). In 1999, a Nipah virus Asia and as of early 2006, to Europe, Africa, and the outbreak in Malaysia destroyed the swine industry Middle East. while simultaneously creating massive public panic Three FAO/OIE regional meetings on HPAI resulting from human fatalities (37). The 2003 (Bangkok, February 2004; Ho Chi Minh City, Feb- severe acute respiratory syndrome (SARS) outbreak ruary 2005; Kuala Lumpur, March 2005), held in infected several hundred people in large parts of collaboration with WHO and the participating coun- South and Southeast Asia and Canada. International tries, acknowledged the prompt response by the travel and tourism were severely curtailed by the international community in mobilizing emergency outbreak of SARS in Asia. The disease took over a funds and technical support to tackle the regional year to be brought under control, costing the subre- crisis and alerted all nations to review their emer- gion over $30 billion (22, 25, 30). Over the past gency preparedness plans. While signifi cant prog- several decades, an average of one newly emerging ress was made in Asia to address the ongoing crisis, disease per year has been identifi ed, of which 75% it was not possible to avoid spillover of the virus to have been of the zoonotic type. Their transboundary other continents. The detailed dynamics of the trans- nature highlights that no country can count itself mission and spread of the H5N1 virus remain poorly exempt from such diseases. understood, and in many regions veterinary capacity The effective, sustained control of such animal is inadequate to carry out the necessary comprehen- diseases, leading to their elimination where feasible sive active surveillance and fi eld mobilization 564 Avian Influenza programs to implement appropriate detection, pre- 9. Protecting global human health and well-being is vention, and disease control measures. Greater polit- a responsibility of the international community. ical commitment is needed in many countries, and international donors need to be more forthcoming to Zoonotic Risk provide additional resources in all aspects of disease The H5N1 strain currently affecting numerous coun- control. Among a number of recommendations tries in three continents has also proved to be fatal emerging from the Ho Chi Minh City meeting, the to humans. Little is known of the H5N1 infection most important and signifi cant one was the call for status in the human population because subclinical the development of a global strategy linked to com- infections could not have been detected and no sys- prehensive disease control plans, supported by sub- tematic serological evaluation has been conducted. stantial fi nancial resources to tackle the HPAI It is not possible to predict when the virus might problem country by country in a coordinated manner. reach an adaptive level to allow for human-to-human In response, FAO and OIE, under the initiative enti- transmission, but as the virus becomes endemic in tled Global Framework for the Progressive Control domestic poultry in some countries, the potential for of Transboundary Animal Diseases (GF-TADs) and new variants due to constantly changing genotype in collaboration with WHO, have developed a global of H5N1 will increase, and the risk of pandemic strategy for HPAI control (13). This chapter is infl uenza could become greater (6, 25). Prepared- broadly based on this strategy paper. ness plans and strategy for human infl uenza epidemics are beyond the scope of this chapter; however, Why a Global Strategy? should a human pandemic occur, the disruption of The key reasons for developing and implementing a civil society and potential fatalities across the globe global strategy for the control of H5N1 HPAI are would be catastrophic. The best prevention mea- outlined: sures to preclude human infections are to confront the zoonotic pathogen at its domestic animal 1. HPAI is a highly infectious and dynamically source. evolving disease that can potentially spread rapidly and widely across countries and Expansion of Highly Pathogenic continents. Avian Infl uenza 2. Certain HPAI viral infections can be zoonotic The rapid spread of H5N1 HPAI in Southeast Asia with a potential to cause a global human in 2003–2004 has been attributed to expanding trade pandemic. in poultry and poultry products and failure to detect 3. HPAI has emerged and spread rapidly as a con- and report the disease early. In late 2005 and in the sequence of regional and intercontinental trade in fi rst 6 months of 2006, the disease spread over 35 poultry. countries, with H5N1 HPAI virus reported in wild- 4. AI viruses may be transported widely and quickly life and/or terrestrial birds (6, 7, 19, 23, 31). by migratory birds, along fl yways and in resting The risk that H5N1 HPAI may be carried along or nesting areas. migratory bird fl yways to areas beyond the origi- 5. HPAI results from low pathogenic avian infl u- nally infected region was incontrovertibly shown enza (LPAI) viruses, which are present in many when H5N1 was fi rst reported beyond its Southeast wild bird species throughout the world (14, 17, Asian source, in Kazakhstan, Mongolia, Siberia, 18, 23, 38). Thus all countries in the world are Turkey, and Romania. In western Europe, most potentially at risk of being infected. countries identifi ed at least one case of H5N1 virus 6. HPAI threatens local, regional, and international in wild birds with only a few detecting spillover to trade and places the global poultry industry in the domestic poultry. Despite limited spread of the developed and developing worlds at risk. disease in western Europe, the negative economic 7. HPAI negatively impacts the livelihoods of mil- repercussions due to transient market collapse on lions of people, especially the rural poor. poultry meat and products such as eggs, even in an 8. HPAI outbreaks are beyond the scope and uninfected country, were great (22). The risk of resources of a single country or region to regular incursion of H5N1 HPAI virus in Europe, control. the Middle East, and Africa, and possibility of its 25 / Global Strategy for Highly Pathogenic Avian Influenza 565 introduction in the Americas in the foreseeable the demand for day-old chicks decreased by more future, remains. than 40%, and the demand for poultry feed was reduced by up to 45%. In Vietnam and Cambodia, International Markets Have Caused HPAI to the prices of nonpoultry meats rose up to 30% when Spread Rapidly live bird markets were disrupted by HPAI and The dynamic regional trade in poultry and poultry remained high even after the poultry markets recov- products in Asia and elsewhere, often unregulated, ered, taking the purchase of poultry meat out of a serious cause for concern due to constant risk of reach for low-income consumers (22). disease spread. The danger of international spread The vast majority of the poor live in rural areas of HPAI is also increased by the dynamics of and depend on mixed farming systems that include regional and international legal and illegal move- some level of poultry production. The total number ments of products and movement of people. There of poor people in the currently affected countries in is also a large global trade in ornamental birds, much Southeast Asia dependent on poultry is estimated at of which is illegal. Their role in HPAI virus trans- between 136 and 210 million (Table 25.1). mission is not fully elucidated. These conditions apply not only to HPAI but also to other TADs. A THE STRATEGY global approach to avian infl uenza, therefore, will The global strategy aims to progressively control have relevance to strategic control of other livestock and eradicate H5N1 HPAI from the domestic poultry diseases, including zoonoses. sector in Asia, Africa, Middle East, and Europe and to prevent incursion of the virus in countries that are Economic Impact and Poultry Trade are in currently not infected. The strategy will also contrib- Jeopardy ute to the overall goal of promoting viable poultry Over 150 million poultry were destroyed as the production in the world, enhancing a robust regional result of the 2003 and 2004 HPAI outbreaks in Asia. and international trade in poultry and poultry prod- The global tally through late 2006 was estimated at ucts, increasing safety of poultry products, and 240 million poultry. The direct and indirect eco- improving the livelihoods of all poultry sector stake- nomic impact, while still being evaluated, exceeds holders, especially those of the rural poor. US$12 billion. Trade in poultry at the domestic, regional, and international levels has been severely Guiding Principles for a Successful affected. The total losses in gross domestic product Implementation of a Global Strategy for HPAI accruing from the damaged poultry sector in Asia The following broad guiding principles were used alone amounted to $10 billion. If the direct health in developing a global vision for the control of risk impact from AI in birds is added to the overall HPAI: negative impact problem on livestock and the drop in tourism, economic losses would be considerably 1. Commitment—The prevention, control, and erad- higher, even if the human incidence of infl uenza ication of HPAI from the domestic poultry sector infections of avian origin were to remain limited. are considered a global public good function, requiring strong national, regional and interna- The Livelihoods of the Rural Poor are tional commitment at political, fi nancial, and Threatened technical levels. Despite remarkable progress in addressing extreme 2. Multidisciplinary—HPAI control programs hunger and poverty, the number of the world’s poor require a multidisciplinary approach to integrate remains high. Some 80% of the poor live in rural technical, social, political, policy, and regulatory areas and the vast majority are dependent on agri- issues in addressing a complex problem. culture for their livelihood. For poor households 3. Broad collaboration—The strategy is inclusive depending for their livelihood on poultry, HPAI has and will use a wide range of collaborators in meant the loss of income and food security. A 2004 addressing the problem. FAO survey found that in seriously affected areas of 4. Adaptable and knowledge based—The strategy Indonesia, more than 20% of the permanent indus- is fl exible and will incorporate new information trial and commercial farm workers lost their jobs, and technologies as they become available, and 566 Avian Influenza

Table 25.1. Example of ﬁ ve targeted countries for HPAI control and population engaged in backyard poultry production in infected countries of Southeast Asia. Human Rural Populations (millions) Population Per Capita Population in Dealing With Poultry— Country (millions) People/km2 Income (US$) Rural Areas (%) Assumptions at 60% and 80%

Lao PDR 6 24 310 4.8 (80) 2.9 3.8 Cambodia 12 71 280 9.6 (80) 5.8 7.7 Indonesia 212 117 710 148.4 (70) 89.0 119.0 Vietnam 81 247 430 60.8 (75) 36.5 48.6 Thailand 62 121 1980 37.2 (60) 22.3 29.7 Total 373 136.5 208.8 In Southeast Asia, as in many parts of the world, poverty is common in urban and periurban areas and the poultry backyard sector is of considerable size and importance. Adapted from World Development Report (39).

will respond to changing political and social integrated, densely populated commercial poultry environment. production operation, usually with one breed of 5. Concern of people’s livelihoods—The strategy chickens being raised and high biosecurity. This will take into account the interests of the liveli- sector has a high level of biosecurity and trades hoods of the rural poor who are the most vulner- internationally. Sector 2 is also densely populated, able and often the most affected. with one type of fowl for commercial purposes 6. Economically sustainable—The strategy will but primarily for national level with moderate bio- encourage equitable poultry sector growth condi- security. Sector 3 is also a commercial operation but tions through a combination of activities that will has minimal biosecurity and focuses on markets that benefi t the poor as well as stimulate market-based are more local. This sector usually produces more economic growth. than one poultry species. This sector is perhaps the most diverse of the four FAO defi nitions. Last, Approach sector 4 is the backyard, free-ranging poultry pro- The strategy is based on a sound epidemiological duction system with nonexistent biosecurity. Poor approach to control HPAI and on optimal prepared- farmers are the main keepers of poultry in this ness to prevent further spread of HPAI in Asia, sector. Each holding has usually low numbers of eastern Europe and Caucasus, the Middle East, sub- poultry and are widely dispersed in rural, urban, or Saharan Africa, and the rest of the world. It recog- periurban areas. Keepers of fi ghting cocks or sport nizes that complete eradication of the H5N1 HPAI pigeons on rooftop roosts could be considered sector virus is not possible if a maintenance host in wild 4 operators. birds is identifi ed. The approach to disease control options also takes The strategy takes into account a range of poultry into consideration the range of epidemiological sce- production systems that exist globally in considering narios that exist in different poultry production and developing HPAI prevention, control, and erad- systems in the affected countries. The epidemio- ication options. FAO has attempted to group and logical scenarios can broadly range from high inci- classify various poultry production systems based dence of disease with frequent outbreaks in poultry, on the level of biosecurity applied for disease control to low incidence with variable fl ock immunity, to (Table 25.2). Such classifi cation is helpful in describ- sporadic disease outbreaks. A number of combina- ing a target production sector and establishes a tions of disease control options are available to common understanding of disease control options. control HPAI, depending on the stage that different According to this classifi cation, poultry produc- countries and farming systems have reached along a tion sector 1 is described as a highly industrialized, continuum of variable disease states (10). Disease Table 25.2. Characteristics of different poultry production systems (FAO classifi cation). Poultry Production Systems Village or Backyard Production Commercial Poultry Production Industrial Poultry Poultry (Including Open Farmed Characteristic Production Large Scale Small Scale Ducks, Unregulated Fighting Cocks)

Production System Sector 1 Sector 2 Sector 3 Sector 4 Biosecurity High Medium Low Low Market outputs Export and urban Urban/rural Live urban/rural Rural, urban, and peri-urban areas Dependence on market for High High High Medium to high inputs Dependence on market High High High Medium access Location Near capital and major Near capital and Smaller towns and rural Outdoors cities major cities areas 567 Type of confi nement Indoors Indoors Indoors/Part-time outdoors Not confi ned Housing Closed Closed Closed/Open Minimal (except fi ghting cocks) Contact with other poultry None None Yes Yes Contact with domestic None None Yes Yes ducks Contact with other None None Yes Yes domestic birds Contact with wildlife None None or minimal Yes Yes Veterinary services Company veterinary care Pays for veterinary Possibly pays for Irregular to poor service veterinary service Source of medicine and Market Market Market Government, Market, irregular to vaccine none Source of technical Company and associates Sellers of inputs Sellers of inputs Government extension service if any information Source of fi nancing Banks and own Banks and own Banks and private Private, occasionally Banks Breed of poultry Commercial Commercial Commercial/Indigenous Native Food security of owner High High High Variable 568 Avian Influenza scenarios can be arbitrarily grouped under the fol- categories (freedom from infection in defi ned com- lowing six broad categories: partments) while ensuring that the countries free of disease continue to remain free from HPAI 1. High disease incidence—High virus load; disease (re)incursion and, if countries detect disease intro- spread to new areas, with or without human duction, early control is undertaken. This would infections; little or no immunity in terrestrial allow countries to conduct unrestricted and safe poultry populations; carrier duck populations, if trade in poultry and poultry products in local, present, are a known source of infection regional, and international markets, in accordance 2. Medium to low disease incidence—High virus with the OIE recommendations in the Terrestrial load; the disease does not readily spread to new Code. areas, no human infections; disease endemic in To fulfi ll this objective, a stepwise and phased smallholder poultry sector (sector 4); variable disease control program with time-frames ranging fl ock immunity depending on vaccination effi - from immediate to short term (1 to 3 years), short to cacy and coverage, where used; carrier duck res- medium term (4 to 6 years), and medium to long ervoirs are a known source of infection; wild bird term (7 to 10 years) for the affected countries is transmission thought to be important proposed and strengthen detection, prevention, and 3. Low level of disease incidence—Low virus load; preparedness capacity in countries at risk in the short highly susceptible poultry population; carrier term. Given the great diversity of HPAI conditions ducks, if present, probably not important; low in the target countries and regions, these time-frames poultry density will vary from country to country and will depend 4. Freedom from infection in certain compartments on a number of factors, such as the current disease and zones—Low virus load; highly susceptible situation, political and fi nancial commitment, disease poultry population; disease incidence present in control options currently being undertaken, avail- smallholder sector in certain areas; commercial able disease control capacity, and, in the long term, poultry farms are HPAI free and implement strict ability to maintain sustained vigilance and emer- biosecurity measures; carrier duck population are gency preparedness. a potential source of infection The key components within each of the time- 5. Freedom from infection after stamping-out— framed programs for strengthening of veterinary Highly susceptible domestic poultry population; infrastructure include the following: at risk if highly pathogenic virus(es) are reintroduced; duck reservoirs not considered a high 1. Enabling legislation risk 2. Emergency management structure for disasters 6. Freedom from infection without history of HPAI (i.e., HPAI incursion) infection—Highly susceptible domestic poultry 3. Surveillance and epidemiology (including risk population; likely rapid spread of HPAI in poultry analysis) if disease introduced; ducks reservoirs not con- 4. Laboratory services (diagnostic) sidered important; occurrence of migratory bird 5. Inspection, quarantine, border control, movement resting areas along migratory fl yways management 6. Communications (including disease recognition, Although all areas or countries in the “freedom safe food handling practices, and behavioral from infection categories” are at risk, countries with changes of risk practices weak disease detection, control and prevention 7. Rehabilitation and restructuring of poultry pro- capacity are at a higher risk than those that have duction systems well-structured veterinary system capacity for fi eld 8. Emergency preparedness and contingency investigation, laboratory detection, and early planning response. 9. Strategic research and development (to include The objective of this broad approach is to progres- epidemiological studies, improved diagnostics sively shift the majority of infected areas or coun- and vaccines, and socioeconomic impacts of tries toward epidemiologically improved disease various control options) 25 / Global Strategy for Highly Pathogenic Avian Influenza 569

OPPORTUNITIES FOR CONTROLLING lates. The OIE/FAO Collaborating Centers and the HPAI Reference Laboratories for avian infl uenza have actively supported efforts to control the ongoing crisis, Diagnostic Methods and Tools and recently OIE and FAO created the OIE-FAO A number of diagnostic methods and tools are avail- Network of Infl uenza Expertise (OFFLU). One of the able for identifi cation, typing, and characterization of aims of the OFFLU network (www.offl u.net) is to AI viruses; these include virus isolation methods, have experts and collaborators available to provide serological tests (agar gel immunodiffusion, hemag- technical assistance to countries and governments in glutination and hemagglutination inhibition tests, need (9). In addition, an important thrust is to sustain and ELISAs), “pen-side” rapid detection tests, highly diagnostic and surveillance networks that promote sensitive and specifi c PCR-based tests, and geno/ regular exchange of disease information and harmoni- pathotyping through molecular sequencing tech- zation of standards. These capacities have under- niques. Active, targeted surveillance following the pinned in the past several successful disease control diagnosis of HPAI infection, followed by at-source programs in Asia, Europe, and Africa for other dis- culling of infected birds and strict biosecurity mea- eases, such as rinderpest and foot and mouth disease. sures, have been the mainstay for the control and eradication of the disease. Diagnostic tests for the Vaccines and Vaccination characterization of AI viruses or specifi c immune AI vaccines have been in use for several decades to response are sensitive and well defi ned in the Manual protect against low and high pathogenic strains of of Diagnostic Tests and Vaccines for Terrestrial avian infl uenza viruses (1, 2, 4, 21, 33, 34). Vaccina- Animals published by the OIE (26, 27). Newer diag- tion, when used as part of a wider strategy to control nostic tools that provide reliable results within the disease, has been an extremely effective tool in minutes and can be used at the farm or the laboratory controlling and eradicating the disease. Recent suc- have recently become commercially available. While cessful application of vaccines to control HPAI in some of the newer products have not been fully vali- Italy (4) and Mexico (21) incorporated additional dated, in general they are able to detect group-specifi c well-defi ned control measures that included early (A) viral antigen from tracheal or cloacal swabs or reporting, culling, enforcement of strict biosecurity, other tissues if the titer of virus is suffi ciently high. and compensation to farmers. Vaccination reduces Several companies have recently begun to produce virus shedding and, when properly applied, can be these quick antigen detection tests specifi cally for H5 of signifi cant benefi t in controlling the rapid spread virus infection. These tests generally have poor to of the disease and reducing risk of human infection. moderate sensitivity and are more useful at the fl ock More recently, wide-scale use of vaccines to control level (samples from several birds showing clinical HPAI in Vietnam was deployed in conjunction with signs or dead birds), as positivity in all samples is not other measures such as culling of infected fl ocks and required to make a diagnosis. Such advantages in those possibly incubating the disease, closure of live rapid diagnostic tools should not replace the more bird markets, bans on open duck rearing, public edu- rigorous and standardized techniques that character- cation, and shifts to centralized slaughtering facili- ize the virus fully, including sequencing genes of ties with a dramatic reduction in HPAI incidence in relevance and fi eld epidemiological data. domestic poultry and humans. The diagnostic capacity at national level is being Vaccination is likely to become an important and continually enhanced, particularly in response to the complementary constituent of any HPAI control H5N1 HPAI ongoing crisis, but still far from achiev- strategy, as massive culling increasingly becomes ing widespread profi ciency in most nations. To better socially unacceptable and represents signifi cant provide diagnostic capabilities, a number of centers fi nancial hardship to the farming communities in are being recognized as regional laboratories provid- countries where compensation is not adequately pro- ing diagnostic support for HPAI control. At a more vided. The logistics and human resources to carry international level, the reference laboratories used by out proper humane culling operations in a timely OIE and FAO continue to provide support for rapid manner also represent a diffi culty that many coun- diagnosis and detailed characterization of virus iso- tries are ill prepared to address. 570 Avian Influenza

Decision to deploy vaccination as part of a larger farm. The choice of disease management strategy HPAI control and eradication strategy needs to be must address human health risk. carefully evaluated on a case-by-case basis. Risk of transmission to humans (in the case of zoonotic Vaccines avian viruses), the rate of spread of the disease, the OIE and FAO have made recommendations for the level of endemicity, the presence or absence of use of OIE-approved AI vaccines, and several such ducks as a virus reservoir, the types of farming vaccines are commercially available. If formulated systems, the capacity of veterinary services, fi nan- in accordance with the Manual of Diagnostic Tests cial and infrastructure resources, and an understand- and Vaccines published biannually by the OIE and ing of the benefi ts and costs of various options are used according to the FAO and OIE recommenda- some of key issues that need be considered. tions, these vaccines would provide excellent pro- For countries with a large industrial production tection against clinical disease in chickens by sector involved in export, the benefi ts of vaccinating reducing mortality and production losses. Vaccina- this sector may be outweighed by the costs associated tion of poultry also reduces the virus load in the with loss of export markets. Under these conditions, environment and thereby the risk of infection to the most appropriate approach may be to enhance poultry and humans. According to the OIE, AI-vac- biosecurity and establish an infection-free industrial cinated poultry are not excluded from the export production compartment that, subject to satisfactory trade, although specifi c technical guidelines must be surveillance data, may regain access to export markets followed to ensure that the vaccine is being applied for the industrial sector before the country regains properly and monitored effectively. HPAI free status. The use of vaccination per se should Postvaccination serology and virology and the use not exclude a country or compartment from export, of sentinel domestic ducks and chickens are essen- providing that the importing country is satisfi ed as to tial measures to monitor effi cacy of vaccination the biological safeguards and surveillance results pre- program. When eradication plans include a pro- sented by the exporting country in accordance with grammed withdrawal of vaccination, serological OIE Terrestrial Animal Health Code (27). monitoring to differentiate infected from vaccinated In some countries, small farm or household animals (5), based on the principle of using neur- poultry production provides food security for poor aminidase heterologous vaccines, could be adopted families, and it is virtually impossible to prevent to differentiate immune response to a vaccine from exposure of domestic chickens to AI virus(es) from that of infection (“differentiating infected from vac- wild birds or poultry moving within live bird market cinated animals” [DIVA]) (5, 35, 36). It is important systems. In these cases, blanket vaccination of back- when devising a DIVA approach that fi eld studies yard poultry (sector 4) and fl ocks on small farms in be conducted to determine which infl uenza viruses areas at risk (FAO sector 2 and even sector 3) may (LPAI) are circulating, so that an appropriate vaccine be an important element in an integrated strategy to selection or design is made if postvaccination serol- manage and progressively eradicate infection. Focus ogy is based on heterologous neuraminidase antigen of the vaccination program could sometimes be best response and/or through the use and monitoring of applied at the source of production—at the hatcher- marked sentinel birds. Alternatively, serology based ies, where day-old chicks (or young goslings) receive on NS1 protein will identify infected birds, while their fi rst dose prior to being delivered to the farms, birds immunized with any subtype of inactivated AI where additional vaccination takes place during vaccines are negative for antibodies to NS1 protein their growth cycle. and can be used as a DIVA strategy in areas without The probability of human infection with a zoo- circulation of LPAI viruses (36). A more accurate notic HPAI virus may be signifi cant in a household and timely refl ection of virus circulation, which setting where children are often involved in the col- would aid in better disease control and elimination, lection of eggs, plucking, and preparation of car- is undertaking testing of select daily poultry deaths casses for human consumption. There may also be for virus by real-time reverse transcriptase–poly- signifi cant risks of human exposure in other settings, merase chain reaction (RRT-PCR) or antigen capture such the husbandry of fi ghting cocks or during the ELISA, as this would quickly identify infected birds depopulation of infected fl ocks on an industrial in a vaccinated population (32). 25 / Global Strategy for Highly Pathogenic Avian Influenza 571

Ensuring vaccine quality control in production or North America (Mexico, United States, and Canada). importation is a signifi cant issue for many countries To date, H5N1 HPAI outbreaks in Asia have been and should form part of the national intervention stamped out in Hong Kong, Japan, the Korean Pen- strategies of infected as well as at risk countries. In insula, and Malaysia. Thailand, after 14 diffi cult addition, the use of vaccines in nontarget species months starting in early 2003, made tremendous would require that studies be undertaken to ensure progress in controlling the disease through intensive effi cacy and potency issues and the establishment of farm level surveillance by village volunteers, strict appropriate vaccination regimens for these species. biosecurity measures, and early culling of infected poultry. The disease has now been almost eradicated Vaccine Banks in the commercial poultry sector (sectors 1, 2, and Preplanning for the need of emergency vaccine pur- 3; see Table 25.2) and probably pushed back into chases have required that a tendering process be village poultry and free-ranging domestic ducks in undertaken as has been the case for H5 homologous the Central Plain. Most of these AI-eradicated coun- and heterologous vaccines used during 2004–2006. tries have the resources to deploy the necessary However, the establishment of vaccine banks on control measures and provide improved veterinary location for regional or continental use with an services and surveillance to support HPAI control. improved understanding of circulating viruses, pre- While these success stories provide encouragement diction of antigenic shift, or stabilization of immu- that concerted efforts do pay off in the control of nodominant antigens in the formulated vaccines has HPAI, they also clearly point to the high level of not been done. With heightened production of AI investment required to support HPAI prevention, vaccines from the private sector, it is recommended detection, and control, which is not always available that a relationship based on the tendering process be to countries less endowed with the necessary human established between an institution (i.e., an interna- or physical resources. tional or regional organization or a ministry of agriculture) and the commercial company. The agreement National and Regional Commitment to Control would likely encompass, at a cost, an emergency HPAI need with an effi cacious and safe vaccine formulated Without the necessary political and fi nancial com- to meet or exceed OIE standards. mitment by affected countries and regional organi- If vaccine banks were to be established to face a zations, HPAI will be very diffi cult to overcome for potential incursion of an HPAI virus such as H5N1, certain countries where outbreaks are reported. It some questions remain. For instance, how does one has been highly encouraging to witness that at-risk country assess risk prior to withdrawing vaccines and affected Asian countries by H5N1 have priori- from the bank? How would participating countries tized HPAI as the most important TAD that is cur- contribute to the maintenance of the vaccine bank rently threatening their livestock sectors. Almost all and replacement of used vaccines? If international affected and at-risk countries have made strong or regional organizations are in charge of the vaccine commitments to support long-term efforts to control bank, how are country requests prioritized without H5N1 HPAI. The regional organizations such as being perceived as giving preferential treatment to Association of Southeast Asia Nations (ASEAN) one member over another? and South Asia Association for Regional Coopera- Currently, no international or regional vaccine tion (SAARC) have prioritized HPAI as a trans- banks exist for AI, as they do for some other TADs, boundary zoonotic disease of the highest signifi cance. but some individual countries have their own H5 and Recently, the ASEAN HPAI Task Force was H7 vaccine banks such as the United States. endorsed at the ministerial level to start plans for the long-term control of HPAI. SAARC, with FAO/OIE Control is Feasible: Learning from Success collaboration, is currently considering regional Stories program for the control of TADs. The tools, general methodologies, and approaches outlined in this chapter have been successfully used International Commitment to Control HPAI by several countries to control and eradicate HPAI A joint FAO/OIE initiative to control TADs glo- infections in Europe (Italy and the Netherlands) and bally (GF-TADs) has been endorsed by both 572 Avian Influenza organizations and OIE delegates (13). It has also or regional HPAI control or prevention interven- been agreed that the HPAI crisis should be addressed tions should support strengthening of such frame- under this initiative. Following the HPAI outbreaks, works to create the necessary enabling environ- FAO and OIE responded rapidly in mobilizing ments. In addition, these countries support major fi nancial resources and technical support to control poverty reduction goals and recognize that control the disease. In the fi rst 15 months since the fi rst of HPAI and other TADs will have a signifi cant H5N1 HPAI outbreaks were reported, country- positive impact on increased livestock production, specifi c support was provided to all the infected greater access to regional and world markets, and countries and subregional networks were estab- improved livelihoods for the rural poor in their lished. This regional network approach is to be emu- countries. lated elsewhere around the world with support from the international organizations, regional entities, and CONSTRAINTS AND CHALLENGES TO donor governments. Again, FAO and OIE have HPAI CONTROL established a Crisis Management Center that focuses There are many constraints to controlling HPAI, on the emergency event (i.e., regional occurrence which vary tremendously and mainly depend on the with potential global spread of a TAD), inaugurated particular country and its farming system. in mid 2006, to better serve the world in rapid deployment of experts in assisting member states in Inadequate Veterinary Services—A Major technical and strategic issues for prevention and Weakness containment of HPAI or other TADs. The OFFLU The veterinary services in several affected and at- network supports the international organizations in risk countries around the globe were, and some these endeavors through the provision of such sci- remain, inadequately equipped to deal with the entifi c professionals and practical expertise. scope, severity, and rapid spread of H5N1 HPAI. Industrialized countries in western Europe and Control measures and approaches vary signifi cantly North America, as well as countries in Asia such as in confi ned commercial (sectors 1 and 2) and semi- Japan, Korea, Australia, and New Zealand, have confi ned or free-housed rural poultry production invested heavily in imposing strict measures to systems (sectors 3 and 4). As a result, the disease control TADs but are also increasingly recognizing has become endemic, especially in the smallholder the fact that the source of many of these infections poultry sector in some countries. is in developing countries. In these countries, gov- The veterinary services also vary greatly in capa- ernments and the livestock farming communities bility and resources. Many countries will require have limited resources to control animal diseases. substantial fi nancial resources to upgrade their This recognition is leading the “disease-free” coun- capacity and train personnel to more effectively tries to focus on controlling HPAI at source rather support their national programs and participate than on building disease-free barriers. actively in regional efforts to control HPAI. In several countries, the necessary policies to establish Policy Issues common ground between public and private health Many affected or at-risk countries now recognize the service providers are unavailable to effectively need to strengthen their regulatory policy frame- support economically viable disease control for both works to enforce animal disease control measures smallholder farmers and small-scale processors and and support formal intraregional and global trade traders. Common constraints to effective disease and are realigning their veterinary regulations and detection and control in countries with weak veteri- policies to meet their agreements under the World nary infrastructure include the following: Trade Organization and OIE standards. These mechanisms include quality and evaluation of veterinary 1. Poultry farming in the region is predominantly a services, animal quarantine, institutional reforms, rural or backyard enterprise with little regulatory guidelines and recommendations for trade in live- oversight. stock and livestock products, certifi cation for 2. Animal disease information is generally lacking, exports, and designation of disease-free zones and and investigations of animal disease outbreaks compartments. Where needed, longer-term national are not thoroughly conducted or reported. 25 / Global Strategy for Highly Pathogenic Avian Influenza 573

3. Levels of consumer and farmer awareness remain mately 3 weeks. Innovative approaches to deal with low. the inability to manage a village as an epidemio- 4. Minimal emergency preparedness planning logical unit should incorporate the use of emergency exists. vaccines in contiguous households within the village, 5. Human resource capacities for human and animal active daily surveillance by owners, animal health health are limited. technicians, or offi cial veterinarians, because even 6. Intersectoral and interagency coordination is quality vaccine requires 10 to 14 days before protec- limited—for instance, veterinary regulatory ser- tive immunity develops. An intensive program of vices networking with wildlife specialist or fi nan- owner and community awareness must ensue, espe- cial planning for humanitarian crises arising from cially in terms of improved hygiene, poultry care, disease outbreaks. and the lapse between vaccination and protection. Vaccinated poultry that show signs of disease must Stamping-Out and Biosecurity Measures are be destroyed and compensation made. Diffi cult to Implement One of the most important aspects of HPAI control More Epidemiological Expertise is Needed and prevention is the application of stamping-out It is vital that countries and regions are able to incor- combined with proper indemnity to owners and porate epidemiological studies linked to disease biosecurity measures. The concept, preventing the control programs to generate quantitative and geo- spread of virus from infected premises (biocontain- referenced data on infection and transmission ment), and measures requiring the prevention of dynamics. Such information can provide a sound infectious agents from gaining entrance to unin- basis for disease investigation techniques and pre- fected premises (bioexclusion) have been very dif- vention and for the control of HPAI. However, in fi cult to practice in several countries in affected most countries, even basic epidemiological exper- regions. Particularly, wherever the disease has tise remains weak, and more modern epidemiologi- become endemic and widespread in the smallholder cal methodologies and tools such as GIS, database poultry sector and domestic ducks, standard biose- management, and analysis are not available. Records curity measures are less effective. The lack of capac- and archives with fundamental information as to ity or regulatory enforcement power to stamp out or location and operation of slaughtering and process- practice even basic biosecurity measures is one of ing plants, distribution centers, marketplaces, local the most important reasons of persistence of the butcheries, or other market chain information are disease and its spread. lacking at central and provincial offi ces. Culling of infected or potentially infected birds on a well-circumscribed farm or commercial operation Inadequate Disease Information Systems can be conducted in a systematic manner with ease. The importance of a harmonized disease informa- More diffi cult has been to implement culling in tion system linked to disease surveillance and epi- village household poultry considered an epidemio- demiological programs in countries and regions is logical unit. That is, if at least one household poul- clearly recognized. The OIE animal disease infor- try fl ock or backyard poultry system is affected, the mation system requirements, the application of village as a whole should be depopulated from which is mandatory for OIE member countries, are poultry. Although the principle for village-wide often not closely followed mostly due to lack of depopulation has merit, its application has proved to proper infrastructure. FAO’s national technical be socially and economically unacceptable in the cooperation projects have supported disease- current H5N1 HPAI crisis in many countries. As reporting systems to improve national decision such, HPAI has not come under containment, with making and meet reporting obligations to the OIE. new outbreaks being reported or recrudescence of There are also additional needs to form a subre- infection occurring when repopulation (i.e., intro- gional network for the sharing and analysis of duction of susceptible day-old chicks) takes place disease information coordinated through a subre- before cleaning and disinfection operations are com- gionally based epidemiology support unit. Such plete and a waiting period within a prescribed time infrastructure will be an important element in the in which premises should be vacant for approxi- control of HPAI. 574 Avian Influenza

Domestic Ducks are Important AI Virus limited access to veterinary care, preventive treat- Reservoirs ment, information and advice, or access to training Since mid-2004, several studies, including a retroac- or resources for even the minimal infrastructure tive analysis of the HPAI virus evolution and fi eld changes to enhance biosecurity (i.e., coops, fencing, evaluation of its spread in China, Vietnam, and or footbaths). There are also concerns that some Thailand, have shown that domestic ducks can be an genotypes of H5N1 HPAI strains might have adapted important reservoir host of the H5N1 HPAI virus (7, to backyard indigenous terrestrial poultry in the 16). The nature of farming systems in some coun- same way as observed in domestic ducks. A similar tries, where domestic ducks are moved in fl ocks situation may also exist in Pakistan with the H7 and over long distances from province to province to H9 strains. Such a situation will increase the com- feed on harvested rice fi elds, played a major role in plexity of controlling the disease, particularly in the the transmission and maintenance of the H5N1 large section of backyard population of poultry in HPAI virus. These traditional practices have made the affected countries. the conventional control measures of culling of infected fl ocks, imposition of biosecurity, and active Wildlife Reservoirs surveillance most diffi cult without strong enforce- Epidemiological studies suggest that wild birds can ment supported by enabling regulations. likely play a role in the transmission of H5N1 HPAI Signifi cant correlations were found between the viruses to domestic poultry, and vice versa (3, 8, 38). presence of free-grazing ducks and HPAI outbreaks The capacity of wild birds to carry H5N1 viruses in domestic poultry in Thailand. Twenty percent of presents a major diffi culty in applying biosecurity clinically normal ducks in the Mekong Delta of measure aiming at the avoidance of contacts between Vietnam were found to harbor the H5N1 HPAI domestic poultry and wild birds. Eradication of a virus. Thus, in the case of H5N1 HPAI, prevention known AI virus in the wild bird population is likely of the intermingling of ducks and domestic poultry not achievable, nor should governments or produc- was key to signifi cantly reduce HPAI transmission ers divert resources to attempt to do so. Focus should (16, 17). Alternatively, targeted control and elimina- be on decreasing the risk of poultry contact with tion of HPAI infection in domestic ducks, either by potential carriers among the wild birds. Although culling of infected animals and by introducing the role of wild birds in the epidemiology of LPAI disease-free domestic ducks, or by strategic vaccina- viruses is well known, the ecology and transmission tion of domestic ducks, would likely reduce or even dynamics of H5N1 HPAI viruses are far from com- eliminate that source of infection. Both Vietnam and plete. Certain species of wild birds appear to be Thailand took these fi ndings and incorporated them highly susceptible to some H5N1 HPAI viruses, into their disease mitigation and prevention strate- such as the bar-headed goose (Anser indicus) in Asia gies. In countries with substantial duck populations, or the mute swan (Cygnus olor) in Europe. It has stamping-out and control will be more diffi cult to also been hypothesized that certain wild birds can achieve. Conversely, countries with low-density serve as a “bridge species” intermingling with poultry populations, such as Cambodia and Laos, waterfowl found in a number of habitats that range will have a geographic and structural advantage in from open wetlands, forest, and even poultry farms. controlling the disease. Whether these “bridge species” act as biological or mechanical carriers of H5N1 HPAI viruses is not Disease Has Become Endemic in Some known but is currently being investigated (14, 19, Countries 20). Implementing control measures in countries with a predominantly (free-ranging) smallholder poultry Failure to Base Disease Control Planning on sector, in which the disease has dispersed widely, is Socioeconomic Impact Assessments a major challenge in the face of limited veterinary Sound economic impact assessments of HPAI infrastructure. One of the continuing dangers of control are essential to determine national economic resurging HPAI outbreaks lies in the smallholder and social intervention priorities, as well as the cost- farming systems where the disease is most diffi cult benefi t of implementing different strategies in to control. Smallholder animal producers have control programs. Such assessments should take into 25 / Global Strategy for Highly Pathogenic Avian Influenza 575 consideration trade issues, poverty reduction targets, a central secretariat. This amount of initial funds for veterinary and livestock development programs, a 6-year period also included the direct support to socioeconomic impact and economic policies, and OIE and FAO reference centers, provision of funds environmental impact and should seek strong con- for applied research competitive grants, and $6 sideration to the needs of the poor. Such analysis is million for emergency contingencies. The sum was presently lacking in most countries, and therefore designed without a crisis at hand. Under the current the targeting of disease control programs through environment of the H5N1 HPAI pandemic in birds, well-developed policy frameworks remains largely with disease continuing to trickle in Asia, Europe, empirical (22, 30). Middle East, and Africa, it is projected that over US$500 million would be required for disease control Weak Linkages with Public Sector worldwide. This estimate does not consider the actual Weak linkages between veterinary service, technical purchase and administration of quality vaccines or and planning departments, between ministries of the national expenditures required for rehabilitation agriculture, environment and natural resources or restructuring of the poultry sector. (potential wildlife issues), human health, and fi nance Although some aspects of GF-TAD have received hamper long-term planning for infectious disease extra budgetary support for FAO, OIE, and WHO, control. Given the zoonotic and transboundary current fi nancial resources have primarily focused natures of certain AI viruses, appropriate linkages on providing critically needed supplies, equipment, and policies need to be in place among a number reagents, and implementation of technical support. of ministries, and groups at the national (e.g., pro- However, the ongoing crisis of H5N1 HPAI, the vincial, district, community, and farmer levels), increasing spread of the disease, and the increasing regional, and international levels to enhance coordi- number of human cases, combined with evidence nation of HPAI control (11–13). that there are pockets of endemicity in some countries, necessitate a long-term commitment of sig- Sustainable Long-term Regional Coordination nifi cantly higher levels of fi nancial support if HPAI is Required is to be controlled worldwide. Through international support and under the GF- TADs framework, subregional networks on diagno- IMPLEMENTATION OF THE STRATEGY sis, surveillance, policy, and economics have been The strategy is being implemented at three levels: established as the fi rst regional coordination mecha- national, regional, and international. The interna- nism in the control of HPAI. Eventually, regional tional organizations understand and respect that organizations such as those listed in Table 25.3 nations are sovereign and that such a global vision should contribute to the management of such net- must be with the commitment of country member- works for long-term sustainability through the tech- ship. Thus, FAO and OIE need to work with coun- nical inputs from the FAO and OIE. The establishment tries and their regional representatives through of the OIE/FAO Animal Health Centers following technical and operational advice, advocacy for donor the GF-TAD initiatives would provide support to support, and policy reform. OIE member countries those regional specialized organizations that lack the have the obligation to follow OIE standards as they depth in technical and human resources to deliver pertain to quality veterinary services (28). strategic inputs on their own during initial stages. A description of the 14 main components and Suffi cient funding is essential for the long-term sus- activities for the global strategy follows. tainability of these coordinating efforts (13). National Level: Development of National HPAI Financial Resources Remain Inadequate Control Strategies and Programs When endorsed by the OIE General Session, the GF- The cornerstone of the global approach is to develop TAD initiative was estimated at some US$80 million national strategies in all regions. In turn, the national to set up a worldwide network for disease intelli- plans for specifi c disease control should be consis- gence, that is, the FAO/OIE/WHO’s Global Early tent with the global strategy. To this end, the inter- Warning System for TADs and zoonoses, including national organizations (FAO and OIE in the at least 13 global support units in all continents, and veterinary arena) should assist countries to develop Table 25.3. Regional and specialized organizations as existing and potential partners to the FAO/OIE Global Framework for the Pro- gressive Control of TADs and the Global Plan for the Progressive Control of HPAI. Name Acronym Member States Headquarters/URL

African Union/ AU/IBAR Algeria, Angola, Benin, Botswana, Burkina Faso, Burundi, Cameroon, Cape Verde, AU in Addis Ababa, Interafrican Central African Republic, Chad, Comoros, Côte d’Ivoire, Democratic Republic of Ethiopia Bureau for Congo, Djibouti, Egypt, Eritrea, Ethiopia, Equatorial Guinea, Gabon, Gambia, www.africa-union.org Animal Ghana, Guinea, Guinea Bissau, Kenya, Lesotho, Liberia, Libya, Madagascar, IBAR in Nairobi, Kenya Resources Malawi, Mali, Mauritius, Mauritania, Morocco, Mozambique, Namibia, Niger, www.au-ibar.org Nigeria, People’s Republic of Congo, Republic of South Africa, Rwanda, São Tomé and Principe, Senegal, Seychelles, Sierra Leone, Somalia, Sudan, Swaziland, Tanzania, Togo, Tunisia, Uganda, and Zambia Inter-Governmental IGAD Djibouti, Eritrea, Ethiopia, Kenya, Somalia, Sudan, and Uganda Djibouti, Djibouti Authority on www.igad.org Development Arab Organization AOAD Algeria, Bahrain, Djibouti, Egypt, Iraq, Jordan, Libyan Jammahiryah, Kingdom of Khartoum, Sudan

576 for Agricultural Saudi Arabia, Kuwait, Lebanon, Mauritania, Morocco, Oman, Palestine, Qatar, www.aoad.org Development Somalia, Sudan, Syria, Tunisia, United Arab Emirates, and Yemen Economic ECOWAS Benin, Burkina Faso, Cape Verde, Côte d’Ivoire, Gambia, Ghana, Guinea, Guinea Abuja, Nigeria Community of Bissau, Liberia, Mali, Mauritania, Niger, Nigeria, Senegal, Sierra Leone, and www.ecowas.int West African Togo States Southern African SADC Angola, Botswana, Democratic Republic of Congo, Lesotho, Madagascar, Malawi, Gaborone, Botswana Development Mauritius, Mozambique, Namibia, Republic of South Africa, Swaziland, United www.sadc.int Community Rep. of Tanzania, Zambia, and Zimbabwe Union du Maghreb UMA Morocco, Tunisia, Algeria, and Mauritania Rabat, Morocco Arabe www.maghrebarabe.org Arab Gulf GCC Bahrain, Kuwait, Oman, Qatar, Kingdom of Saudi Arabia, and United Arab Riyadh, Saudi Arabia Coordination Emirates www.gcc-sg.org Council Pan American PAHO Antigua and Barbuda, Argentina, Bahamas, Barbados, Belize, Bolivia, Brazil, Washington, D.C., USA Health Canada, Chile, Colombia, Costa Rica, Cuba, Dominica, Dominican Republic, www.paho.org Organization Ecuador, El Salvador, Grenada, Guatemala, Guyana, Haiti, Honduras, Jamaica, Mexico, Nicaragua, Panama, Paraguay, Peru, Saint Lucia, St. Vincent and the Grenadines, St. Kitts and Nevis, Suriname, Trinidad and Tobago, USA, Uruguay, and Venezuela Inter-American IICA North America—Canada, Mexico, and USA San Jose, Costa Rica Institute for www.iica.int Cooperation on Agriculture Central region—Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, and Panama Caribbean—Antigua and Barbuda, Bahamas, Barbados, Dominica, Dominican Republic, Grenada, Guyana, Haiti, Jamaica, Saint Lucia, St. Kitts and Nevis, St. Vincent and the Grenadines, Suriname, and Trinidad and Tobago Andean region—Bolivia, Colombia, Ecuador, Peru, and Venezuela Southern region—Argentina, Brazil, Chile, Paraguay, and Uruguay Europe—Spain Organización OIRSA Mexico, Guatemala, El Salvador, Honduras, Nicaragua, Costa Rica, Panama, Belize, San Salvador, El Salvador Internacional de and Dominican Republic www.oirsa.org Sanidad Agropeacuaria Caribbean CARICOM Antigua and Barbuda, Bahamas, Barbados, Belize, Dominica Grenada, Guyana, Georgetown, Guyana

577 Community Haiti, Jamaica, Montserrat, St. Kitts and Nevis, Saint Lucia, St. Vincent and The www.caricom.org Grenadines, Suriname, and Trinidad and Tobago South Asian SAARC Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan, and Sri Lanka Kathmandu, Nepal Association for www.saarc-sec.org Regional Cooperation Economic ECO Islamic State of Afghanistan, Azerbaijan Republic, Islamic Republic of Iran, Tehran, Iran Cooperation Republic of Kazakhstan, Kyrgyz Republic, Islamic Republic of Pakistan, Republic www.ecosecretariat.org Organization of Tajikistan, Republic of Turkey, Turkmenistan, and Republic of Uzbekistan Association of ASEAN Brunei Darussalam, Cambodia, Indonesia, Laos, Malaysia, Myanmar, Philippines, Jakarta, Indonesia Southeast Asian Singapore, Thailand, and Vietnam. ASEAN + 3 (includes ASEAN countries, plus www.aseansec.org Nations PR China, Republic of Korea, and Japan) European Union/ EU/EC Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, Brussels, Belgium European France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Lithuania, http://europa.eu/ Commission Luxembourg, Malta, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, the Netherlands, and United Kingdom European EU-FMD Albania, Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Rome, Italy Commission for Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Israel, Italy, www.fao.org/ag/againfo/ the Control of Lithuania, Luxembourg, FYR Macedonia, Malta, The Netherlands, Norway, commissions/en/eufmd/ Foot-and-Mouth Portugal, Poland, Romania, Slovenia, Spain, Sweden, Switzerland, Turkey, United eufmd.html Disease Kingdom, and Yugoslavia 578 Avian Influenza their own country-specifi c disease control plans. A systems. Ensure timely disease reporting to inter- typical country-specifi c project should adopt a task national and regional organizations, including force approach, guided by a national steering com- neighboring nations and trading partners. mittee to support a comprehensive HPAI control 9. Strengthen links between technical and planning program. The function of the task force is to coor- departments and between ministries of agricul- dinate and monitor a sound disease control program. ture, human health, environment, and fi nance to The task force is expected to be led by the veterinary improve capacity for long-term strategic plan- services with strong linkages with ministries of ning and response to emergency situations. health, environment, planning, and fi nance; public works; institutions of security; extension services; Regional Level: Subregional Cooperation and community workers; and nongovernment organiza- Collaboration tions (NGOs). The national steering committee The cornerstone of the regional strategy is through should be composed of key decision makers and the establishment of strong and effi cient regional stakeholders, particularly of the private sector, and coordination units constituted to ensure that the may include national experts, research institutes, disease control plans are implemented in a system- and NGOs. Important areas of focus at national level atic, coordinated, and phased manner. These regional are as follows: coordination units are to support country-specifi c studies and to fi nance efforts to control HPAI. Several 1. Baseline evaluation of the veterinary services and subregional HPAI support units are currently in infrastructure for self-evaluation or under the operation in each of the three subregions of southeast guidance of the Performance, Vision and Strat- Asia, east Asia, and south Asia; two in eastern Europe egy for Veterinary Services (28). These evalua- and Caucasus; one in the Middle East; and four in tions are guided by OIE-accredited assessors Africa. An additional four are being planned in Latin using the performance, vision, and strategy as a America and the Caribbean. All these can become tool to identify needs and gaps compared with the the regional support units sought in GF-TAD initia- OIE Code. tive. The focal points for these units are expected to 2. Strengthen legal and institutional frameworks to be regional specialized organizations or, where create an enabling environment for supporting needed, the OIE representations or FAO regional or control or prevention of HPAI, as well as other subregional offi ces as OIE/FAO Animal Health TADs. Centers at the service of member nations, as described 3. Strengthen national veterinary services, on the in GF-TAD (13). Through these units, subregional basis of gap analysis following the evaluation of networks for disease surveillance, diagnosis, and veterinary service. information and for policy development and eco- 4. Develop and implement effective HPAI control nomic impact assessment will be further strength- programs. ened and followed up by specifi c international 5. Develop prevention measures for the introduc- organization projects (i.e., FAO Technical Coopera- tion of HPAI in countries currently free from the tion Program projects Special Fund for Emergency disease. and Rehabilitation Activities) or donor-supported 6. Provide socioeconomic impact assessments on programs. Other important issues that the regional disease control strategies and, where necessary, networks will address will include the following: provide an objective assessment of the impacts on the different stakeholders. 1. Institutionalized and intersectoral coordination 7. Prepare contingency and emergency prepared- 2. Good governance in program management ness plans for each country. Plan and execute 3. Capacity in diagnosis, surveillance, and epide- fi eld test exercises and immediately take correc- miological analysis tives measures to rectify defi cits. 4. Capacity in policy and socioeconomic impact 8. Improve capacity at the national level in diagno- assessment sis, epidemiology, disease surveillance, and early 5. Public awareness and communication detection and reporting, and disease information 6. Research and development 25 / Global Strategy for Highly Pathogenic Avian Influenza 579

International Level: Global Coordination Development of Regional Projects in New The global nature of HPAI necessitates countries to Regions at Immediate Risk engage in internationally agreed plans for the control The international strategy outlines the prevention of TADs. An international support facility to coor- activities in the event of HPAI introduction. In case dinate the implementation of the strategy is a pre- the disease is newly introduced, an adequate and requisite, as prescribed under the GF-TAD rapid response is necessary in preventing economic mechanism, the FAO and OIE global initiative to impact for the poultry sector, minimizing the chances control TADs and certain zoonoses. The main for HPAI endemicity, and thereby preventing risk to responsibility of such a facility will be to human health.

1. Forge a cohesive partnership among the three Epidemiology-Based Control Measures international organizations: FAO, OIE, and The lack of reliable epidemiological information, WHO. and the sound analysis thereof, has hampered the 2. Coordinate the subregional networks. development of rational, targeted disease control 3. Fully develop the Global Early Warning System measures in many countries. Thus, well-structured (GLEWS) to enable better integration and analy- epidemiological studies and surveillance programs ses on the emergence of new infectious diseases, will be integrated with the disease control measures, including HPAI. which will then be adjusted and improved as new 4. Expand the FAO and OIE Crisis Management information becomes available. Center in Rome when disease events of interna- The following country-specifi c risk-based sur- tional importance extend the capacity for routine veillance strategies will be used: assistance to member countries in their disease control activities. 5. Provide technical and operational assistance to 1. Thorough knowledge of poultry census and pro- subregional networks through the OIE and FAO ducers categorized at the lowest administrative epidemiology collaborating centers, World Ref- level erence Laboratories, including OFFLU. 2. Establishment of case defi nition for HPAI in 6. Play a strategic role in coordinating research in poultry species improved tools for HPAI control. 3. Identifi cation of factors governing infection 7. Provide a global vision for HPAI control dynamics should complement simple case strategy. fi nding 8. Mobilize and allocate resources for HPAI control 4. Determination of potential disease transmission and prevention through active donor liaison pathways along the production and market activities. chains 5. Molecular characterization of HPAI virus strains Given the interface between animal and human from birds and other animals to determine geo- disease and the concern as to food safety issues, graphical locations and genetic changes strengthening the partnership between FAO, OIE, 6. Evaluation of the level of human exposure in and WHO will be important. A number of common different circumstances to determine risks of activities, such as disease information sharing, joint human-to-human transmission fi eld investigations, epidemiological studies, sharing of virus strains, contingency planning, and public Participatory methodologies involving farmers, awareness, are essential. Such a partnership is to be veterinary paraprofessionals, and community work- proactive in the organization and implementation ers is to be used extensively, given the fact the major of joint regional and international workshops and control targets are the small-scale and semicommer- meetings with public and private sectors of mem- cial poultry production systems 3 and 4 (see Table ber countries and enhance cooperation among the 25.3). Comprehensive surveillance programs are to various OIE/FAO and WHO collaborating centers be planned and implemented jointly with the public and reference laboratories. health personnel. 580 Avian Influenza

Disease Information Systems essential. This will include fi eld workers involved in A uniform disease information system should exist disease identifi cation and outbreak investigations, for the veterinary services as part of their control farm workers involved in culling, and laboratory programs to provide better analytical capacity, above workers tasked in sample handling, virus isolation, and beyond meeting the reporting obligations and agent diagnosis. Adequate resources are to be required by the OIE. The system should be linked allocated for biological safety hoods in laboratory with rapid and standardized methods of routine settings and appropriate personal protective equip- analysis of surveillance data, which will demon- ment (PPE) for fi eld or laboratory use. strate important changes in the HPAI and other Disease Control Programs and Poverty disease situations, and promptly supply this infor- Alleviation mation to fi eld personnel. The activation of such Many of the world’s poor rely on small-scale or systems should go hand in hand with training in backyard poultry raising for their livelihood. This epidemiological principles so that sound data an - demographic group has limited or no access to alysis becomes possible for improved decision veterinary care delivery. In the global strategy, making. particular support for these low-income groups is Targeting the Source considered: In developing country-specifi c HPAI control strate- 1. Improving animal health services at the village gies and programs, the broad principle of targeting level by means of organizing community-based the disease at source of infection will be applied. In early warning networks, utilizing the existing the case of H5N1 HPAI, this refers predominantly pool of veterinary paraprofessional village to the smallholder poultry sector or a domestic duck workers, or their establishment population, a major carrier host reservoir. Wild birds 2. Increasing farmers’ general awareness through are also implicated as reservoirs of AI viruses, but simple biosecurity guidelines on infectious the global strategy emphasizes the importance of disease prevention and control using publications enhancing biosecurity (bioexclusion) by promoting in local languages ways to keep wild birds away from poultry (i.e., 3. Implementation of mechanisms to compensate screens on coops or windows, the use of nets, seg- farmers for losses and improve early reporting regation of species, and other interventions). and rapid implementation of disease containment Elimination of the virus source from backyard measures poultry will be a diffi cult and long-term task, espe- 4. Providing access to credit or microfi nance as a cially in poor countries with limited resources. tool for rehabilitation as an alternative to direct Where duck farming is important, the strategy compensation, which some countries may not be underlines disease control options, including restruc- able to afford turing of domestic duck farming systems to separate 5. Developing farmer groups and/or associations to domestic ducks from terrestrial poultry and wild help improve awareness and dissemination of birds, strategic culling of infected domestic ducks, information and an increased voice in local and and progressively enhancing fl ock immunity through national matters systematic vaccination campaigns to reduce the viral load in an environment and virus shedding. The Restructuring and Rehabilitation of the Poultry short- to medium-term task of controlling the disease Sectors by reducing virus circulation in the industrial poultry Restructuring the poultry sector may be an important production sector, large-scale breeder units, and strategy to guard against the damaging effects of medium to small-sized commercial units is logisti- HPAI but is also one of the most complicated inter- cally far more feasible. ventions to be undertaken requiring understanding of the whole socioeconomic system. Restructuring Personal Safety Issues requires different approaches at different levels Due to the infectious nature of certain HPAI virus within a poultry sector possibly requiring a range of to humans, particularly H5N1 and H7N7, training of processes in varied areas in a single country, by people in biosecurity and biosafety procedures is virtue of the differences in their poultry sector 25 / Global Strategy for Highly Pathogenic Avian Influenza 581 infrastructures, marketing characteristics, backyard Collaboration with Stakeholders versus commercial poultry production, and socio- The multidimensional problems associated with economic impact. Restructuring should be seen as a HPAI and other TAD infections necessitate collabo- gradual process, affecting the various segments of ration from a wide range of stakeholders within each the sector in different ways and at different rates. country. These include Because of these variations, only the general principles that may be undertaken are outlined: 1. Various sectors such as veterinary service, planning, fi nance, agriculture, health, environment, 1. Rationale for restructuring should be based on a public works and transport, livestock depart- well-defi ned socioeconomic impact analysis, ments, security, national research institutions, taking into account the interests of all stake- and diagnostic laboratories holders. 2. Private sector professionals and allied industries 2. Government commitment with full support from (e.g., large poultry production companies, feed stakeholders is necessary and must follow a long- mills, drug and vaccine companies, breeder term strategy. farms, farmers’ associations, private veterinari- 3. Livelihoods of smallholder poultry farmers, who ans and paraprofessionals, and farmer involve- represent the majority of poultry in many HPAI- ment at the grass roots level), which have a affected countries, should be taken into account. responsibility and a role in providing timely and 4. Market forces should drive the restructuring accurate disease information to their clients strategy, taking into account commercial and through awareness messages, product descrip- smallholder poultry producers. tions and their availability, and direct contact 5. Public and private sectors should collaborate and through sales representatives and technical be transparent in the implementation of restruc- staff turing strategies. 3. In cases of zoonotic disease, one of the more 6. Restructuring should be an integral part of an important linkages will be between the animal overall disease control strategy that includes health and the public health sector. Disease pre- biosecurity, vaccination, zoning and/or compart- vention and control plans, and surveillance pro- mentalization, should follow OIE and FAO grams, could be jointly authored and conducted guidelines, and should take into account issues of according to the mandates provided by country human and food safety. legislation. 7. Public awareness should be promoted to gain support from producers, consumers, government agencies, private sector institutions, and other Capacity Building stakeholders. The development of a robust veterinary service with a sustainable human resource base is one of the most Compartmentalization and Zoning important objectives of country-specifi c emergency Given the current epidemiological characteristics disease control strategies. There is a great variation of H5N1 HPAI in various affected countries, com- in capacity to deal with serious outbreaks of infec- plete eradication may not be achieved in the next 5 tious disease across the world, and the capacity to 10 years and then only in poultry. Therefore, building needs to be tailored to specifi c circum- compartmentalization and zoning concepts, as stances prevailing in each country. Therefore, it is described in the OIE Terrestrial Animal Health crucial that the needs of a country be evaluated for Code, will be an important tool in preventing rein- short-, mid-, and long-term planning in developing fection and assisting the recovery of marketing training objectives. The PVS evaluation tool devel- opportunities for many affected countries, especially oped by the OIE, in collaboration with the Inter- those with potential poultry export industries. Thus, American Institute for Cooperation on Agriculture in considering disease prevention and control strate- (IICA), serves such a purpose (28). Planning and gies, progressive control focuses on developing training should be incorporated at the early stages of disease-free compartments and zones, made safe a professional’s career or even during their academic from reinfection. preparation. 582 Avian Influenza

Applied Research 9. The appearance of new endemic reservoirs of While a range of methodologies and tools are avail- domestic poultry will be minimized. able to control HPAI, a number of aspects of AI 10. Risk of human pandemic will be progressively ecology are not clearly understood and, should they minimized, and safe trade in poultry reestab- be researched, could contribute to improving inter- lished. national, regional, and local strategy development or implementation. The key among these issues are IMPACTS The successful implementation of the global strat- 1. To elucidate the role of wild birds, backyard egy will directly impact the livelihoods of millions indigenous poultry, or swine in maintenance and of commercial, smallholder, and backyard poultry transmission of AI viruses farmers and their stakeholders, and contribute sig- 2. To determine the effi cacy of AI vaccines in nifi cantly to the achievement of the Millennium various species of birds other than chickens Development Goals, especially those focused on 3. To develop novel diagnostic methodologies and infection diseases, hunger, and poverty alleviation. improved technologies for enhanced vaccine effi - It is expected that these outcomes will positively cacy and delivery impact poverty reduction of resource-poor small- 4. To assess the role of vaccination in reducing holder farmers, improve food safety, and decrease virus shedding in a fi eld setting health hazards for consumers and provide better 5. To determine the appropriate strains to be used market opportunities for poultry producers at all in vaccine formulation in each country economic levels. 6. To identify major risk factors for transmission of zoonotic AI viruses to humans from domestic Budgetary Needs poultry Of the total indicative budget foreseen for global 7. To identify socioeconomic issues and market- HPAI intervention, between 80% and 90% should driven forces that play in disease dissemination be allocated to country-specifi c activities, between 10% and 15% to regional activities, and less than A PROMISING OUTLOOK 5% to international activities such as coordination, Successful implementation of the global strategy global epidemiological analysis, and tracking and would result in the following major outputs. early warning systems and 2% for investigation and research. Generally, of the total budget, 65% will be 1. HPAI spread in poultry will be contained, and earmarked for disease control (laboratory upgrad- spill-over transmission to other species (including, fi eld surveillance, biosecurity, vaccination strat- ing humans) minimized. egies and campaigns), 25% for training and capacity 2. HPAI incidence in poultry will be progressively building, 5% for institution building such as policy reduced. development and socioeconomic impact assessment, 3. Progressive HPAI eradication in all com- and 5% for support public awareness programs. mercial farming systems and zones will be achieved. CONCLUSION 4. Introduction or establishment of HPAI viruses Of all the known HPAI outbreaks recorded around will be prevented in noninfected countries. the world, no viral strain had such a wide geo- 5. Emergency preparedness plans for HPAI and graphical and social impact of the H5N1 HPAI other incursions of TADs will be improved. virus. 6. National, subregional policies on HPAI preven- The international strategy for HPAI as developed tion and control will be available and imple- by the leading international animal health organiza- mented through a strengthened veterinary tions (FAO and OIE) is biased to the experience of service. various genotypes of H5N1 since its fi rst description 7. Enhanced HPAI control capacity will be in 1997 and its epidemic character of 2003–2006. developed. All other HPAI outbreaks dwindle in comparison 8. Improved understanding of virus ecology will with the events starting in 2003 and the effects of be available. H5N1 HPAI on the poultry sector, repercussion to 25 / Global Strategy for Highly Pathogenic Avian Influenza 583 human health, biological diversity and conservation, oil emulsion vaccines. American Journal of Vet- and socioeconomic impact. Arguably, the human erinary Research 40:165–169. pandemic during the fi rst part of 20th century due to 3. Capua, I., and D.J. Alexander. 2006. The challenge H1N1 virus was responsible for millions of human of avian infl uenza to the veterinary community. illness and death as well as social and civil society Avian Pathology 35:189–205. 4. Capua, I., S. Marangon, P.M. Dalla, and U. Can- upheaval; it was not derived from an HPAI virus but tucci. 2000. Vaccination for avian infl uenza in an LPAI virus of possible avian origin. Italy. Veterinary Record 147:751. Broadly, the international strategy for HPAI takes 5. Capua, I., C. Terregino, G. Cattoli, F. Mutinelli, on a complex matrix of situations. In one instance, and J.F. Rodriguez. 2003. Development of a DIVA it must understand the different poultry sectors (differentiating infected from vaccinated animals) (FAO sectors 1 thought 4) and it must have compo- strategy using a vaccine containing a heterologous nents for prevention and emergency preparedness, neuraminidase for the control of avian infl uenza. detection and early response, and rehabilitation and Avian Pathology 32:47–55. restructuring of an affected poultry sector. It must 6. Chen, H., G.J.D. Smith, K.S. Li, J. Wang, X.H. be inclusive of commercial and trade aspects as well Fan, J.M. Rayner, D. Vijaykrishna, J.X. Zhang, as protect the small producer or family farm from L.J. Zhang, C.T. Guo, C.L. Cheung, K.M. Xu, L. Duan, K. Huang, K. Qin, Y.H.C. Leung, W.L. Wu, being eliminated from its livelihood sustenance. The H.R. Lu, Y. Chen, N.S. Xia, T.S.P. Naipospos, international strategy must have not only a global K.Y. Yuen, S.S. Hassan, S. Bahri, T.D. Nguyen, view but also regional, national, and local interven- R.G. Webster, J.S.M. Peiris, and Y. Guan. 2006. tion measures in a continuum. The strategy also Establishment of multiple sublineages of H5N1 incorporates short-term (immediate-term) needs as infl uenza virus in Asia: implications for pandemic well as mid- and long-term vision. Last, the strategy control. Proceedings of the National Academy of at all levels (international to local) should incorpo- Sciences U S A 103:2845–2850. rate specifi c elements: (1) enabling legislation; (2) 7. Chen, H., G.J.D. Smith, S.Y. Zhang, K. Qin, J. emergency management structure for disasters (i.e., Wang, K.S. Li, R.G. Webster, J.S.M. Peiris, HPAI incursion); (3) surveillance and epidemiology and Y. Guan. 2005. H5N1 virus outbreak in migra- (including risk analysis); (4) laboratory services tory waterfowl: a worrying development could help to spread this dangerous virus beyond its (diagnostic); (5) inspection, quarantine, border stronghold in southeast Asia. Nature 436:191– control, and movement management; (6) communi- 192. cations (including disease recognition, safe food 8. Deogu, M., M.A. De Marco, I. Donatelli, L. Cam- handling practices, and behavioral changes of risk pitelli, and E. Catelli. 2003. Ecological aspects of practices); (7) production rehabilitation and restruc- infl uenza A virus circulation in wild birds of the turing for the different sectors—industry and house- western Palearctic. Veterinary Research Commu- hold; (8) emergency preparedness and contingency nications 27(Suppl. 1):101–106. planning; and (9) research and development (viro- 9. Edwards, S. 2006. OFFLU Network on Avian logical, diagnostic, vaccine, epidemiological, and Infl uenza. Emerging Infectious Diseases 8:1287– socioeconomic issues). These elements are essential 1288. for this specifi c strategy, but most importantly they 10. Food and Agriculture Organization of the United Nations. 2004. Recommendations on the Preven- will prepare the global community to better manage tion, Control and Eradication of Highly Pathogenic any serious emerging disease in the future. Avian Infl uenza (HPAI) in Asia, September 2004. Available at http://www.fao.org/docs/eims/ REFERENCES upload/165186/FAOrecommendationsonHPAI. 1. Alexander, D.J. 1993. Orthomyxovirus infections. pdf. p. 59. In: McFerran J.B. & McNulty M.S. (eds.) Viral 11. Food and Agriculture Organization of the United Infections of Vertebrates, Volume 3: Viral Infec- Nations and World Organization for Animal tions of Birds. Elsevier: Amsterdam, pp. 287– Health. 2005. A Global Strategy for the Progres- 316. sive Control of Highly Pathogenic Avian Infl uenza 2. Brugh, M., C.W. Beard, and H.D. Stone. 1979. (HPAI). May 2005. Available at http://www.fao.org/ Immunization of chickens and turkeys against docs/eims/upload/189432/AI_Global%20Strategy. avian infl uenza with monovalent and polyvalent doc. p. 55. 584 Avian Influenza

12. Food and Agriculture Organization of the United 22. McLeod, A., N. Morgan, A. Prakash, and J. Hin- Nations and World Organization for Animal richs. 2006. Economic and social impacts of avian Health. 2005. A Global Strategy for the Progres- infl uenza. Food and Agriculture Organization of sive Control of Highly Pathogenic Avian Infl uenza the United Nations. Available at http://www.fao. (HPAI). September 2005. Available at http://www. org/AG/AGAInfo/subjects/en/health/diseases- fao.org/docs/eims/upload//210745/Glo_pro_ cards/cd/documents/Economic-and-social- HPAI_oct05_en.pdf. p. 85. impacts-of-avian-infl uenza-Geneva.pdf. Accessed 13. Food and Agriculture Organization of the United August 1, 2006. Nations and World Organization for Animal 23. Munster, V.J., A. Wallensten, C. Baas, G.F. Rim- Health. 2004. The Global Framework for the Pro- melzwaan, M. Schutten, B. Olsen, A.D.M.E. gressive Control of Transboundary Animal Dis- Osterhaus, and R.A.M. Fouchier. 2005. Mallards eases. Available at http://www.fao.org/ag/againfo/ and highly pathogenic avian infl uenza ancestral programmes/en/empres/documents/docs/GF- viruses, Northern Europe. Emerging Infectious TADsMay2004.pdf. pp. 1–40. Diseases 11:1545–1551. 14. FAO/OIE. 2006. International Scientifi c Confer- 24. National Wildlife Health Centre. 2006. Referenced ence on Avian Infl uenza and Wild Birds, Rome, Reports of Highly Pathogenic Avian Infl uenza Italy, May 30–31, 2006. Available at http://www. H5N1 in Wildlife and Domestic Animals. Availa- fao.org/docs/eims/upload/213826/AI_recomman- ble at http://www.nwhc.usgs.gov/disease_infor- dationswildbirds.pdf. pp. 1–8. mation/avian_influenza/affected_species_chart. 15. Gale, P. 2006. BSE risk assessments in the UK: a jsp. Accessed April 1, 2006. risk tradeoff? Journal of Applied Microbiology 25. Newcomb, J. 2005. Economic Risks Associated 100:417–427. with an Infl uenza Pandemic (Prepared Testimony 16. Gilbert, M., P. Chaitaweesub, T. Parakamaswonga, before the United States Senate Committee on S. Premashthira, T. Tiensin, W. Kalpravidh, H. Foreign Relations) November 9, 2005. BioEco- Wagner, and J. Slingenbergh. 2006. Free-grazing nomic Research Associates (Bio-ERA). Available ducks and highly pathogenic avian infl uenza, at http://www.senate.gov/~foreign/testimony/2005/ Thailand. Emerging Infectious Diseases 12:227– NewcombTestimony051109.pdf. p. 4. Accessed 234. July 15, 2006. 17. Gilbert, M., X. Xiao, J. Domenech, J. Lubroth, V. 26. OIE (World Organization for Animal Health). Martin, and J. Slingenbergh. 2006. Anatidae migra- 2004. In: Manual of Diagnostic Tests and Vaccines tion in the Western Paleartic and spread of highly for Terrestrial Animals (Mammals, Birds and pathogenic avian infl uenza H5N1 virus. Emerging Bees), 5th Ed. OIE: Paris. Infectious Diseases 12:1650–1656. 27. OIE (World Organization for Animal Health). 18. Ito, T., K. Okazaki, Y. Kawaoka, A. Takada, 2005. Available at http://www.oie.int/eng/normes/ R.G. Webster, and H. Kida. 1995. Perpetuation mmanual/A_00037.htm. Accessed August 15, of infl uenza A viruses in Alaskan waterfowl 2006. reservoirs. Archives of Virology 140:1163– 28. OIE (World Organization for Animal Health) and 1172. IICA (Inter-American Institute for Cooperation on 19. Kou, Z., F.M. Lei, J. Yu, Z.J. Fan, Z.H. Yin, C. Agriculture). 2006. Performance, Vision and Strat- Jia, X, K.J. Xiong, Y.H. Sun, X.W. Zhang, X.M. egy for Veterinary Services. OIE: Paris. Available Wu, X.B. Gao, and T. Li, X. 2005. New genotype at http://www.oie.int/downld/Prep_conf_Avian_ of avian infl uenza H5N1 viruses isolated from tree inf/A_Final_PVS.pdf. sparrows in China. Journal of Virology 79:15460– 29. OIE (World Organization for Animal Health) 15466. and FAO (Food and Agriculture Organization). 20. Kwon, Y.K., S.J. Joh, M.C. Kim, Y.J. Lee, J.G. 2005. Ensuring Good Governance to Address Choi, E.K. Lee, S.H. Wee, H.W. Sung, J.H. Kwon, Emerging and Re-Emerging Animal Disease M.I. Kang, and J.H. Kim. 2005. Highly pathogenic Threats. Available at http://www.oie.int/downld/ avian infl uenza in magpies (Pica pica sericea) in Good_Governance/A_good_gouvernance.pdf. South Korea. Journal of Wildlife Diseases 41:618– OIE: Paris, France. p. 24. 623. 30. Otte, M.J., R. Nugent, and A. McLeod. 2004. 21. Lee, C.W., D.A. Senne, and D.L. Suarez. 2004. Transboundary animal diseases: Assessment of Effect of vaccine use in the evolution of Mexican socioeconomic impacts and institutional responses. lineage H5N2 avian infl uenza virus. Journal of Livestock Policy Discussion Paper No. 9. FAO: Virology 78:8372–8381. Rome, Italy, p. 46. 25 / Global Strategy for Highly Pathogenic Avian Influenza 585

31. Pattnaik, B., A.K. Pateriya, R. Khandia, C. Tosh, 35. Swayne, D.E., M. Garcia, J.R. Beck, N. Kinney, S. Nagarajan, S. Gounalan, H.V. Murugkar, B.P. and D.L. Suarez. 2000. Protection against diverse Shankar, N. Shrivastava, P. Behera, S. Bhagat, J. highly pathogenic avian infl uenza viruses in S.M. Peiris, and H.K. Pradhan. 2006. Phylogenetic chickens immunized with a recombinant fowl analysis revealed genetic similarity of the H5N1 pox vaccine containing an H5 avian infl uenza avian infl uenza viruses isolated from HPAI out- haemagglutinin gene insert. Vaccine 18:1088– breaks in chickens in Maharashtra, India with 1095. those isolated from swan in Italy and Iran in 2006. 36. Tumpey, T.M., R. Alvarez, D.E. Swayne, and D.L. Current Science 91:77–81. Suarez. 2005. Diagnostic approach for differentiat- 32. Swayne, D.E., and B. Akey. 2005. Avian infl uenza ing infected from vaccinated poultry on the basis control strategies in the United States of America. of antibodies to N1S, the nonstructural protein of In: R.S. Schrijver and G. Koch (eds.). Avian Infl u- infl uenza A virus. Journal of Clinical Microbiol- enza Prevention and Control, Wageningen UR ogy 43:676–683. Frontis Series. Springer: Dordrecht, the Nether- 37. Uppal, P.K. 2000. Emergence of Nipah virus in lands, pp. 103–130. Malaysia. Annals of the New York Academy of 33. Swayne, D.E., J.R. Beck, M.L. Perdue, and C.W. Sciences 916:354–357. Beard. 2001. Effi cacy of vaccines in chickens 38. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. against highly pathogenic Hong Kong H5N1 avian Chambers, and Y. Kawaoka. 1992. Evolution and infl uenza. Avian Diseases 45:355–365. ecology of infl uenza A viruses. Microbiological 34. Swayne, D.E. 2003. Vaccines for List A poultry Review 56:152–179. diseases. Emphasis on avian infl uenza. In: F. 39. World Bank. 2003. World Development Report Brown and J. Roth (eds.). Vaccines for OIE List A 2004: Making Services Work for Poor People. and Emerging Animal Diseases. Developments in World Bank and Oxford University Press: Wash- Biologicals (Basel). 114:201–212. ington, DC, p. 288. Index

Adaptation Anseriformes, 7. See also Aquatic birds HP AI H5N1 virus civets from, 112– of anseriformes to AI, 32 AI adaptation with, 32 13 of charadriiformes to AI, 32 AI distribution among species of, 43, HP AI H5N1 virus control strategies of human infl uenza and pathogenicity, 45 of, 271–74 32–33, 458 AI isolation among species of, 44t HP AI H5N1 virus from, 251–53 of pigs to AI and pathogenicity, 32– reservoirs in, 46 HP AI H5N1 virus outbreaks in, 33 Antigenic drift 218t of poultry to AI transmission, human infl uenza epidemics caused HP AI H5N1 virus westward 68–69 by, 455 movement from, 262–65, 563–65 of turkeys to AI transmission, 69 human infl uenza vaccination and, HP AI in poultry impacting food and Adjuvants, 417, 421. See also Oil 14 economic livelihood in, 546–47 adjuvants poultry and, 14–15 humans and HP AI H5N1 virus from, Africa. See also Egypt; South Africa Antigenic shift 110–11 HP AI H5N1 virus control strategies human infl uenza from, 15 mammal and avian summary data for of, 274–75 human infl uenza pandemics from, HP AI H5N1 virus from, 101t HP AI H5N1 virus outbreaks in, 461–62 mammals and lesions from HP AI 218t pigs impacted by, 15–16 H5N1 virus from, 111t HP AI in poultry impacting poultry impacted by, 15 mammals with HP AI H5N1 virus economics in, 545t Antiviral drugs, 24–25, 461 from, 109–10 Agar gel immunodiffusion assay Aptosis, 91 MDTs for HP AI H5N1 virus from, (AGID) Aquatic birds 100–101 AI confi rmation through, 300 LP AI and HP AI between poultry MDTs in chickens with HP AI H5N1 for antibody detection, 305 and, 72f virus from, 103t principles of, 301–2 LP AI reservoirs in, 63–64 MDTs in ducks with HP AI H5N1 seven-well pattern used for test by, Asia. See also Hong Kong; North virus from, 106t 301f Korea; Vietnam mink and HP AI H5N1 virus from, AGID. See Agar gel immunodiffusion Australia and risks posed by HP AI 112 assay H5N1 virus from, 248 pathobiology of HP AI from, 100– AI. See Avian infl uenza clades of HP AI H5N1 virus from, 102 Aldehydes, 398 254t pigs and HP AI H5N1 virus from, Alkaline hydrolysis dogs and HP AI H5N1 virus from, 111 cost associated with, 348 112 poultry diseases and writers from, disposal methods of poultry carcasses ducks and HP AI H5N1 virus from, 151 through, 347–48 102, 104–5, 104f, 107f, 109–10f, seals and HP AI H5N1 virus from, mobile unit for, 348f 266–67 111–12 Amino acid ducks and necrosis from HP AI sparrows and HP AI H5N1 virus HA representative isolate subtypes H5N1 virus from, 108f from, 105f and sequence of, 7f ducks pathobiology groups with HP wild birds and HP AI H5N1 virus HP AI and PCS insertions of, AI H5N1 virus from, 106t from, 105, 107–9 28–29 felids and HP AI H5N1 virus from, Audit. See Biosecurity audit nucleotide accumulated insertions 112 Australia. See also New South Wales; resulting in codon of, 28 fowl plague spread across, 173–74 Queensland; Victoria PCS of HA in Australian H7 subtype genetic studies on HP AI H5N1 virus amino acid sequence around PCS of and sequence of, 242t from, 253–55, 257–58 HA of H7 subtype in, 242t purine stretches duplication giving Hong Kong and disease from HP AI control strategies for HP AI in, 247– rise to, 29 H5N1 virus from, 259–62 48 virulence and PCS changes in, 27 HP AI control strategies for targeted HA similarities in H7 subtype in, Animal welfare, 314 countries in, 566t 246t

Australia (continued) survival time of AI in environment Germany and spreading of fowl HP AI H5N1 virus from Asia and plan for, 356–57 plague from, 169 impacting, 248 tasks and circumstances for, 373 Greve and recording of fowl plague HP AI H7 subtype origins in, 246–47 template for small producer and plan at, 168–69 HP AI in, 239 for, 356t Burnet, F.M., 163 HP AI in wild birds in, 239–40 unusual population plans for, 361 HP AI outbreaks and lessons learned USA control strategies for poultry Calcium hydroxide, 399 in, 245 with, 515–17 Calcium oxide, 399 HP AI variations in clinical disease vaccination and high level of, 431 California, 518f in, 245–46 vaccination as alternative to, 353 Canada Austria, 167–68 Vietnam and costs of vaccination chickens and lesions from outbreak of Avian infl uenza (AI). See related topics and, 539–40 HP AI H7N3 virus in, 207 wild bird movement patterns and, 362 HP AI H5N9 virus in, 198–99 Baudet, E.A.R.F., 150 wild birds transmission of AI and HP AI H7N3 virus and disposal Belgium, 231 plan for, 358 method of composting in, 343f Biosecurity. See also Biosecurity audit; Biosecurity audit HP AI H7N3 virus control strategies Biosecurity risk assessment defi nition of, 369 of, 208–9 advice for, 77 explanation of, 379 HP AI H7N3 virus outbreak in, 207– alternatives to, 353 general considerations of, 371 9 birds linking population considered justifi cation for, 370 human infections of HP AI H7N3 for, 359–60 Biosecurity risk assessment. See also virus in, 209

communication importance for Biosecurity risk assessment form Carbon dioxide (CO2) regional, 364 AI outbreak observations used for, euthanasia of poultry with, 311–12 control strategies challenge with HP 374t euthanasia of wild birds in a chamber AI and implementation of, 573 application of results from, 377–79 with compressed, 326 cost concerns of poultry farms and, basis for, 372t euthanasia of wild birds through 355, 361–62 compartmentalization supported from culling bag with frozen, 326–28 defi nition of, 391 results of, 378–79 mass depopulation and euthanasia economics of poultry and, 554–55 complexes and scores from, 380f, from caged-house methods and, educational programs and training in, 382f 322–23 495 complexes managed with help of, mass depopulation and Water-based elements of, 354 379–81 foam compared to gassing with, goals of, 353 contract growers rewarded with low 320–21 history of, 76, 370 scores in, 379 mass depopulation of fl oor-reared HP AI and regional quarantines for, defi nition of, 369 poultry by free standing panel 364–65 educational programs and training in, enclosure and, 316–17 HP AI fought with, 290 495 mass depopulation of fl oor-reared humans and personal safety issues experimental data and retrospective poultry by live haul cage for, 580 data aiding, 373 enclosures and, 319–20 LP AI fought with, 290 farm manager presence needed for, mass depopulation of fl oor-reared LP AI outbreak in poultry and control 377 poultry by plastic tenting and, 317– strategies of, 364 history of, 370 18 LPMs and domestic ducks creating individual questions contributing to, mass depopulation of fl oor-reared risks in, 361 381f poultry by whole house gassing of, outbreak’s importance to, 359 justifi cation of, 370 319 plan development for, 354–56 methods for, 372 mass depopulation through, 313 populations associated with disease poultry spreading AI and, 373 Cats. See Felids risk and plan for, 358–59 practical use of, 378t C&D. See Cleaning and disinfection poultry and concerns with forming standardization of, 371 Cell-mediated immunity (CMI) regional, 363–64 Biosecurity risk assessment form human infl uenza recovery and, 458– poultry and sources of information AREA section in, 376 59 on, 496t farm identifi cation in, 375–76 overview of, 408–9 poultry and voluntary movement FARM section in, 376–77 Centanni, E., 149 control programs of, 362–64 HOUSE section in, 377 Cervical dislocation, 325–26 poultry transmission of AI and plan Blum, F., 156 Charadriiformes, 7. See also Aquatic for, 357–58 Boehmer, F., 156 birds; Shorebirds poultry travel between LPMs and Brieg, A., 150 AI adaptation with, 32 risks with, 359–60 Brunswick poultry exhibition AI distribution among species of, 43, risk factors determined for, 355, 374– chicken breeders fate at, 169t 45 75, 375t fowl plague at, 168–70 AI isolation among species of, 44t Index 589

AI subtype diversity in, 47 salmonella reduction time- PPE increased for, 392, 403, 473 reservoirs in, 46 temperature guidelines for, 505t preparation following AI event for, Chiba, 173 vaccination for HP NAI in, 504t 392–93 Chickens Chile QACs for, 397–98 Brunswick poultry exhibition and fate HP AI H7N3 virus control strategies safety aspects of disinfection in, 399 of breeders of, 169t in, 206–7 selection criteria for disinfectant in, Canada and HP AI H7N3 virus HP AI H7N3 virus outbreak in, 205– 399–400 causing lesions in, 207 6 soaps and detergents used in, 394, Chile and HP AI H7N3 virus causing lesions in chickens from outbreak of 396 lesions in, 206 HP AI H7N3 virus in, 206 sodium carbonate for, 399 Columella on farming of, 149–50 Chu, C.M., 161 sodium dichlorotriazine trione for, Doyle on fowl plague spread by Citric acid, 398 397 commercial trade of, 170 Civets, 112–13 sodium hydroxide for, 398–99 environmental persistence of AI on, Clades special considerations with, 402–3 70 of Asian HP AI H5N1 virus, 254t terminology for, 391–92 European farming of, 149–50 HP AI H5N1 virus and spatial worker health and safety for, 403 fowl plague clinical signs in, 155t, distribution of, 257f Cleavage 193f HP AI H5N1 virus and WHO chickens and variations of AI fowl plague control strategies with classiﬁ cation of, 253 replication from HA and, 88–89t vaccination of, 175–76 Cleaning and disinfection (C&D) of HA0 of HP AI H5N1 virus, 258t fowl plague gross pathology for, aldehydes for, 398 of HA and HP AI cellular 155–56, 156t approved disinfection for, 494t pathobiology, 11 fowl plague through commercial calcium hydroxide for, 399 of HA and LP AI cellular trade of, 170–71 calcium oxide for, 399 pathobiology, 11 global production and trade of, chemical disinfectants recommended of HA of HP AI, 88 499 for, 395t of HA of LP AI, 88 HA cleavage responsible for citric acid for, 398 CMI. See Cell-mediated immunity

variations in AI replication in, 88– control strategies for poultry facilities CO2. See Carbon dioxide 89t with, 522 Columella, L.I.M., 149–50 history of, 59–60 cost of, 550 Compartmentalization housing during Roman era for, 60 cresols for, 397 biosecurity risk assessment used for HP AI and lesions in, 93t disinfectant use guidelines in, 399 supporting, 378–79 HP AI and vaccines protecting, 413– disinfection process in, 402 global strategy with zoning and, 581 14t educational programs training for, Complexes HP AI H5N1 virus and post 493 biosecurity risk assessment for intranasal inoculation of, 91f EPA registered disinfectants and managing, 379–81 HP AI H5N2 virus creating lesions active ingredients for, 395t biosecurity risk assessment scores for, in, 200f equipment for, 402–3 380f, 382f HP AI outbreak causing massive ethanol used for, 396 Composting mortality in, 199f feed system and, 403 costs associated with, 345 HP AI replication in nasal epithelium hypochlorites for, 397 disposal methods of poultry carcasses of, 90f inorganic acids for, 398 through, 342–45 HP studies in, 11 iodine used for, 396 HP AI H7N3 virus in Canada and immunology and vaccination of peroxygen compounds used for, 396 disposal method of, 343f ducks and geese compared to, phenols used for, 397 test results for, 344t 415 planning, 393 Virginia and, 343–45 incubation period for AI in, 69 post disinfection actions with, 402 Contract growers, 379 infectious period for AI in, 69 poultry facilities and dry cleaning in, Controlled marketing, 529 lesions from fowl plague in, 193t 400–401 Control strategies. See also Biosecurity; LP AI and age based mortality rate poultry facilities and response Death; Mass depopulation of, 13 procedures with, 391–92 of Africa for HP AI H5N1 virus, LP AI and embryonation of eggs poultry facilities and routine, 393 274–75 from, 4 poultry facilities and water system of Asia for HP AI H5N1 virus, 271– LP AI and lesions in, 93t cleaning in, 400 74 LP AI and turkeys compared to, 75– poultry facilities and wet cleaning in, of Australia for HP AI, 247–48 76 401–2 of Canada for HP AI H7N3 virus, MDTs from HP AI H5N1 virus with poultry facilities with LP AI and, 208–9 Asian lineage for, 103t 529–30 C&D in poultry facilities for, 522 MDTs from HP AI in other poultry poultry producers and chemical of Chile for HP AI H7N3 virus, compared to, 92–93 methods for, 394 206–7 590 Index

Control strategies (continued) HP AI and targeting of source for, Cost costs and economics impacted by, 580 of alkaline hydrolysis, 348 538–39 HP AI in Asian targeted countries of biosecurity and concerns of poultry costs associated with movement and, and, 566t farms, 355, 361–62 549–50 HP AI in poultry and Vietnam costs of C&D, 550 decreasing host susceptibility in, and benefi ts of, 540t of composting, 345 291 immunity for, 292 of control strategies for movement, diagnostics and surveillance in, 290– international implementation for, 579 549–50 91 for LP AI, 287–88 of disposal methods, 550 diagnostics and surveillance methods LP AI and cost of, 292–93 economics of control strategies and, for HP AI and, 569 LP AI and identifi cation of sources 538–39 disease information systems for, for, 517–18 economics of poultry rehabilitation 580 LP AI and specifi c measures for eggs and, 538–39 educational programs for, 289–90, for, 526 HP AI control strategies and, 293 485, 522–523 LP AI and specifi c measures for of incineration, 342 epidemiology-based measures for, humans for, 526–27 of landfi ll, 340 579 LP AI and specifi c measures for LP AI control strategies and, 292–93 of Europe for HP AI H5N1 virus, infected birds for, 525–26 of on-site burial, 338 274–75 LP AI and specifi c measures for of poultry and loss of production, FAO role in, 294 manure for, 526 550–51 for fowl plague and poultry trade, LP AI and specifi c measures for poultry vaccines comparisons with, 174 vehicles and equipment for, 527 427 for fowl plague and vaccination of LP AI and vaccination for, 530 reporting and confi rmation with chickens, 175–76 LP AI movement in poultry identifi ed effective animal health information for fowl plague and vaccination of for, 518 system and, 549 geese, 175 LP AI outbreak in poultry and reporting and confi rmation with genetic resistance and, 291–92 biosecurity for, 364 laboratory staff and equipment and, goals of, 289 LP AI response measures for, 525 549 history of successful, 571 LP AI spreading to different of in situ plasma vitrifi cation, 349 for HP AI, 288–89 populations and prevention with, vaccines and, 530 HP AI and challenge of countries 527–28 Vietnam biosecurity and vaccination with current disease endemic for, of Mexico for HP AI H5N2 virus, and, 539–40 574 205 Vietnam control strategies for HP AI HP AI and challenge of ducks as of Middle East for HP AI H5N1 in poultry and benefi ts and, 540t reservoirs for, 574 virus, 275 Cresols. See Cresylic acid HP AI and challenge of economic national implementation for, 575, 578 Cresylic acid (Cresols), 397 assessments for, 574–75 of The Netherlands for HP AI H7N7 Culling bag, 326–28 HP AI and challenge of economic virus, 230–31 inadequacies for, 575 OIE role in, 294 Death, 311 HP AI and challenge of inadequate poultry and exposure risk level Diagnostics and surveillance methods. disease information systems for, identifi cation for, 521–22 See also Agar gel immunodiffusion 573 poultry in USA and, 513 assay; Elisa antibody testing HP AI and challenge of inadequate poultry in USA and biosecurity for, advanced analysis in, 304 epidemiological expertise for, 573 515–17 AI assay characteristics in, 300t HP AI and challenge of inadequate poultry population cross-over and, antibody detection in, 304–5 veterinary services for, 572–73 521 antigen detection immunoassays in, HP AI and challenge of public sector poultry with confi ned populations 301 for, 575 and, 519–20 control strategies and, 290–91 HP AI and challenge of sustainable poultry with infection and elimination direct detection of AI in clinical long-term regional coordination for, 291 samples for, 456 for, 575 poultry with nonconfi ned populations educational programs covering, 488– HP AI and challenge of wild birds as and, 520–21 89 reservoirs for, 574 regional implementation for, 578 HI assay used in, 302–3 HP AI and challenge with biosecurity of Texas for HP AI H5N2 virus, HP AI control strategies and, 569 implementation for, 573 210–11 LP AI illegal sources and, 523–24 HP AI and cost of, 293 of USA for fowl plague, 195 MN used for, 457 HP AI and international commitment of USA for HP AI H5N2 virus, 201–2 molecular/nucleic acid-based tests in, to, 571–72 vaccination programs and, 429–31, 302, 456–57 HP AI and national and regional 552–53, 569–70 NI assay used in, 303 commitment to, 571 vaccines and proper use in, 570–71 rapid point of care tests for, 456 Index 591

RT-PCR assay for, 456–57 HP studies in, 11 Educational programs sample collection, processing, and immunology and vaccination of AI information and sources for, 487t handling for, 305 chickens compared to geese and, basic requirements and key sequence analysis in, 304 415 components for, 485 VI and, 299–301, 456 LP AI and age based mortality rate biosecurity risk assessment training in vivo Pathotyping in, 304 of, 13 in, 495 Disposal methods LP AI in domestic, 99 biosecurity training in, 495 cost of, 550 maintenance cycle for AI in, 48f C&D training through, 493 educational programs considerations Minnesota turkeys with AI and HA control strategies and necessity of, with poultry carcass and, 491, 493 subtypes in AI of, 517t 289–90, 485, 522–23 history of poultry carcasses and, 333– necrosis from HP AI H5N1 virus of diagnostics and surveillance methods 35 Asian lineage and, 108f covered in, 488–89 HP AI H7N3 virus in Canada and wild birds commingling with disposal methods for poultry composting for, 343f domestic, 74 carcasses and considerations of, LP AI H7N1 virus infected poultry 491, 493 carcasses and, 334t Eckroade, Robert, 199f emergency preparedness and response poultry carcasses and alkaline Economics training in, 489, 490t hydrolysis for, 347–48 control strategies for HP AI and evaluating target audience need for, poultry carcasses and comparative challenge of assessments for, 574– 485–86 analysis of, 350–51 75 HP AI H5N1 virus infecting humans poultry carcasses and composting for, control strategies for HP AI and and focus of, 486, 488 342–45 challenge of inadequacies for, 575 LP AI and HP AI information for, poultry carcasses and incineration for, cost of poultry rehabilitation 486 341–42 impacting, 538–39 mass depopulation and euthanasia poultry carcasses and in situ plasma costs associated with control considerations in, 489, 491 vitrifi cation for, 348–49 strategies impacting, 538–39 vaccination strategies included in, poultry carcasses and introduction to disease phases and issues with society 493, 495 options for, 335–36 and, 538t Egypt, 543f poultry carcasses and landfi ll for, for global strategy, 582 Electrocution 338–41 HP AI creating international market mass depopulation and euthanasia poultry carcasses and on-site burial shocks and impacting, 544 from caged-house methods and, for, 336–38 HP AI creating USA market shocks 323 poultry carcasses and rendering for, and impacting, 544–46 mass depopulation of poultry with, 345–47 HP AI H5N1 virus impacting, 565 313–14 poultry carcasses and sources of HP AI impacting African poultry and, Elisa antibody testing, 305 information on, 494t 545t Emus, 104f Dixon, Edmund Saul, 59 HP AI in poultry impacting Asian England Dogs, 112 food and livelihood in, 546–47 HP AI H5N1 virus outbreak in, 221 Domestic birds, 9. See also Poultry HP AI in poultry impacting Middle HP AI H7N3 virus outbreak in, 219 Doyle, T.M. Eastern food and livelihood in, 546 HP AI H7N7 virus outbreak in, fowl plague spread through human infl uenza effecting, 541 219–21 commercial chicken trade and, 170 humans contracting disease from Environmental persistence ND differentiation from fowl plague birds minimized and benefi ts for, on chicken of AI, 70 and studies of, 159, 161, 164, 170 537 on poultry of AI, 70 Ducks market shock impacting, 538 on wild birds of AI, 48 AI infection spatial and temporal poultry and HP AI long term Environmental Protection Agency variation in, 47 prevention impacting, 555–57 (EPA), 392, 395t biosecurity risks of LPMs and poultry biosecurity impacting, 554–55 EPA. See Environmental Protection domestic, 361 poultry losses impacting Agency control strategies and challenges of compensation in, 551–52 Ercolani, G.B., 165 HP AI reservoirs in, 574 poultry productivity improved Ethanol, 396 history of, 61 through avoiding disease and Europe. See also Austria; Belgium; HP AI H5N1 virus of Asian lineage benefi ts in, 537–40 Germany; Great Britain; Italy; The and, 102, 104–5, 104f, 107f, 109– poultry restocking impacting, 553–54 Netherlands; Turkey 10f, 266–67 reporting and confi rmation chicken farms in, 149–50 HP AI H5N1 virus of Asian lineage enforcements and benefi ts in, 548– fowl cholera legislation for, 176 and MDTs for, 106t 49 fowl plague and third epizootic in, HP AI H5N1 virus of Asian lineage USA and fowl plague impacting, 196 170 and pathobiology groups for, 106t vaccination and considerations with, fowl plague epidemics in, 164t HP AI in domestic, 99 552–53 fowl plague legislation for, 176 592 Index

Europe (continued) control strategies and role of, 294 Greve and Brunswick poultry fowl plague scientifi c progress in, global strategy and regional exhibition recording of, 168–69 148–49 organizations and partners of OIE histopathology of, 156–57 fowl plague spreading through, 171– and, 576–77t history of, 145–46 72 HP AI H5N1 virus and response of history reexamined for, 150 Gerlach on fowl plague in, 171 OIE and, 562 HP AI H5N9 virus differentiation HP AI H5N1 virus control strategies poultry sectors defi ned by, 71, 567t from, 198 of, 274–75 Fowl cholera HP AI mistaken for, 166 HP AI H5N1 virus outbreaks in, 218t European legislation for, 176 ILT differentiation from, 161 rendering used in, 347 fowl plague differentiation from, 158t intoxications compared to, 158–59 Euthanasia history of, 157–58 Italy and fi rst epizootic of, 164–66 educational programs considering Fowl plague. See also Chiba Italy and second epizootic of, 166–67 methods of mass depopulation and, anamnesis and, 152 Kleine and Möllers fi ndings on, 154, 489, 491 Asia and spreading of, 173–74 157 mass depopulation and caged-house Austria and, 167–68 Künnemann fi ndings on, 154

methods with CO2 for, 322–23 bacterial examinations of, 157 Lode and Gruber on, 150, 167–68 mass depopulation and caged-house Baudet on, 150 Maggiora and Valenti ﬁ ndings on, methods with electrocution for, biological properties of, 163–64 154, 167 323 Brieg on, 150 Nakamura and Iwasa ﬁ ndings on, 154 mass depopulation compared to, 309– Brunswick poultry exhibition and, natural and experimental hosts of, 10 168–70 152, 153t, 154 poultry and accepted methods of, Burnet and Ferry studies on virus ND differentiation from, 159–61, 160t 311–12 replication of, 163 New York City LPM and, 192

poultry and CO2 used for, 311–12 chicken commercial trade spreading, Perroncito observations on, 145, 149– wild birds and cervical dislocation 170–71 51, 154, 161–63, 165–66 for, 325–26 chickens and clinical signs of, 155t, Pfenninger and Metzger studies on,

wild birds and compressed CO2 in a 193f 155–56 chamber for, 326 chickens and lesions from, 193t poultry and, 124–25 wild birds and culling bag with chickens gross pathology with, 155– poultry and quarantine phases for,

frozen CO2 for, 326–28 56, 156t 174–75 wild birds and injectable anesthetics chicken vaccination attempt control poultry cleansing and disinfection for, for, 324 strategies for, 175–76 175 wild birds and methods for, 324 Chu and ND differentiation from, 161 poultry trade and control strategies Exposure. See also Exposure risk index clinical signs of, 154–55 for, 174 poultry and AI sources of, 67f contagiousness of, 161 Reinacher and Weiss studies on, 163 poultry and control strategies of risk diagnosis criteria in 1920s for, 197t research achievements on, 148t level identifi cation of, 521–22 Doyle and commercial chicken trade septicemic spirochetosis of poultry to AI, 67–68 spreading, 170 differentiation from, 159, 159t turkeys infected with infl uenza Doyle and ND differentiation from, Straub reports on, 165 viruses from pigs and, 10 159, 161, 164 survival time inside and outside host Exposure risk index (ERI), 521 Ercolani and Piana studies in, 165 of, 163 Europe and spreading of, 171–72 terminology for, 146–47, 146t FAO. See Food and Agriculture Europe and third epizootic of, 170 USA and, 191–92, 197–98 Organization European epidemics of, 164t USA and C&D for, 196 Federal Insecticide, Fungicide, and European legislation for, 176 USA and control strategies for, 195 Rodenticide Act (FIFRA), 392 European scientifi c progress for USA and diagnosis of, 194–95 Felids, 112 diseases including, 148–49 USA and economic implications of, Ferry, J.D., 163 experimental transmission of, 161–62 196 FIFRA. See Federal Insecticide, experimental transmission USA and experimental studies of, Fungicide, and Rodenticide Act reevaluation and for, 162 193–94 Finch fowl cholera differentiation from, USA and lesions and clinical signs HP AI H5N1 virus fi ve days post 158t of, 192–93 intranasal inoculation in zebra, geese gross pathology with, 156 USA and source theories on, 196–97 102f geese vaccination attempt control USA quarantines for, 195–96 necrosis from HP AI H5N1 virus in strategies for, 175 virus N discovered through studies house, 103f Gerlach on Europe and spreading of, of, 172–73 necrosis from HP AI H5N1 virus in 171 virus replication of, 162–63 zebra, 100f Germany impacted from Brunswick visibility for, 163 Food and Agriculture Organization poultry exhibition and, 169 von Ostertag fi ndings on, 154 (FAO) Gratzl and Köhler on, 150 Freese, R., 157 Index 593

Geese African control strategies for HP AI humans infected with HP AI and, 453 fowl plague control strategies of and, 274–75 humans with HP AI and Asian vaccination of, 175 Asian control strategies for HP AI lineage of, 110–11 fowl plague gross pathology for, 156 and, 271–74 LBM spreading HP AI and, 266 history of, 61 Asian HP AI and westward mammal and avian summary data for HP AI H5N1 virus in, 102–3f movement of, 262–65, 563–65 Asian lineage of HP AI and, 101t HP AI in domestic, 99 Australia and risks posed by Asian mammals and experimental infections immunology and vaccination of HP AI and, 248 with HP AI and, 113–14 chickens compared to ducks and, chicken post intranasal inoculation mammals with HP AI and Asian 415 with HP AI and, 91f lineage of, 109–10 LP AI in domestic, 99 civets with HP AI and Asian lineage mammals with lesions from HP AI Gerlach, F., 171 of, 112–13 and Asian lineage of, 111t Germany clades of Asian HP AI and, 254t MDTs for chickens with HP AI and fowl plague at Brunswick poultry clade spatial distribution for HP AI Asian lineage of, 103t exhibition spreading throughout, and, 257f MDTs for ducks with HP AI and 169 diagnostic tests for HP AI and, 270 Asian lineage of, 106t HP AI H7N7 virus outbreak in, 223 dogs with HP AI and Asian lineage MDTs for HP AI and Asian lineage HP AI H7N7 virus spread to, 231 of, 112 of, 100–101 Giemsa, Gustav, 148 ducks with HP AI and Asian lineage Middle Eastern control strategies for GISN. See Global Infl uenza of, 102, 104–5, 104f, 107f, 109– HP AI and, 275 Surveillance Network 10f, 266–67 mink with HP AI and Asian lineage Global Infl uenza Preparedness Plan, economics impacted by outbreak of of, 112 462 HP AI and, 565 mutation possibilities of, 33 Global Infl uenza Surveillance Network educational programs focus on necrosis in ducks with HP AI and (GISN), 459 humans infected with HP AI and, Asian lineage of, 108f Global strategy. See also Control 486, 488 necrosis in emus with HP AI and, strategies England and outbreak of HP AI and, 104f capacity building for, 581 221 necrosis in house fi nch with HP AI compartmentalization and zoning for, European control strategies for HP AI and, 103f 581 and, 274–75 necrosis in zebra fi nch with HP AI disease control programs and poverty FAO and OIE responding to HP AI and, 100f alleviation for, 580 and, 562 pathobiology groups for ducks with economics for, 582 felids with HP AI and Asian lineage HP AI and Asian lineage of, 106t FAO and OIE regional partners and of, 112 pathobiology of Asian lineage of HP organizations for, 576–77 future outlook of HP AI and, 275–77 AI and, 100–102 for HP AI, 561–63 geese with HP AI and, 102–3f pathology of HP AI and, 269–70 HP AI and approach of, 566, 568–69 genetic studies on Asian HP AI and, pigs with HP AI and Asian lineage HP AI and guiding principals of 253–55, 257–58 of, 111 implementing, 565–66 global strategy reasoning for HP AI poultry spreading HP AI and, 265– HP AI H5N1 virus and reasons for, and, 564 66, 268, 500 564 HA0 cleavage site for HP AI and, prepandemic vaccines for HP AI and, impacts of, 582 258t 460–61 outlook of positivity for, 582 HA antigenetic change in HP AI and, protein deletions in, 259 poultry sector restructuring and 258–59 Scotland and outbreak of HP AI and, rehabilitation for, 580–81 HA genetic drift in HP AI and, 256f 217, 219 research applied to, 582 HA PCS variations in, 33, 34–35t, 35 seals with HP AI and Asian lineage stakeholder collaboration for, 581 Hong Kong and disease from Asian of, 111–12 Glycosylation, 30 HP AI and, 259–62 seasonal effects on spreading of HP Gratzl, E., 150 HP AI and global impact of, 33 AI and, 269 Great Britain. See also England; HP and host susceptibility with, 46 sparrows with HP AI and Asian Scotland human infections from poultry with lineage of, 105f HP AI outbreaks and positions in, HP AI and, 466–70, 472–73 virulence increasing for, 35 221f humans and distribution of HP AI WHO clade classifi cations for HP AI HP AI outbreaks in, 221–22 and, 465f and, 253 Greece, 151 humans and epidemic curve of HP AI wild bird and poultry transmission of Greve, L, 168–69 and, 468f HP AI and, 64 humans and female infections of HP wild bird maintenance and H5N1 virus AI and, 471f transmission of HP AI and, 50–51 Africa and Asia and Europe humans and male infections of HP AI wild bird preventative measures for outbreaks of HP AI and, 218t and, 471f humans against HP AI and, 51 594 Index

H5N1 virus (continued) Italy and eradication of HP AI and, H7 subtype. See also H7N1 virus; wild bird species with, 50t 228 H7N2 virus; H7N3 virus; H7N4 wild bird spreading HP AI and, 267– Italy and spreading of HP AI and, virus; H7N7 virus 68 225–26, 227t amino acid sequence around PCS of wild birds with experimental Italy and wild birds infected with HP HA of Australian, 242t infections of HP AI and, 49–50 AI and, 228 Australia and origins of HP AI and, wild birds with HP AI and Asian poultry distribution in Italy with HP 246–47 lineage of, 105, 107–9 AI and, 226f HA similarities in Australian, 246t wild birds with natural infections of H7N2 virus, 462–63 pathogenicity of, 10 HP AI and, 49 H7N3 virus RNA recombination leading to zebra fi nch fi ve days post intranasal Canada and control strategies for HP outbreaks in, 29 inoculation with HP AI and, AI and, 208–9 H9N2 virus, 462–63 102f Canada and human infections with HA0, 258t H5N2 virus HP AI and, 209 Hemagglutinin (HA). See also HA0 chickens and lesions from HP AI and, Canada and outbreak of HP AI and, AI classifi ed by subtypes of, 62 200f 207–9 amino acid sequence of representative Italy and outbreak of HP AI and, chickens and lesions from Canadian isolates for subtypes of, 7f 224–25 outbreak of HP AI and, 207 Australian H7 subtype and amino LP AI H5N2 virus outbreak chickens and lesions from Chilean acid sequence around PCS of, 242t similarities to outbreak of HP AI outbreak of HP AI and, 206 birds and subtype distribution of, 63t and, 203f Chile and control strategies for HP chickens and variations of AI Mexico and control strategies for HP AI and, 206–7 replication from cleavage of, 88– AI and, 205 Chile and outbreak of HP AI and, 89t Mexico and LP AI H5N2 virus 205–6 cleavage of HP AI in, 88 changing to HP AI and, 204–5 disposal method of composting in cleavage of LP AI in, 88 Mexico and outbreak of LP AI and, Canada for HP AI and, 343f elements essential to AI of, 26f 204 England and outbreak of HP AI and, glycosylation infl uencing poultry and experimental studies of 219 pathogenicity of, 30 HP AI and, 204t human illness caused by HP AI and, H5N1 virus variations at PCS of, 33, South Africa and outbreak of HP AI 464–66 34–35t, 35 and, 222–23 Pakistan and outbreak of HP AI and, H7 subtype in Australia and Texas and control strategies for HP 224 similarities in, 246t AI and, 210–11 poultry in Queensland infected by HP AI cellular pathobiology and Texas and outbreak of HP AI and, outbreak of HP AI and, 244 cleavage of, 11 209–10 poultry in Victoria infected by HP AI H5N1 virus and antigenetic USA and control strategies for HP AI outbreak of HP AI and, 243–44 change of, 258–59 and, 201–2 H7N4 virus, 244–45 HP AI H5N1 virus and genetic drift USA and LP AI and, 202, 204 H7N7 virus of, 256f USA and LP AI beginning for, 199– Belgium and spreading of HP AI and, HP and changes in, 12 200 231 human infl uenza and SA binding to, USA and LP AI changing to HP AI England and outbreak of HP AI and, 457–58 in outbreak of, 200–201 219–21 LP AI and PCS of, 26–27 USA history of HP AI and, 199 Germany and outbreak of HP AI and, LP AI cellular pathobiology and H5N3 virus, 222 223 cleavage of, 11 H5N8 virus, 223–24 Germany and spreading of HP AI Minnesota turkeys AI detection and H5N9 virus and, 231 ducks AI subtypes of, 517t Canada and HP AI and, 198–99 human illness caused by HP AI and, mutations at PCS of, 27–28, 28t fowl plague differentiation from HP 464–66 pathogenicity of AI and role of, 25 AI and, 198 The Netherlands and control PCS and lab manipulation of, 30 H5 subtype. See also H5N1 virus; strategies for HP AI and, 230–31 PCS changes and problems in, 35–36 H5N2 virus; H5N3 virus; H5N8 The Netherlands and humans infected vaccines and in vitro expressed, 422 virus; H5N9 virus with HP AI and, 231–32 vaccines and in vivo expressed, 422– pathogenicity of, 10 The Netherlands and outbreak of HP 24 H7N1 virus AI and, 229–30 vaccine strain selection and, 425–26 disposal methods of poultry carcasses The Netherlands and pigs infected viral assembly process and, 5–6 infected with LP AI and, 334t with HP AI and, 232–33, 500 viral infection and role of, 4–5 Italy and clinical fi ndings of HP AI North Korea outbreak of HP AI and, Hemagglutinin inhibition assay (HI and, 226, 228 233 assay), 302–3 Italy and emergence of HP AI and, poultry in Victoria infected by HI assay. See Hemagglutinin inhibition 225 outbreak of HP AI and, 241–43 assay Index 595

High pathogenicity (HP) chickens and lesions from Chilean control strategies of diagnostics and Africa and Asia and Europe outbreak of H7N3 virus AI with, surveillance methods for AI with, outbreaks of H5N1 virus AI with, 206 569 218t chickens and lesions from H5N2 control strategies with international African control strategies for H5N1 virus AI with, 200f commitment for AI with, 571–72 virus AI with, 274–75 chickens and studies of, 11 control strategies with national and African economics impacted by Chile and control strategies for H7N3 regional commitment for AI with, poultry infected by AI with, 545t virus AI with, 206–7 571 in AI and embryonation of chicken Chile and outbreak of H7N3 virus AI diagnostic tests for H5N1 virus AI eggs, 4 with, 205–6 with, 270 AI documented epidemics with, 129– civets with H5N1 virus of Asian disposal method of composting in 34t lineage AI with, 112–13 Canada for H7N3 virus AI with, amino acid insertions at PCS for AI clades of Asian H5N1 virus AI with, 343f with, 28–29 254t dogs with H5N1 virus of Asian aptosis produced by AI with, 91 clade spatial distribution for H5N1 lineage AI with, 112 aquatic birds and poultry and virus AI with, 257f ducks and AI with, 99 infections LP AI and AI with, 72f cleavage of HA of AI with, 88 ducks and studies in, 11 Asian control strategies for H5N1 cleavage of HAO of H5N1 virus AI ducks with H5N1 virus of Asian virus AI with, 271–74 with, 258t lineage AI with, 102, 104–5, 104f, Asian food and economic livelihood control strategies and challenge of 107f, 109–10f, 266–67 impacted by poultry infected by AI biosecurity implementation for AI economics impacted by outbreak of with, 546–47 with, 573 H5N1 virus AI with, 565 Asian H5N1 virus AI and westward control strategies and challenge of economics of international market movement and, 262–65, 563–65 countries with current endemic of shocks from AI with, 544 Asian H5N1 virus AI with, 251–53 AI with, 574 economics of poultry and long term Australia and AI with, 239 control strategies and challenge of prevention of AI with, 555–57 Australia and control strategies for AI ducks as reservoirs for AI with, economics of USA market shocks with, 247–48 574 from AI with, 544–46 Australia and lessons learned from control strategies and challenge of educational programs focus on outbreaks of AI with, 245 economic assessments for AI with, humans infected by H5N1 virus AI Australia and origins of H7 subtype 574–75 with, 486, 488 AI with, 246–47 control strategies and challenge of educational programs providing Australia and risks posed by Asian economic inadequacies for AI with, information on LP AI and AI with, H5N1 virus AI with, 248 575 486 Australia and variations in clinical control strategies and challenge of England and H7N3 virus AI with, disease of AI with, 245–46 inadequate disease information 219 Belgium and spreading of H7N7 systems for AI with, 573 England and H7N7 virus AI with, virus AI with, 231 control strategies and challenge of 219–21 biosecurity and regional quarantines inadequate epidemiological England and outbreak of H5N1 virus for AI with, 364–65 expertise for AI with, 573 AI with, 221 biosecurity ﬁ ghting AI with, 290 control strategies and challenge of European control strategies for H5N1 Canada and control strategies for inadequate veterinary services for virus AI with, 274–75 H7N3 virus AI with, 208–9 AI with, 572–73 FAO and OIE response to H5N1 Canada and H5N9 virus AI with, control strategies and challenge of virus with, 562 198–99 public sector for AI with, 575 felids with H5N1 virus of Asian Canada and human infections with control strategies and challenge of lineage AI with, 112 H7N3 virus AI with, 209 sustainable long-term regional food safety risks with LP NAI and Canada and outbreak of H7N3 virus coordination for AI with, 575 NAI with, 506–7 AI with, 207–9 control strategies and challenge of fowl plague and AI with, 166 challenges of AI which is, 16 wild birds as reservoirs for AI fowl plague differentiation from chicken and post intranasal with, 574 H5N9 virus AI with, 198 inoculation with H5N1 virus AI control strategies cost for AI with, future outlook of H5N1 virus AI with, 91f 293 with, 275–77 chicken mortality in outbreak of AI control strategies for AI with, geese and AI with, 99 with, 199f 288–89 geese and H5N1 virus AI with, 102– chickens and lesions from AI with, control strategies for targeted 3f 93t Asian countries with AI with, genetic studies on Asian H5N1 virus chickens and lesions from Canadian 566t AI with, 253–55, 257–58 outbreak of H7N3 virus AI with, control strategies for targeting source Germany and outbreak of H7N7 virus 207 of AI with, 580 AI with, 223 596 Index

High pathogenicity (HP) (continued) Italy and outbreak of H5N2 virus AI The Netherlands and control Germany and spreading of H7N7 with, 224–25 strategies for H7N7 virus AI with, virus AI with, 231 Italy and spreading of H7N1 virus AI 230–31 global nature of AI with, 123, 135–36 with, 225–26, 227t The Netherlands and humans infected global strategy approach for AI with, Italy and wild birds infected with with H7N7 virus AI with, 231–32 566, 568–69 H7N1 virus AI with, 228 The Netherlands and outbreak of global strategy for AI with, 561–63 LBM spreading H5N1 virus AI with, H7N7 virus AI with, 229–30 global strategy implementation 266 The Netherlands and pigs infected guiding principals for AI with, LP AI giving way to AI with, 73–74 with H7N7 virus AI with, 232–33, 565–66 LP AI H5N2 virus outbreak 500 global strategy reasoning for AI with, similarities to outbreak of H5N2 North Korea and outbreak of H7N7 564 AI with, 203f virus AI with, 233 Great Britain and outbreaks of AI LP compared to, 3 Pakistan and H7N3 virus AI with, with, 221–22 mammal and avian summary data for 224 Great Britain and positions of Asian lineage of H5N1 virus AI pathobiology groups for ducks with outbreaks of AI with, 221f with, 101t H5N1 virus of Asian lineage AI of H5N1 virus and global impact, 33 mammals and experimental infections with, 106t of H5N1 virus and host susceptibility, with H5N1 virus AI with, 113–14 pathobiology of Asian lineage of 46 mammals with H5N1 virus of Asian H5N1 virus AI with, 100–102 HA antigenetic change in H5N1 virus lineage AI with, 109–10 pathology of H5N1 virus AI with, AI with, 258–59 mammals with lesions from H5N1 269–70 HA changes associated with, 12 virus of Asian lineage AI with, pigs with H5N1 virus of Asian HA cleavage and cellular 111t lineage AI with, 111 pathobiology of AI which is, 11 MDTs for chickens with H5N1 virus poultry and gross lesions from AI HA genetic drift in H5N1 virus AI of Asian lineage AI with, 103t with, 93–94, 93–96f, 96 with, 256f MDTs for ducks with H5N1 virus of poultry and microscopic lesions from history of AI with, 128, 135 Asian lineage AI with, 106t AI with, 96–98f, 97t, 98 Hong Kong and disease from Asian MDTs of chickens compared to other poultry and replication of AI with, 88 H5N1 virus AI with, 259–62 forms of poultry for AI with, 92– poultry and syndromes infl icted by AI human illness caused by H7N3 virus 93 with, 87–88 AI with, 464–66 MDTs of H5N1 virus of Asian poultry clinical signs of AI with, 92– human illness caused by H7N7 virus lineage AI with, 100–101 93 AI with, 464–66 measures for stamping out AI with, poultry distribution in Italy with human infections from poultry with 547 H7N1 virus AI with, 226f H5N1 virus AI with, 466–70, 472– Mexico and control strategies for poultry eggs pasteurization time with 73 H5N2 virus AI with, 205 infections of NAI with, 506t humans and distribution of H5N1 Mexico and LP AI H5N2 virus poultry experimental studies with virus AI with, 465f changing to H5N2 virus AI with, H5N2 virus AI with, 204t humans and epidemic curve of H5N1 204–5 poultry in New South Wales infected virus AI with, 468f Middle Eastern control strategies for by outbreak of H7N4 virus AI humans and female infections of H5N1 virus AI with, 275 with, 244–45 H5N1 virus AI with, 471f Middle Eastern food and economic poultry in Queensland infected by humans and male infections of H5N1 livelihood impacted by poultry outbreak of H7N3 virus AI with, virus AI with, 471f infected by AI with, 546 244 humans and recent infections of AI mink with H5N1 virus of Asian poultry in Victoria infected by with, 454–55t, 464 lineage AI with, 112 outbreak of H7N3 virus AI with, humans and risk of transmission of molecular and biological features of 243–44 AI with, 500–501 AI which is, 10–11 poultry in Victoria infected by humans infected with H5N1 virus nasal epithelium of chicken and outbreak of H7N7 virus AI with, with, 453 replication of AI with, 90f 241–43 humans with H5N1 virus of Asian necrosis in ducks with H5N1 virus of poultry spreading H5N1 virus AI lineage AI with, 110–11 Asian lineage AI with, 108f with, 265–66, 268, 500 Ireland and outbreak of H5N8 AI necrosis in emus with H5N1 virus AI poultry tissue and cellular sites of with, 223–24 with, 104f viral replication and damage of AI Italy and clinical fi ndings of H7N1 necrosis in house fi nch with H5N1 with, 98–99 virus AI with, 226, 228 virus AI with, 103f poultry trade restrictions from NAI Italy and emergence of H7N1 virus necrosis in zebra fi nch with H5N1 with, 499–500 AI with, 225 virus AI with, 100f poultry trade risks of LP NAI and Italy and eradication of H7N1 virus necrosis produced by AI with, NAI with, 501–2 AI with, 228 90–91 predicting AI which is, 12–13 Index 597 prepandemic vaccines for H5N1 virus wild birds with experimental of fowl plague spread through AI with, 460–61 infections of AI H5N1 virus with, commercial chicken trade, 170–71 public health implications of AI with, 49–50 of fowl plague spread to Austria, 293–94 wild birds with H5N1 virus of Asian 167–68 ratites and AI with, 99–100 lineage AI with, 105, 107–9 of fowl plague terminology, 146–47 recommended measures for wild birds with natural infections of of fowl plague virus replication, 162– responders to AI with, 473 AI H5N1 virus with, 49 63 regulation of AI with, 126 zebra fi nch fi ve days post intranasal of fowl plague visibility, 163 Scotland and outbreak of H5N1 virus inoculation with H5N1 virus AI of Geese, 61 AI with, 217, 219 with, 102f of global nature of AI, 124 seals with H5N1 virus of Asian Hirst, G.K., 161 of horses and AI, 64 lineage AI with, 111–12 Histopathology, 156–57 of HP AI, 128, 135 seasonal effects on spreading of History of HP AI arising from LP AI, 73–74 H5N1 virus AI with, 269 of biosecurity, 76, 370 of HP AI H5N1 virus from Asia, South Africa and H5N2 virus AI biosecurity and importance of 251–53 with, 222–23 outbreaks in, 359 of HP AI H5N1 virus from Asia and South Africa and H5N3 virus AI of biosecurity risk assessment, 370 genetic studies, 253–55, 257–58 with, 222 of broad homosubtypic protection of of HP AI H5N1 virus in Asia and sparrows with H5N1 virus of Asian poultry vaccines, 426 disease in Hong Kong, 259–62 lineage AI with, 105f of chickens, 59–60 of HP AI H5N1 virus in Asia and species variation in response to AI of Chile and HP AI H7N3 virus westward movement of virus, 262– with, 90 outbreak, 205–6 65, 563–65 Texas and control strategies for of composting in Virginia, 343–45 of HP AI H5N1 virus outbreak in H5N2 virus AI with, 210–11 of control strategies with successful England, 221 Texas and outbreak of H5N2 virus AI outcomes, 571 of HP AI H5N1 virus outbreak in with, 209–10 of disposal methods of poultry Scotland, 217, 219 Turkey and timeline for outbreak in carcasses, 333–35 of HP AI H5N2 virus in USA, 199 AI with, 548t of ducks, 61 of HP AI H5N2 virus outbreak in USA and H5N2 virus AI with, 199 of European chicken farms, 149–50 Italy, 224–25 USA and LP AI H5N2 virus of European scientifi c progress for of HP AI H5N2 virus outbreak in changing to AI with, 200–201 fowl plague and other diseases, South Africa, 222–23 USA control strategies for H5N2 148–49 of HP AI H5N2 virus outbreak in virus AI with, 201–2 of fowl cholera, 157–58 Texas, 209–10 vaccination for chickens infected with of fowl plague, 145–46 of HP AI H5N3 virus outbreak in NAI with, 504t of fowl plague and bacterial South Africa, 222 vaccination for poultry infected with examinations, 157 of HP AI H5N8 virus outbreak in NAI with, 503–4 of fowl plague and fi rst epizootic in Ireland, 223–24 vaccines for AI with, 407, 432–33 Italy, 164–66 of HP AI H5N9 virus in Canada, vaccines protecting chickens from AI of fowl plague and reexamination, 198–99 with, 413–14t 150 of HP AI H7N3 virus outbreak in vaccines protection assessment and of fowl plague and second epizootic Canada, 207–9 AI with, 413–15 in Italy, 166–67 of HP AI H7N3 virus outbreak in Vietnam costs and benefi ts for control of fowl plague and third epizootic in England, 219 strategies on poultry with AI with, Europe, 170 of HP AI H7N3 virus outbreak in 540t of fowl plague differentiation from Pakistan, 224 WHO clade classifi cation for H5N1 ND, 159–61 of HP AI H7N7 virus outbreak in virus AI with, 253 of fowl plague in Brunswick poultry England, 219–21 wild bird and poultry transmission of exhibition, 168–70 of HP AI H7N7 virus outbreak in AI H5N1 virus with, 64 of fowl plague in LPM of New York Germany, 223 wild bird maintenance and City, 192 of HP AI in Australia, 239 transmission of AI H5N1 virus of fowl plague in USA, 191–92, 197– of HP AI in wild birds, 125–26 with, 50–51 98 of HP AI outbreaks in Great Britain, wild bird preventative measures for of fowl plague in USA and economic 221–22 humans against AI H5N1 virus implications, 196 of HP AI regulation, 126 with, 51 of fowl plague in USA and source of HP AI risk of transmission to wild birds and AI with, 99, 125–26 theories, 196–97 humans, 500–501 wild birds in Australia and AI with, of fowl plague research achievements, of HP AI through documented 239–40 148t epidemics, 129–34t wild birds spreading AI H5N1 virus of fowl plague spreading through of human illness caused by HP AI with, 267–68 Europe, 171–72 H7N3 virus, 464–66 598 Index

History (continued) educational programs focus on HP AI vaccines and new strategies for, 459– of human illness caused by HP AI H5N1 virus infecting, 486, 488 60 H7N7 virus, 464–66 history of birds and, 59 Humoral immunity, 408, 458–59 of human infections of HP AI H5N1 HP AI and recent infections of, 454– Hypochlorites, 397 virus from poultry, 466–70, 472–73 55t, 464 of human infl uenza, 65 HP AI H5N1 virus distribution in, ILT. See Infectious Laryngotracheitis of humans and birds, 59 465f Immune system, 409 of incineration in Virginia, 341–42 HP AI H5N1 virus epidemic curve Immunology. See also Cell-mediated of landfi lls in Virginia, 338–41 for, 468f immunity; Humoral immunity; of LP AI in poultry, 125 HP AI H5N1 virus from poultry Immune system; Mucosal of LP AI in wild birds, 125–26 infecting, 466–70, 472–73 immunity of LP AI outbreaks in Minnesota HP AI H5N1 virus infecting, 453 AI virus infection and host in terms poultry, 514–15, 516t HP AI H5N1 virus infecting females of, 411 of LP AI regulation, 126 of, 471f antigen response in, 409–11 of man-made systems and examples HP AI H5N1 virus infecting males assessment of, 433 of LP AI, 127–28 of, 471f components of innate immunity in, of Mexico and LP AI H5N2 virus HP AI H5N1 virus with Asian 410 outbreak, 204 lineage in, 110–11 vaccination of chickens compared to of OIE, 145 HP AI H7N3 virus causing illness in, ducks and geese for, 415 of pigs and AI, 64–65 464–66 vaccine protection of poultry and of poultry AI vaccines, 416 HP AI H7N3 virus outbreak in basis from, 412 of poultry and fowl plague, 124–25 Canada infecting, 209 vaccines and vaccination for poultry of poultry and other bird trade HP AI H7N7 virus causing illness in, and basis in, 408 restrictions and AI isolations, 502– 464–66 Inactivation 3 HP AI H7N7 virus outbreak in The NAI in poultry products and methods of poultry art and writing, 151–52 Netherlands infecting, 231–32 for, 504–5 of poultry diseases and writers from HP AI risk of transmission to, 500– poultry facilities and dry heat for AI ancient Greece, 151 501 and, 393–94 of poultry diseases and writers from LP AI and recent infections of, 454t poultry facilities and steam for AI Asia, 151 LP AI causing illness in, 462–64 and, 394 of poultry diseases and writers from wild birds with HP AI H5N1 virus poultry facilities and UV light for AI Rome, 151 and preventative measures for, 51 and, 393 of poultry’s commercial era, 60, 71 Human infl uenza procedures for, 70–71 of rendering in Europe, 347 age and attack rates of, 455–56 Incineration of rendering in Virginia, 346 antigenic drift and vaccination with, box incinerator diagram for, 341f of TADs of zoonotic nature, 563–64 14 costs associated with, 342 of terminology for AI, 126–27 antigenic drift causing epidemics of, disposal methods of poultry carcasses of turkeys, 60–61 455 through, 341–42 of USA and AI outbreaks, 514 antigenic shift causing pandemic of, Virginia and, 341–42 of USA and civil freedoms, 147 461–62 Incubation period, 69 of USA and LP AI H5N2 virus antigenic shift impacting, 15 Infectious Laryngotracheitis (ILT), 161 outbreak, 202, 204 CMI contributing to recovery from, Infectious period, 69 of USA science and technology, 147– 458–59 Infl uenza viruses. See also Global 48 economic effects of, 541 Infl uenza Preparedness Plan; of vaccines for HP AI, 407, 432–33 fear of, 540–41 Global Infl uenza Surveillance of vaccines for LP AI, 407–8, 432 GISN and formulating vaccines for, Network; Human infl uenza; of vaccine strain selection, 425 459 Notifi able avian infl uenza of virulence changes in AI, 25 HA binding to SA in, 457–58 life cycle of, 4–6 of wild birds and AI, 43 history of, 65 nomenclature of, 4 of wild birds and domestic ducks humoral immunity contributing to poultry infected by, 10 commingling, 74 recovery from, 458–59 species specifi city of, 8–9 Hong Kong, 259–62 pathogenicity and adaptation of, 32– turkeys exposed to pigs and, 10 Horses, 64 33, 458 viral diversity mechanisms of, 13–14 HP. See High pathogenicity pigs as mixing vessel for AI and, 5 viral protein and, 5–6 Human(s). See also Human infl uenza public health impact of, 453, 455–56 virulence infl uenced by mutation of biosecurity and personal safety issues RNA viruses subpopulations proteins in, 30 of, 580 resistance to antiviral drugs for, wild birds and, 6–8 economic benefi ts of minimizing 24–25, 461 wild birds and separation in North disease from birds spreading to, vaccination annually for prevention American v. Eurasian types of, 537 of, 459 6–7 Index 599

Injectable anesthetics, 324 in mammals from HP AI H15N1 control strategies and specifi c Inorganic acids, 398 virus of Asian origin, 111t measures for eggs with AI with, Inorganic compounds, 396 poultry with HP AI and gross, 93–94, 526 In situ plasma vitrifi cation 93–96f, 96 control strategies and specifi c cost associated with, 349 poultry with HP AI and microscopic, measures for humans with AI with, disposal methods of poultry carcasses 96–98f, 97t, 98 526–27 through, 348–49 poultry with LP AI and gross, 92 control strategies and specifi c system diagram of, 349f poultry with LP AI and microscopic, measures for infected birds with AI Interspecies transmission 92 with, 525–26 of AI, 62f, 87 USA fowl plague clinical signs and, control strategies and specifi c common cases of, 68 192–93 measures for manure with AI with, Intoxication, 158–59 Liposomes, 421–22 526 In vivo Pathotyping, 304 Live bird market (LBM) control strategies and specifi c Ireland, 223–24 AI distribution through, 9–10 measures for vehicles and Italy HP AI H5N1 virus spreading through, equipment with AI with, 527 fowl plague and fi rst epizootic in, 266 control strategies cost for AI with, 164–66 Live poultry marketing (LPM), 71 292–93 fowl plague and second epizootic in, biosecurity and risks of domestic control strategies for AI with, 287–88 166–67 ducks in, 361 control strategies for poultry HP AI H5N2 virus outbreak in, 224– biosecurity and risks of poultry travel movement and AI with, 518 25 between, 359–60 control strategies of biosecurity for HP AI H7N1 virus and clinical fowl plague in New York City and, poultry outbreak of AI with, 364 fi ndings in, 226, 228 192 control strategies of vaccination for HP AI H7N1 virus emergence in, 225 frequency of AI in, 75 AI with, 530 HP AI H7N1 virus eradication in, LP AI introduced into commercial control strategies preventing 228 poultry through, 74–75, 123–24 spreading to different populations HP AI H7N1 virus infected poultry LP AI prevention for, 523 of AI with, 527–28 and distribution in, 226f ND compared to AI in, 372–73 diagnostics and surveillance methods HP AI H7N1 virus infecting wild species commingling and AI in, 76 for illegal sources of AI with, 523– birds in, 228 Lode and Gruber, 150, 167–68 24 HP AI H7N1 virus spreading in, 225– Low pathogenicity (LP) disposal methods of poultry carcasses 26, 227t AI regulation with, 126 infected with H7N1 virus AI with, Iwasa, T., 154 aquatic birds and poultry and 334t infections HP AI and AI with, ducks age based mortality rate when Kaupp, B.F., 152 72f exposed to AI which is, 13 Klee, Robert, 152 aquatic birds as reservoirs for AI ducks and AI with, 99 Kleine, F.K., 154, 157 with, 63–64 educational programs providing Koch, Robert, 148–49 biosecurity fi ghting AI with, 290 information on HP AI and AI with, Köhler, H., 150 California turkey farms and 486 Künnemann, O., 154 movement of AI with, 518f features of AI with, 128 C&D for poultry facilities with AI food safety risks with HP NAI and Landfi ll with, 529–30 NAI with, 506–7 costs associated with, 340 cellular pathobiology and HA geese and AI with, 99 disposal methods of poultry carcasses cleavage of AI which is, 11 global nature of AI with, 123–24, through, 338–41 challenges of AI which is, 16 135–36 problems with, 339 chicken eggs embryonation and AI HA PCS for AI with, 26–27 Virginia and history with, 338–41 with, 4 host susceptibility for AI with, 46 LBM. See Live bird market chickens age based mortality rate HP AI H5N2 virus outbreak Legislation, 176 when exposed to AI which is, 13 similarities to outbreak of H5N2 Lesions chickens and lesions from AI with, AI with, 203f chickens and fowl plague causing, 93t HP AI originating from, 73–74 193t chickens compared to turkeys in HP compared to, 3 chickens and HP AI creating, 93t terms of AI with, 75–76 human illness caused by AI with, chickens and HP AI H5N2 virus cleavage of HA of AI with, 88 462–64 creating, 200f controlled marketing for poultry humans and recent infections of AI chickens and HP AI H7N3 virus in fl ocks infected with AI with, 529 with, 454t Canada causing, 207 control strategies and identifying LPM and commercial poultry chickens and HP AI H7N3 virus in sources of AI with, 517–18 introduced to AI with, 74–75, 123– Chile causing, 206 control strategies and response to 24 chickens and LP AI creating, 93t sources of AI with, 525 LPM and prevention of AI with, 523 600 Index

Low pathogenicity (LP) (continued) LP. See Low pathogenicity poultry that is ﬂ oor-reared and CO2 man-made systems and examples of LPM. See Live poultry marketing with live haul gage enclosures for, AI with, 127–28 Lush, D., 161 319–20

mass depopulation for poultry fl ocks poultry that is fl oor-reared and CO2 infected with AI with, 528–29 M2 protein, 4, 5 with plastic tenting for, 317–18 measures for stamping out AI with, Maggiora, A., 154, 167 poultry that is fl oor-reared and 548 Maintenance cycle methods for, 315–16 Mexico and HP AI H5N2 virus for ducks with AI, 48f PPE used in, 314–15 changing from H5N2 virus AI for poultry with AI, 69–70, 75–76 strategy of, 310–11 with, 204–5 for wild birds with AI, 48–49 water-based foam accepted methods Mexico and outbreak of H5N2 virus Mammals. See also Civets; Felids; for, 321

AI with, 204 Humans; Mink; Pigs; Seals water-based foam compared to CO2 Minnesota poultry and outbreaks of AI infections in, 64–65 gassing for, 320–21 AI with, 514–15, 516t avian immune system compared to worker health concerns with, 314–15 molecular and biological features of immune system of, 409 Matrix gene, 8f AI which is, 10–11 HP AI H5N1 virus experimental MDTs. See Mean death times poultry and, 125 infections in, 113–14 Mean death times (MDTs) poultry and clinical signs of AI with, HP AI H5N1 virus with Asian for chickens with HP AI H5N1 virus 91–92 lineage and lesions in, 111t with Asian lineage, 103t poultry and global nature of AI with, HP AI H5N1 virus with Asian for ducks with HP AI H5N1 virus 127 lineage and summary data for with Asian lineage, 106t poultry and gross lesions from AI avian species and, 101t of HP AI H5N1 virus with Asian with, 92 HP AI H5N1 virus with Asian lineage, 100–101 poultry and isolated AI with, 65 lineage in, 109–10 of HP AI in chicken compared to poultry and microscopic lesions from Man-made systems. See also Live other poultry, 92–93 AI with, 92 poultry marketing Metzger, E., 155–56 poultry and replication of AI with, 88 LP AI examples in, 127–28 Mexico poultry and syndromes inﬂ icted by AI poultry introduced to AI through, 71– HP AI H5N2 virus control strategies with, 87 72 of, 205 poultry eggs pasteurization time with reservoirs of AI in, 514 LP AI H5N2 virus changing to HP infections of NAI with, 507t Mann, G., 157 AI H5N2 virus in, 204–5 poultry tissue and cellular sites of Market shocks LP AI H5N2 virus outbreak in, 204 viral replication and damage of AI economics impacted by, 538 Microneutralization test (MN), 457 with, 92 HP AI and international economics Middle East poultry trade risks of HP NAI and of, 544 HP AI H5N1 virus control strategies NAI with, 501–2 HP AI and USA economics of, 544– of, 275 poultry vaccines for live AI with, 46 HP AI in poultry impacting food and 422–23 Mass depopulation. See also Death economic livelihood in, 546 ratites and AI with, 99 animal welfare issues with, 314 Mink, 112

recommended measures for CO2 use in, 313 Minnesota responders to AI with, 473 educational programs considering HA subtypes in AI of ducks and serological surveillance and sentinel methods of euthanasia and, 489, 491 detection of AI in turkeys from, birds detecting AI with, 435–36 euthanasia and caged house methods 517t

species variation in response to AI with CO2 for, 322–23 LP AI outbreaks in poultry in, 514– with, 90 euthanasia and caged house methods 15, 516t turkeys and sources of AI with, 72f with electrocution for, 323 turkeys and use of vaccines in, 532f USA and accidental sources of AI euthanasia compared to, 309–10 Möllers, B., 154, 157 with, 523 information sources on methods of, Morphology, 3–4 USA and H5N2 virus AI with, 202, 492t Mucosal immunity, 411–12 204 LP AI infected poultry ﬂ ocks and, Mutations USA and H5N2 virus beginning as 528–29 H5N1 virus and possibilities for, 33 AI with, 199–200 poultry and electrocution for, 313–14 HA PCS and, 27–28, 28t USA and HP AI H5N2 virus poultry and selection criteria for, importance and effects of, 24–25 changing from AI with, 200–201 312–13 RNA viruses and ease of, 24 vaccines for AI with, 407–8, 432 poultry and SOPs for, 315 virulence of inﬂ uenza viruses

vaccines protection assessment and poultry that is ﬂ oor-reared and CO2 inﬂ uenced by proteins and, 30 AI with, 413–14 gassing of whole house for, 319

wild birds and AI with, 99, 125–26 poultry that is ﬂ oor-reared and CO2 NA. See Neuraminidase wild birds and prevention of AI with, with free standing panel enclosure NAHEMS. See National Animal Health 523 for, 316–17 Emergency Management System Index 601

NAI. See Notifi able avian infl uenza poultry trade risks related to HP NAI human infl uenza adaptation and, 32– Nakamura, N., 154 and LP of, 501–2 33, 458 Nasal epithelium, 90f trade risk mitigation for countries free NA impacting virulence and, 31 National Animal Health Emergency of, 503 NS impacting virulence and, 31 Management System (NAHEMS), vaccination for chickens infected with pigs AI adaptation and, 32–33 489 HP of, 504t polymerase complex impacting National Poultry Improvement Plan vaccination for poultry infected with virulence and, 31–32 (NPIP), 123, 488 HP of, 503–4 virulence confl icting with, 71 ND. See Newcastle disease NPIP. See National Poultry Pattern recognition receptors (PRRs), Necrosis Improvement Plan 409–10 in ducks with HP AI H5N1 virus of NS. See Nonstructural gene PCS. See Proteolytic cleavage site Asian lineage, 108f Nucleotides, 28 Peroxygen compounds, 396 in emus with HP AI H5N1 virus, Perroncito, Edward, 145, 149–51, 154, 104f Offi ce Internationale des Epizooties 161–63, 165–66, 165f in house fi nch with HP AI H5N1 (OIE) Personal protective equipment (PPE) virus, 103f control strategies and role of, 294 C&D and increased level of, 392, HP AI producing, 90–91 global strategy and regional 403, 473 in zebra fi nch with HP AI H5N1 organizations and partners of FAO mass depopulation and use of, 314– virus, 100f and, 576–77t 15 Negri, A., 157 history of, 145 Pesticide Product Label System (PPLS), The Netherlands HP AI H5N1 virus and response of 392 HP AI H7N7 virus and control FAO and, 562 Petényi, J.S., 165 strategies of, 230–31 OIE. See Offi ce Internationale des Petri, Julius Richard, 148 HP AI H7N7 virus outbreak in, 229– Epizooties Pfenninger, W., 155–56 30 Oil adjuvants, 421 Phenols, 397 HP AI H7N7 virus outbreak infecting On-site burial Piana, o.V., 165 humans in, 231–32 costs associated with, 338 Pigs HP AI H7N7 virus outbreak infecting cross section and longitudinal section antigenic shift impacting, 15–16 pigs in, 232–33, 500 of, 337f history of AI in, 64–65 Neuraminidase (NA), 4 disposal method of poultry carcasses HP AI H5N1 virus with Asian viral assembly process and, 5–6 through, 336–38 lineage in, 111 virulence and pathogenicity problems with, 338 HP AI H7N7 virus outbreak in The implications of, 31 in Virginia, 336–38 Netherlands infecting, 232–33, 500 Neuraminidase inhibition assay (NI Ostriches. See Ratites human infl uenza and AI viruses assay), 303 Outdoor rearing, 76 mixed in, 5 Newcastle disease (ND) infl uenza viruses and turkeys Chu and fowl plague differentiation Pakistan, 224 exposure to, 10 from, 161 PAMPs. See Pathogen-associated pathogenicity and AI adaptation in, Doyle and fowl plague differentiation molecular patterns 32–33 from, 159, 161, 164 Pasteur, Louis, 148, 157 Point of care test. See Rapid point of fowl plague differentiation from, Pathobiology care tests 159–61, 160t concepts of, 66–67 Polymerase complex, 31–32 LPMs and AI compared to, 372–73 ducks infected with HP AI H5N1 Poultry. See also Brunswick poultry New South Wales, 244–45 virus of Asian lineage and groups exhibition; Chickens; Ducks; Live New York City, 192 for, 106t poultry marketing; National Poultry NI assay. See Neuraminidase inhibition of HP AI H5N1 virus with Asian Improvement Plan; Turkeys assay Lineage, 100–102 AI isolations after trade restrictions Nonstructural gene (NS), 31 HP AI HA cleavage and cellular, 11 for other birds and, 502–3 North Korea, 233 LP AI HA cleavage and cellular, 11 AI transmission effi ciency in water Notifi able avian infl uenza (NAI) poultry with AI and concepts of, 66f birds v., 514 food safety risks with HP NAI and terms of, 66t ancient Greek writers on diseases in, LP of, 506–7 Pathogen-associated molecular patterns 151 inactivation methods for poultry (PAMPs), 409–10 antigenic drift and, 14–15 products with, 504–5 Pathogenicity. See also High antigenic shift impacting, 15 poultry eggs pasteurization time with pathogenicity; Low pathogenicity art and writing on, 151–52 infections of HP of, 506t glycosylation infl uencing HA and, Asian food and economic livelihood poultry eggs pasteurization time with 30 impacted by HP AI in, 546–47 infections of LP of, 507t of H5 subtype, 10 Asian writers on diseases in, 151 poultry trade restrictions related to of H7 subtype, 10 biosecurity and voluntary movement HP of, 499–500 HA role in AI and, 25 control programs for, 362–64 602 Index

Poultry (continued) disposal method of landfi ll for HP AI clinical signs in, 92–93 biosecurity cost concerns of farms carcasses from, 338–41 HP AI H5N1 virus spreading through, with, 355, 361–62 disposal method of on-site burial for 265–66, 268, 500 biosecurity plan and transmission of carcasses from, 336–38 HP AI H5N2 experimental studies AI through, 357–58 disposal method of rendering for on, 204t biosecurity regional plan concerns carcasses from, 345–47 HP AI H7N1 virus distribution in for, 363–64 disposal method options introduction Italy for, 226f biosecurity risk assessment and AI for carcasses from, 335–36 HP AI H7N3 virus outbreak in spreading through, 373 disposal methods and sources of Queensland infecting, 244 biosecurity risks of travel between information regarding carcasses of, HP AI H7N3 virus outbreak in LPMs with, 359–60 494t Victoria infecting, 243–44 biosecurity sources of information disposal methods for LP AI H7N1 HP AI H7N4 virus outbreak in New for, 496t virus infected carcasses of, 334t South Wales infecting, 244–45 C&D chemical methods for producers disposal methods of carcasses from, HP AI H7N7 virus outbreak in of, 394 333–35 Victoria infecting, 241–43 C&D dry cleaning for facilities with, dry heat for inactivation of AI in HP AI impacting African economics 400–401 facilities with, 393–94 and, 545t C&D for LP AI in facilities with, economic benefi ts of avoiding disease HP AI infl icted syndromes in, 87–88 529–30 and increasing productivity in, HP AI replication in, 88 C&D response procedures for 537–40 HP NAI and LP NAI risks through facilities with, 391–92 economic compensation for loss of, trade of, 501–2 C&D routine for facilities with, 393 551–52 HP NAI and pasteurization times for C&D water system cleaning for economics and cost of rehabilitation eggs of, 506t facilities with, 400 of, 538–39 HP NAI causing restrictions in trade C&D wet cleaning for facilities with, economics of biosecurity for, 554–55 of, 499–500 401–2 economics of long term prevention of humans infected with HP AI H5N1

CO2 used for euthanasia of, 311–12 HP AI for, 555–57 virus from, 466–70, 472–73 commercial era of, 60, 71 economics of restocking, 553–54 immunology as basis for vaccine controlled marketing for LP AI educational considerations with protection of, 412 infected fl ocks of, 529 disposal methods for carcasses of, immunology as basis for vaccines and control strategies and exposure risk 491, 493 vaccination of, 408 level identifi cation for, 521–22 Egypt and layer market chains of, inactivation methods for NAI infected control strategies and LP AI 543f products of, 504–5 movement in, 518 electrocution for mass depopulation infl uenza viruses infecting, 10 control strategies for confi ned of, 313–14 Kaupp and book on, 152 populations of, 519–20 environmental persistence of AI on, Klee and book on, 152 control strategies for nonconfi ned 70 LP AI and gross lesions in, 92 populations of, 520–21 euthanasia accepted methods for, LP AI and HP AI between aquatic control strategies for population 311–12 birds and, 72f cross-over in, 521 exposure sources of AI for, 67f LP AI and microscopic lesions in, control strategies of biosecurity for exposure to AI of, 67–68 92 LP AI outbreak in, 364 FAO defi ned sectors for, 71, 567t LP AI and tissue and cellular sites of control strategies of C&D for fowl plague and, 124–25 viral replication and damage in, 92 facilities with, 522 fowl plague control strategies and LP AI clinical signs in, 91–92 control strategies of eliminating trade of, 174 LP AI global nature of, 127 infected, 291 fowl plague disinfection and LP AI in, 125 cost of loss of production for, cleansing for, 175 LP AI infl icted syndromes in, 87 550–51 fowl plague quarantine phases for, LP AI isolation in, 65 disposal method comparative analysis 174–75 LP AI outbreaks in Minnesota with, for carcasses from, 350–51 global production and trade of, 499– 514–15, 516t disposal method of alkaline 500 LP AI replication in, 88 hydrolysis for carcasses from, 347– global strategy and restructuring and LPM introducing LP AI to 48 rehabilitation for sectors of, 580–81 commercial, 74–75, 123–24 disposal method of composting for HP AI and gross lesions in, 93–94, LP NAI and pasteurization times for carcasses from, 342–45 93–96f, 96 eggs of, 507t disposal method of incineration for HP AI and microscopic lesions in, maintenance cycle of AI in, 69–70, carcasses from, 341–42 96–98f, 97t, 98 75–76 disposal method of in situ plasma HP AI and tissue and cellular sites of man-made systems introducing AI to, vitrifi cation for carcasses from, viral replication and damage in, 71–72 348–49 98–99 market chains of, 542–44 Index 603

mass depopulation by CO2 gassing of various minor species of, 61 economic benefi ts and enforcement whole house for fl oor-reared, 319 Vietnam costs and benefi ts of control for, 548–49 mass depopulation by CO2 with free strategies for HP AI in, 540t farmer awareness for pathways of, standing panel enclosure for fl oor- village poultry introducing AI to 548 reared, 316–17 commercial, 75f Reservoirs mass depopulation by CO2 with live wild birds and transmission of AI in anseriformes, 46 haul cage enclosures for fl oor- within and between, 358f in charadriiformes, 46 reared, 319–20 wild birds infected with HP AI H5N1 of HP AI in ducks and wild birds mass depopulation by CO2 with virus transmitted by, 64 creating challenges for control plastic tenting for fl oor-reared, wild birds introducing AI to, 72–74, strategies, 574 317–18 73f of LP AI in aquatic birds, 63–64 mass depopulation criteria for, 312– PPE. See Personal protective equipment man-made systems as AI, 514 13 PPLS. See Pesticide Product Label in wild birds, 46, 513–14 mass depopulation for LP AI infected System Risk analysis, 369 fl ocks of, 528–29 Protein. See also Hemagglutinin; M2 Risk assessment. See Biosecurity risk mass depopulation methods for fl oor- protein; Neuraminidase assessment reared, 315–16 in AI structure, 3–4 Risk management, 369 mass depopulation SOPs for, 315 H5N1 virus and deletions of, 259 RNA MDTs from HP AI in chickens infl uenza viruses and viral, 5–6 antiviral drugs for human infl uenza compared to other forms of, 92–93 infl uenza viruses virulence infl uenced and resistance of subpopulations of Middle Eastern food and economic by mutations of, 30 viruses in, 24–25, 461 livelihood impacted by HP AI in, Proteolytic cleavage site (PCS) H7 subtype outbreaks from 546 amino acid changes in virulence and, recombination of, 29 mucosal immunity in, 411 27 make up of, 23–24 outdoor rearing risks of AI for, 76 amino acid insertions for HP AI at, mutation ease of viruses in, 24 pathobiology concepts for AI in, 66f 28–29 Rome, 151 production and international trade in H5N1 virus variations at HA and, 33, Rosenthal, W., 157 meat of, 541t 34–35t, 35 Roux, Pierre Emile, 148 production sectors of, 541–42 HA and mutations at, 27–28, 28t RT-PCR assay, 456–57 Roman writers on diseases in, 151 HA and problems from changes at, secondary spread of AI between 35–36 Salmonella, 505t fl ocks of, 75 HA lab manipulation and, 30 SARS. See Severe acute respiratory steam for inactivation of AI in of HA of Australian H7 subtype and syndrome facilities with, 394 amino acid sequence, 242t Savonuzzi, E., 149 system continuum and country LP AI and HA at, 26–27 Scotland, 217, 219 examples of, 542t PRRs. See Pattern recognition receptors Seals, 111–12 Thailand and meat market chains of, Public health implications, 293–94 Septicemic spirochetosis, 159, 159t 543f Purines, 29 Sequence analysis, 304 transmission of AI and adaptation of, Serological surveillance 68–69 Quaternary ammonium compounds LP AI detected by sentinel birds in, USA control strategies for, 513 (QACs), 397–98 435–36 USA control strategies of biosecurity Queensland, 244 vaccination and infected birds for, 515–17 detected through, 436–37 UV light for inactivation of AI in Rapid point of care tests, 456 vaccination success determined facilities with, 393 Ratites. See also Emus through, 435 vaccination for HP NAI in, 503–4 HP AI in, 99–100 Severe acute respiratory syndrome vaccination issues for, 424–25 LP AI in, 99 (SARS), 563 vaccine cost comparisons for, 427 Recycling. See Rendering Shorebirds, 47 vaccine potency for, 416–17 Reinacher, M., 163 Sialic acid (SA), 457–58 vaccines and mass administration for, Rendering Sodium carbonate, 399 427–28 disposal methods of poultry carcasses Sodium dichlorotriazine trione, 397 vaccines and posthatch parental through, 345–47 Sodium hydroxide, 398–99 administration for, 427 Europe and, 347 SOPs. See Standard operational vaccine’s broad homosubtypic fl ow chart for process of, 346f procedures protection for, 426 Virginia and, 346 South Africa vaccines for AI in, 416 Reporting and confi rmation HP AI H5N2 virus outbreak in, 222– vaccines for live LP AI in, 422–23 cost of effective animal health 23 vaccine studies for AI in other birds information system for, 549 HP AI H5N3 virus outbreak in, and, 418–20t cost of laboratory staff and equipment 222 van Heelsbergen and book on, 152 for, 549 Sparrows, 105f 604 Index

Standard operational procedures (SOPs), fowl plague experimental studies in, Vietnam and costs of biosecurity and, 315 193–94 539–40 Steam. See Wet Heat fowl plague in, 191–92, 197–98 virological surveillance after, 434–35 Sterz, I., 157 fowl plague lesions and clinical signs Vaccines Straub, o.V., 165 in, 192–93 adjuvants in, 417, 421 Stubbs, Evan, 199f fowl plague quarantines of, 195–96 banks of, 571 Survival time H5N2 virus beginning as LP AI, categories of, 417 of AI under laboratory conditions, 199–200 control strategies and proper use of, 357f HP AI creating market shocks and 570–71 biosecurity plan and environment impacting economics in, 544–46 cost comparisons of poultry and, 427 impacted by AI and, 356–57 HP AI H5N2 virus and control cost for, 530 fowl plague inside and outside host strategies of, 201–2 criteria for protection with, 412–13 and, 163 LP AI accidental sources in, 523 DNA based, 424 LP AI H5N2 virus outbreak changing HA and selection of strains of, 425– TADs. See Transboundary animal into HP AI in, 200–201 26 diseases LP AI H5N2 virus outbreak in, 202, HP AI and, 407, 432–33 Texas 204 HP AI and chickens protected by, HP AI H5N2 virus control strategies poultry control strategies of, 513 413–14t in, 210–11 science and technology in, 147–48 HP AI and protection assessment of, HP AI H5N2 virus outbreak in, 209– USA. See United States of America 413–15 10 UV light. See Ultraviolet light HP AI H5N1 virus and prepandemic, Thailand, 543f 460–61 TLR. See Toll-like receptor Vaccination human infl uenza and GISN for Toll-like receptor (TLR), 410 biosecurity and alternative of, 353 formulating, 459 Transboundary animal diseases (TADs) biosecurity during, 431 human infl uenza and new strategies defi nition of, 561 control strategies and programs for, with, 459–60 zoonotic nature of, 563–64 429–31, 552–53, 569–70 immunology as basis for protection of Transmission cycle, 47–48 control strategies for fowl plague in poultry with, 412 Turkey (country), 548t chickens and, 175–76 immunology as basis for vaccination Turkeys control strategies for fowl plague in of poultry and, 408 adaptation to AI transmission of, 69 geese and, 175 inactivated whole AI and, 417 California and LP AI moving economics associated with, 552–53 indirect assessment of protection between farms with, 518f educational programs and strategies from, 416 history of, 60–61 for, 493, 495 licensed AI, 431–32 infl uenza viruses and exposure of evaluating, 531 liposomes in, 421–22 pigs on, 10 HP NAI in chickens and, 504t live vectored, 423–24 LP AI and chickens compared to, 75– HP NAI in poultry and, 503–4 LP AI and, 407–8, 432 76 of human infl uenza and antigenic LP AI and protection assessment of, LP AI sources for, 72f drift, 14 413–14 Minnesota ducks’ HA subtypes in AI human infl uenza prevention and management and environmental and detection of AI in, 517t annual, 459 conditions for, 428–29 vaccines used in Minnesota for, identifi cation of infected animals Minnesota turkeys and use of, 532f 532f after, 433–34 oil adjuvants in, 421 immunology of chickens compared to poultry and AI and, 416 Ultraviolet light (UV light), 393 ducks and geese with, 415 poultry and broad homosubtypic United States of America (USA). See justifi cations for, 531–32 protection of, 426 also California; Minnesota; New limitations of, 532–33 poultry and live LP AI and, 422–23 York City; Texas; Virginia LP AI and control strategies of, 530 poultry and mass administration of, AI outbreaks in, 514 mechanics of, 530–31 427–28 civil freedoms in, 147 of poultry and basis from poultry and other birds with AI and control strategies with biosecurity for immunology, 408 studies on, 418–20t poultry in, 515–17 poultry issues with, 424–25 poultry and posthatch parental fowl plague and economical priorities for, 431 administration of, 427 implications on, 196 serological surveillance detecting poultry and potency of, 416–17 fowl plague and source theories of, infected birds after, 436–37 protection measured by interruption 196–97 serological surveillance determining of contact transmission and, 415t fowl plague C&D in, 196 success of, 435 protection of AI and factors fowl plague control strategies of, surveillance after, 433 impacting, 415–16 195 vaccine application concepts for quality control in manufacturing, fowl plague diagnosis in, 194–95 emergency program of, 430f 426–27 Index 605

strain selection of, 425 pathogenicity confl icting with, 71 H5N1 virus found in species of, 50t vaccination program for emergency polymerase complex impacting HP AI H5N1 virus and human and concepts for applying, 430f pathogenicity and, 31–32 preventative measures with, 51 in vitro expressed HA for, 422 Virus isolation (VI), 299–301, 456 HP AI H5N1 virus experimentally in vivo expressed HA for, 422–24 Virus N, 172–73 infected in, 49–50 Valenti, G.L., 154, 167 Von Ostertag, R., 154 HP AI H5N1 virus maintenance and Van Heelsbergen, T., 152 transmission in, 50–51 VI. See Virus isolation Water-based foam, 320–21, 321 HP AI H5N1 virus naturally infected Victoria Weiss, E., 157, 163 in, 49 HP AI H7N3 virus infecting poultry Wet Heat (Steam), 394 HP AI H5N1 virus of Asian lineage in, 243–44 WHO. See World Health Organization and, 105, 107–9 HP AI H7N7 virus infecting poultry Wild birds. See also Anseriformes; HP AI H5N1 virus spread through, in, 241–43 Charadriiformes; Sparrows 267–68 Vietnam AI in, 43 HP AI H7N1 virus in Italy infecting, costs and benefi ts of HP AI control AI isolation among species of, 44–45t 228 strategies for poultry in, 540t AI subtypes in, 46–47 HP AI in, 99, 125–26 vaccination and biosecurity costs of, biosecurity and movement patterns HP AI in Australia and, 239–40 539–40 of, 362 infl uenza viruses found in, 6–8 Viral evolution, 23 biosecurity plan and transmission of infl uenza virus separation between Virchow, Rudolf, 156 AI through, 358 North American v. Eurasian in, Virginia control strategies and challenges of 6–7 composting used in, 343–45 HP AI reservoirs in, 574 LP AI in, 99, 125–26 incineration used in, 341–42 domestic birds transferring AI to, 9 LP AI prevention for, 523 landfi ll history in, 338–41 domestic ducks commingling with, maintenance cycle for AI in, 48–49 map of AI outbreaks in, 340f 74 poultry and HP AI H5N1 virus on-site burial in, 336–38 environmental persistence of AI in, transmitted to, 64 rendering used in, 346 48 poultry and transmission of AI within Virological surveillance, 434–35 euthanasia methods for, 324 and between, 358f Virulence euthanasia through cervical poultry introduced to AI through, 72– amino acid changes at PCS and, 27 dislocation for, 325–26 74, 73f

of H5N1 virus increasing, 35 euthanasia through compressed CO2 research on AI in, 51–52 historical changes in AI and, 25 in a chamber for, 326 reservoirs in, 46, 513–14 mutation of proteins inﬂ uencing euthanasia through culling bag with transmission cycle of AI in, 47–48

inﬂ uenza viruses and, 30 frozen CO2 for, 326–28 World Health Organization (WHO), NA impacting pathogenicity and, 31 euthanasia through injectable 253, 462. See also Global Inﬂ uenza NS impacting pathogenicity and, 31 anesthetics for, 324 Surveillance Network