VIRULENCE AND PATHOGENESIS OF NEWCASTLE DISEASE VIRUS
ISOLATES FOR DOMESTIC CHICKENS
by
GLAUCIA DENISE KOMMERS
(Under the Direction of Corrie C. Brown and Daniel J. King)
ABSTRACT
Newcastle disease virus (NDV) isolates vary greatly in virulence and pathogenicity depending on several factors including the host species and the infecting virus strain. This research was performed in order to investigate the effect of serial passages of several NDV isolates in domestic chickens. Six isolates were recovered from pigeons and six isolates had heterogeneous origin (recovered from chickens, wild, and exotic birds). A monoclonal antibody (MAb) panel revealed that four of the pigeon-origin isolates were the variant pigeon paramyxovirus-1 (PPMV-1) and two of them were avian paramyxovirus-1 (APMV-1) isolates. Pathotyping tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. However, the PPMV-1 were still of moderate virulence for chickens after passages. The fusion protein cleavage site amino acid sequence of all six pigeon-origin (PPMV-1 and APMV-1) isolates was typical of virulent NDVs. Although the results of the pathotyping tests indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and nervous signs in some of the chickens inoculated intraconjunctivally. However, severe lesions were observed mostly affecting the heart and brain after intraconjunctival inoculation with passaged PPMV-1 isolates. Pigeons must be considered seriously as a potential source of NDV infection and disease for commercial poultry flocks. All six heterogeneous-origin isolates were characterized by reactivity to MAbs as members of the APMV-1 serotype. Three isolates showed low virulence for chickens before and after passage and had the F protein cleavage site typical of low virulence viruses. The other three heterogeneous-origin isolates were classified by the pathotyping tests and sequence analysis of the F protein cleavage site as moderate to highly virulent for chickens. An isolate recovered from an exotic dove had marked virulence increase after passage. The exact mechanism for the virulence increase observed with this isolate remains undefined. A pathogenesis study with the chicken-passaged isolates revealed that two of them were highly virulent for chickens, with marked tropism for lymphoid tissues. The results reported here demonstrate the high risk for domestic chickens represented by some NDV- infected non-poultry species. INDEX WORDS: Apoptosis, Avian paramyxovirus-1, Avian virology, Chickens, Immunohistochemistry, In situ hybridization, Newcastle disease, Nucleotide sequence analysis, Pathogenesis, Pigeon paramyxovirus-1, Veterinary Pathology VIRULENCE AND PATHOGENESIS OF NEWCASTLE DISEASE VIRUS
ISOLATES FOR DOMESTIC CHICKENS
by
GLAUCIA DENISE KOMMERS
Médico Veterinário, Universidade Federal de Santa Maria, Brazil, 1987
M.S., Universidade Federal de Santa Maria, Brazil, 1993
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirement for the Degree
DOCTOR OF PHILOSOPHY
ATHENS, GEORGIA
2002 © 2002
Glaucia Denise Kommers
All Rights Reserved. VIRULENCE AND PATHOGENESIS OF NEWCASTLE DISEASE VIRUS
ISOLATES FOR DOMESTIC CHICKENS
by
GLAUCIA DENISE KOMMERS
Approved:
Major Professors: Corrie C. Brown Daniel J. King
Committee: K. Paige Carmichael Barry Harmon Bruce Seal Pedro Villegas
Electronic Version Approved:
Gordhan L. Patel Dean of the Graduate School The University of Georgia May 2002 iv
DEDICATION
I dedicate this work to my family. Your every day prayers and your love made me stronger than I thought I could ever be. Muito obrigado!!!!! v
ACKNOWLEDGMENTS
First of all, I would like to thank God for his care, for his love, and for his answers to all my prayers over the last four years.
I would like to thank my major professors, Drs. Corrie Brown and Jack King, for their expert guidance and patience with me and with this work. Both of them went far beyond their call of duty as major professors, giving me their friendship and care.
I would like to thank all my committee members, Drs. Paige Carmichael, Barry
Harmon, Bruce Seal, and Pedro Villegas for their expert input and for all the time spent reviewing this work. I would like to thank all the faculty of the Department of Pathology for their teaching, patience, time, and for making me want to be an excellent pathologist as all of them are.
I would like to thank all graduate students who shared sometime with me during this journey. My special thanks goes to my first three officemates, Drs. Anapatricia
García, Lucia García-Camacho, and Suzana Tkalcic, and to my last three officemates,
Drs. Yong-Baek Kim, Laura Perkins, and Nobuko Wakamatsu. Without their friendship I probably would not have done the work I came here to do.
I would like to thank all the staff members of the Department of Pathology. My special thanks goes to Amanda Crawford, “my guardian angel”, who always stood by me as a friend and cared for me as a member of her wonderful family. I would also like to thank Erica Behling-Kelly, Joyce Bennett, Phillip Curry, Melissa Scott, and Dr. James
Stanton for their excellent technical assistance and patience in the laboratory. vi
I would like to thank the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq; Brasília, DF - Brazil), for supporting me with a scholarship. I wish to thank the U.S. Poultry and Egg Association and USDA-ARS-CRIS for their support. I also wish to thank Dr. David Swayne for allowing me to do a major part of my work in the Southeast Poultry Research Laboratory (SEPRL - USDA).
I would like to thank all faculty of the Department of Pathology at the
Universidade Federal of Santa Maria (UFSM; Santa Maria – RS, Brazil). My special thanks goes to Dr. Claudio Barros for all his efforts in helping me to be accepted as an
UGA PhD student.
My special thanks also goes to all my friends who were giving me their support, sharing with me their experiences, and making me look forward to achieving my goals along the last four years. This is a very long list represented here by the ones I have met in the USA: Maria Rosa Ferreira, Eleonora and Marcos Machado, Monica and Carlos
Costa, Eugenia and Cármelo de Los Santos, and Carolina Realini. vii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... v
CHAPTER
1 INTRODUCTION...... 1
2 LITERATURE REVIEW...... 5
3 VIRULENCE OF PIGEON-ORIGIN NEWCASTLE DISEASE VIRUS
ISOLATES FOR DOMESTIC CHICKENS...... 52
4 PATHOGENESIS OF SIX PIGEON-ORIGIN ISOLATES OF
NEWCASTLE DISEASE VIRUS FOR DOMESTIC CHICKENS...... 92
5 VIRULENCE OF SIX HETEROGENEOUS-ORIGIN NEWCASTLE
DISEASE VIRUS ISOLATES BEFORE AND AFTER SEQUENTIAL
PASSAGES IN DOMESTIC CHICKENS ...... 124
6 PATHOGENESIS OF CHICKEN-PASSAGED NEWCASTLE
DISEASE VIRUSES ISOLATED FROM CHICKENS, WILD, AND
EXOTIC BIRDS ...... 167
7 CONCLUSIONS...... 201 CHAPTER 1
INTRODUCTION
Newcastle disease (ND) is one of the most important avian diseases because of its potential economic impact on the poultry industry. Virulent ND is a reportable disease in most countries of the world and is reportable as a List A disease for member countries of the Office International des Epizooties (OIE).8 List A diseases are those that are highly transmissible and their occurrence is of major importance for the international trade of animals and animal products. 8 Newcastle disease is caused by an avian paramyxovirus serotype 1 (APMV-1), synonymous with Newcastle disease virus (NDV).1, 2 NDV has been classified according to five pathotypes that relate to the disease signs produced in infected chickens: a) asymptomatic enteric NDV (avirulent viruses); b) lentogenic NDV
(low virulence viruses); c) mesogenic NDV (moderately virulent viruses); d) neurotropic velogenic NDV (NVNDV; highly virulent neurotropic viruses); and e) viscerotropic velogenic NDV (VVNDV; highly virulent viscerotropic viruses).1, 2, 6
There is an international trend that started in 1992 with the ND definition in the
European Union (EU) Council Directive 92/66/EEC,4 valid for countries belonging to the
EU, to separate NDV isolates into two virulence groups: the low virulence viruses
(asymptomatic enteric and lentogens) and the virulent viruses (mesogens and velogens), based on the intracerebral pathogenicity index (ICPI) as a differential test.5
Following this international trend, the OIE established new standards during 1999 with a major change in the official definition of ND.7 ND is now defined as an infection 2 of birds caused by a virulent virus of the APMV-1 serotype. An isolate will be classified as virulent for chickens if it has an ICPI of 0.7 or greater or if it has multiple basic amino acids at the fusion (F) protein cleavage site.7
Currently, the United States Code of Federal Regulations (US-CFR) refers to the presence of “exotic Newcastle disease” (END) as any velogenic ND. END is defined as
“an acute, rapidly spreading, and usually fatal viral disease of birds and poultry.” 3 A major difference in the OIE’s definition for ND is that it refers to an infection with viruses of the APMV-1 serotype, while the US-CFR refers to a disease caused by highly virulent NDVs (velogenic pathotype).
In the USA, isolates that meet the OIE criteria for virulence (but do not meet the US-
CFR criteria) have been recovered almost exclusively from non-poultry sources, including racing pigeons, wild, and exotic birds. The potential threat represented by these isolates for poultry has to be thoroughly investigated because previous observations revealed that NDV isolates from non-poultry species may not show their potential virulence for domestic chickens in conventional pathogenicity tests, until passaged several times in chickens. 1, 2
The difference between the OIE and US-CFR definitions of ND raises several questions. What is the threat to poultry represented by isolates that are classified as virulent by OIE but are not by the US-CFR? Could passage of these isolates in chickens result in increased virulence that justifies using a more severe standard than the US-CFR?
What is the risk of trade sanctions if the USA has to adopt the OIE standards?
The overall hypothesis of this study was that the virulence of pigeon-origin and heterogeneous-origin NDV isolates would increase after passage in domestic chickens. 3
To test this hypothesis, biological and molecular assays were performed to determine the potential of six pigeon-origin and six primarily non-chicken-origin isolates of NDV to cause disease in domestic chickens. These objectives were fulfilled by: a) assessing the virulence of the NDV isolates before and after four serial passages in
chickens through four pathotyping tests: the intracerebral pathogenicity index (ICPI);
the intravenous pathogenicity index (IVPI); the mean death time (MDT); and the
intracloacal pathogenicity test. b) evaluating portions of the viral genome, including the fusion (F) and matrix (M)
genes, before and after passage. c) assessing the pathogenesis of the passaged viruses for chickens inoculated
intraconjunctivaly (a more natural route of infection) through evaluation of clinical
signs, mortality, gross and microscopic lesions, including immunohistochemistry, in
situ hybridization, and apoptosis assays.
REFERENCES
1. Alexander DJ: Newcastle disease and other avian Paramyxoviridae infections. In:
Diseases of Poultry, ed. Calnek BW, Barnes HJ, McDougall LR, and Saif YM, Beard
CW, 10th ed., pp. 541-569. Iowa State University Press, Ames, IA, 1997
2. Alexander DJ: Newcastle disease virus and other avian paramyxoviruses. In: A
laboratory manual for the isolation and identification of avian pathogens, ed. Swayne
DE, Glisson JR, Jackwood MW, Pearson JE, and Reed WM, 4th ed., pp. 156-163.
American Association of Avian Pathologists, Kennett Square, PA, 1998 4
3. Code of Federal Regulations (CFR), Title 9, Parts 1-199, Section 53.1. Cod Fed
Regulat 1:159, 2000
4. Council of the European Communities. Council Directive 92/66/EEC - introducing
Community measures for the control of Newcastle disease. Off J Europ Commun
35:1-20, 1992
5. King DJ: Newcastle disease virus pathotyping: the current emphasis on the ICPI.
Respiratory Diseases of Poultry - AAAP Symposium. Ann Conv Am Vet Med Assoc,
138:38-42, 2001
6. Office International des Epizooties: Manual of standards for diagnostic tests and
vaccines. Chapter 2.1.15 – Newcastle disease. 4th ed., pp. 221-232. OIE, Paris,
France, 2000
7. Office International des Epizooties: Official Acts. Resolution No. XIII - Newcastle
Disease. Off Int Epizoot Bull 111:266-267, 1999
8. Office International des Epizooties: OIE - Disease Classification. List A. Newcastle
disease. URL: www.oie.int/eng/maladies/en_classification.htm, January 10, 2002 CHAPTER 2
LITERATURE REVIEW
NEWCASTLE DISEASE
A. History
The first recorded outbreaks of Newcastle disease (ND) date from 1926 in Newcastle- upon-Tyne, England 43 and on the island of Java, Indonesia.10 However, Alexander 12 mentioned the existence of evidence in the literature that there may have been outbreaks of a ND-like disease before 1926. Possible problems in the assessment of ND outbreaks before 1926 are the lack of distinctive clinical signs of ND and the difficulty in differentiating ND from highly pathogenic avian influenza (HPAI) in the field because both diseases cause similar respiratory signs and high mortality in avian species.12
B. Etiology
B. 1. Classification
Newcastle disease virus (NDV) is synonymous with avian paramyxovirus type 1
(APMV-1).10,11 It has been classified in the order Mononegavirales, family
Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus.10,11,89 However, the complete nucleotide sequence of NDV showed that NDV is only distantly related to other rubulaviruses. It has been proposed that NDV be considered as a member of a new genus within the subfamily Paramyxovirinae.92 Based on matrix (M) protein amino acid 6 sequences, Seal et al 126 observed that NDV separates as a clade more closely related to morbilliviruses than to rubulaviruses. A large-scale study of the phylogenetic relationships among the Paramyxoviridae showed both fusion (F) and M phylogenies were consistent with previous proposals for expanding the taxonomic diversity within the
Paramyxoviridae to include a new genus for NDV.140
NDV is a nonsegmented, single-stranded, negative-sense, enveloped RNA virus.
Viral particles are observed by electron microscopy as pleomorphic, varying from spherical (150-300 nm in diameter) to filamentous (about 100 nm across and of variable length). Projections of approximately 8-12 nm are observed on the viral surface, corresponding to the F and hemagglutinin-neuraminidase (HN) glycoprotein spikes. The
“herring bone” nucleocapsid (about 13-18 nm in diameter) might be seen either free or emerging from disrupted viral particles.10,89
The NDV genome consists of 15,156 nucleotides.98,115 Six major genes encode the structural proteins (in the order 3’-NP-P-M-F-HN-L-5’): NP – nucleoprotein (an
RNA binding nucleocapsid protein); P – phosphoprotein (nucleocapsid associated); M - matrix protein (an unglycosylated envelope protein); F - fusion protein (smaller surface projections); HN – hemagglutinin-neuraminidase (larger projection on virus surface); and
L (large) protein – an RNA-dependent RNA polymerase associated with the nucleocapsid.189,98,144
B. 2. Clinical forms
Specific clinical signs are not observed in birds with ND. The clinical disease might range from subclinical infection to 100 % mortality in a short period of time. Many 7 factors related to the host (species, age, and immune status), virus strain (pathotype, dosage and route of infection), and environmental or social stress can influence the severity and the course of the disease.10,11,26,68
Historically, NDV has been classified according to five pathotypes that relate to the disease signs produced in infected fully susceptible chickens:10,11,104 a) asymptomatic enteric NDV - presence of avirulent viruses in the intestinal tract; b) lentogenic NDV - mild or inapparent respiratory infection with low virulence viruses; c) mesogenic NDV - low mortality, acute respiratory disease, and nervous signs in some birds infected with moderately virulent viruses; d) neurotropic velogenic NDV (NVNDV) - respiratory and neurologic signs with high mortality caused by highly virulent neurotropic viruses; and e) viscerotropic velogenic NDV (VVNDV) - acute lethal infection caused by highly virulent viscerotropic viruses in which hemorrhagic lesions are usually observed in the gastrointestinal (GI) tract.10,11
According to the new ND definition105 (discussed at the end of this chapter), viruses of all five pathotypes would fall into two major groups: “low virulence viruses” (for asymptomatic enteric and lentogenic NDVs) and “virulent viruses” (for mesogenic and velogenic NDVs).
B. 3. Biological and molecular characterization of NDV
Important concerns about NDV are related to the variation in virulence and in the capacity to produce disease and how to differentiate between different types of NDV. 9,12
The conventional diagnosis of ND involves the isolation of the virus, the identification of the virus as NDV, and the pathotyping tests to define the virulence of the isolate.10,11,104 8
Molecular-based techniques have also been used to assess the potential pathogenicity104 and for molecular epidemiology to determine the origin of the isolates and methods of spread during ND outbreaks.2
Traditionally, in vivo tests have been used to make an acceptable assessment of the virulence of NDV isolates for chickens.12 Four pathotyping tests have been employed to differentiate virulence among NDV isolates: a) the intracerebral pathogenicity index
(ICPI) in 1-day-old chicks from specific-pathogen-free (SPF) parents; b) the intravenous pathogenicity index (IVPI) in 6-week-old SPF chickens;10,11 and c) the intracloacal inoculation pathogenicity test in 6-to-8-week-old chickens.10,11,113 The mean death time
(MDT) in 9-to-10-day-old embryonating eggs is another pathotyping test used during the preliminary characterization of NDV virulence. Viruses are characterized as low, moderate, or high virulence based on time to embryo death postinoculation, a more rapid death indicating a more virulent virus.10,11 The ICPI differentiates low virulence lentogens from mesogens of intermediate virulence and highly virulent velogens. In the ICPI test, generally the lentogenic viruses give indices of up to 0.4, asymptomatic enteric viruses usually slightly lower indices, mesogenic vaccines are usually around 1.4 and velogenic viruses 1.7 upwards (maximum of 2.0).9 The IVPI differentiates most mesogens and all lentogens from velogens 9,11 but tends not to show distinction between mesogenic and lentogenic viruses.9 Isolates that produce severe disease and mortality following intracloacal inoculation are identified as velogenic and VVNDV isolates are differentiated from NVNDV by the presence of hemorrhagic lesions evident at necropsy.11,113 9
Although the pathotyping tests have been employed successfully for NDV characterization, they are laborious and there are some limitations and difficulties in interpretation of the results.11 For example, standard ICPI and IVPI tests with PPMV-1 isolates produced compact grouping of the ICPIs but a wide range of IVPIs,17 making the interpretation of the virulence of the viruses for chickens difficult. There is evidence that viruses isolated from birds other than poultry may not show their “true virulence for chickens” in conventional pathogenicity tests10,11 until passaged several times in chickens.11 King 72 reported that the chicken breed in which the tests are performed might also be a source of variability for the results of the pathotyping test. In that study it was demonstrated that SPF White Leghorns were more susceptible than SPF White
Rocks. The accuracy of NDV virulence characterization is extremely important9 because
ND is a reportable disease that might result in international trade restrictions.104,108
The application of panels of monoclonal antibodies (MAbs) 6,15,90,112 was a major advance for the characterization of NDV isolates, although it did not replace the in vivo tests.2 Using conventional serologic tests with polyclonal antibodies, NDV isolates are antigenically similar to the extent that all isolates are included in a single serotype. 11
However, MAbs may detect slight variations in antigenicity and provide an approach to antigenic differentiation and grouping of NDV strains and isolates.2,16 The remarkable uniqueness of the variant pigeon paramyxovirus-1 (PPMV-1) described below was established through panels of Mabs.6,15,16,90,112
A severe disease in pigeons characterized clinically by diarrhea followed by nervous signs that included ataxia, tremors, wing and leg paralysis, and torticollis spread through most of Europe in the early 1980’s.134 The disease was caused by the NDV variant 10
PPMV-1, distinguishable from other members of the APMV-1 group by unique binding patterns to monoclonal antibodies (MAbs).6,20,90,120 The sequence of events related to
PPMV-1 diagnosis and worldwide spread in pigeons is summarized on Table 2.1.
The pigeon-NDV variant was initially reported to be more virulent for pigeons than for chickens.67 Further studies showed that despite the close antigenic identity among them, PPMV-1 isolates have considerable variation in their pathogenicity for chickens.39
Turkeys experimentally infected with PPMV-1 presented similar clinical signs as observed in chickens.66 PPMV-1 infection also has been reported in other birds species such as kestrels (Falco tinnunculus)93 and pheasants (Phasianus colchicus).34
PPMV-1 was recognized as a very important disease-causing agent for domestic poultry during 1984, when approximately 20 outbreaks of ND in non-vaccinated laying hens in Great Britain were found to be caused by the NDV variant PPMV-1. The source of the virus was probably feedstuffs contaminated with droppings and other materials from infected feral pigeons feeding in certain feed stores in Liverpool docks.19,21 After contact with infected pigeons, ND symptoms were also observed in poultry in Austria during the 1980’s. 133
The importance of molecular-based techniques to characterize NDV isolates was recognized by OIE during 1999,105 with the addition of an in vitro test in the definition of
ND.12 The test is based on the identification of multiple basic amino acids at the fusion protein cleavage site to characterize an isolate as virulent.105 According to Alexander,12 allowing an in vitro test for the confirmation of NDV virulence was a major step forward.
However, the permanence of an in vivo test (the ICPI) in the definition is still necessary, primarily because in the case of mixtures of virulent and avirulent viruses, a possible 11
Table 2.1. Sequence of events involving the diagnosis and spread of PPMV-1 viruses worldwide.
Year Events Until 1971 - Cases of natural infection by NDV were reported in pigeons. 91 1971-73 - Epizootic of ND in poultry in Europe. Reported cases of velogenic ND in racing pigeons in Holland, Great Britain, Belgium, and Germany. Pigeons had been infected presumably after contact with diseased poultry. 134 Late 1970’s - A severe disease was described in pigeons in the Middle East as “contagious paralysis” 100 or “viral encephalomyelitis” with unknown etiology 4 (initially believed to be caused by a member of the family Herpesviridae). 5,131 1980 - Classic lentogenic NDV strains isolated from pigeons with respiratory signs.135 Serologically, 7% and 19% of the racing pigeon population in Belgium and France, respectively, were positive for NDV infection without overt clinical disease. 134 1981 - Mediterranean racing pigeons with a disease resembling the neurotropic form of ND were first observed. 134 - The earliest published report of this disease was in two racing pigeons imported into Belgium from Italy. The virus isolated from those pigeons was characterized as a paramyxovirus-1 strain. 134 - The BVC 78 isolate from sick pigeons in the Middle East (with what was called “pigeon herpes encephalomyelitis”) was tentatively described as a pathotype variant of NDV. Years later, BVC 78 was characterized by monoclonal antibodies as possibly the first known isolate of the NDV variant “pigeon paramyxovirus-1” (PPMV-1). 67 1982-83 - The infection with PPMV-1 spread quickly among the racing pigeon population throughout Europe because of mixing during races and extensive trade. 21,22,134 - The disease reached racing pigeon lofts in Great Britain during 1983. 13,20,22,93 1984 - The disease in pigeons reached panzootic proportions. 8,10,12,134 - Approximately 20 outbreaks in domestic fowls in England were caused by the NDV variant PPMV-1, confirmed by MAb binding patterns. 21 - There was evidence suggesting that the viruses passed to domestic fowl via feedstuffs contaminated with droppings and other materials from infected feral pigeons.19,93 - PPMV-1 infection identified in pigeons in the USA112 and has been continuously isolated since then. 112,114,127,130 Since 1985 - The infection in pigeons may be considered worldwide. 134 12 finding, primers may preferentially select the virus of low virulence giving a false result.12
A current challenge is the design of a molecular-based technique that may be able to cover all three aspects of ND diagnosis: virus detection, characterization, including inference of virulence, and epidemiology without some use of in vivo testing.2 A number of approaches that have followed development of the reverse transcriptase polymerase chain reaction (RT-PCR) including restriction enzyme analysis, probe hybridization, and nucleotide sequencing (for cleavage site analysis and epidemiological studies) were reviewed in detail by Aldous & Alexander.2 The first RT-PCR for NDV identification was reported in 1991.64 More recently, an RT-PCR has been developed to detect NDV in tissues and feces samples.51 A nested RT-PCR-ELISA technique for rapid and sensitive
NDV detection from tissue samples was developed by Kho et al.70 A heteroduplex mobility assay (HMA) was developed to aid in further identification of molecular markers as predictors of NDV virulence.27,123 A triple one-step RT-PCR was designed for rapid identification (3-4 h) of NDV isolates as well as to differentiate virulent from avirulent NDV isolates.136 Fluorogenic probes in a PCR assay were used for rapid NDV pathotyping.3 These attempts represent a major advance in the use of molecular-based approaches for NDV diagnosis and characterization. 2
Data about global epidemiology as well as local spread of ND has been obtained through nucleotide sequence and phylogenetic analysis of NDV.38,40,59,75,76,122,124-126,137
Despite the marked genetic diversity of NDV, the analysis of the data obtained by several researchers revealed that viruses sharing temporal, geographical, antigenic or epidemiological parameters tend to fall into specific virus lineages or clades.2,95,124,126 13
B. 4. Virulence and pathogenicity of NDV
The pathogenicity of NDV depends upon the tissue tropism and virulence of the virus.97 There is evidence that NDV uses ubiquitous sialic acids in complex sugars as cell receptors.54,101,103 However, the striking strain-dependent differences in tissue tropism and virulence observed with NDV do not depend upon the virus-receptor interactions but on the presence of cellular proteases required for the activation of the viral fusion glycoprotein precursor (F0).50,52,101,103,109,119 The inactive precursor F0 is synthesized as a single chain molecule. In order for the viral particles to become infectious, F0 has to undergo posttranslational endoproteolysis by host cell proteases.101 The F0 of virulent
NDV strains have an oligobasic cleavage motif 112R/K-R-Q-R/K-R-F117 (R = arginine; K
= lysine; Q = glutamine; F = phenylalanine)48 allowing cleavage by a protease(s) ubiquitously present in cells throughout the body.12,52,101 Furin (or paired basic amino acid-cleaving enzyme; furin/PACE), a subtilisin-like endoprotease detected in mammalian cells, is able to cleave the F0 of virulent NDV.52 The infection is consequently pantropic or systemic.12,101 The presence of a phenylalanine residue at position 117, the N-terminus of the F1 protein, is highly conserved among all virulent
NDV isolates, while the low virulence isolates have a leucine (L)48 residue at that position.37,50
Low virulence strains having monobasic cleavage motif 112G/E-K/R-Q-G/E-R-L117
(E = glutamate; G = glycine)48 undergo F0 cleavage only in a few limited tissue types expressing specific trypsin-like protease(s), hence causing an infection more localized to particular organs as the respiratory and alimentary tracts.12,52,101 Some moderately virulent
PPMV-1 isolates from pigeons 37,145 as well as some isolates from chickens grouped 14 antigenically with PPMV-1 viruses 63 have the sequence 112G-R-Q-K-R-F117 37,39,63,145 or
112R-R-Q-K-R-F117.39
Several studies revealed the importance of the F protein cleavage site amino acid sequence as a determinant of NDV virulence.37,50,52,101 Using reverse genetic techniques,116 a recent study strongly demonstrated that the cleavability of the F protein is a major determinant of NDV virulence.115 By introducing three nucleotide changes in the cDNA, a genetically tagged derivative of the La Sota strain was generated in which the amino acid sequence of the protease cleavage site (111G-G-R-Q-G-R-L117) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (111G-R-
R-Q-R-R-F117). The ICPI of the strain derived from the unmodified cDNA was completely nonvirulent (ICPI = 0.00) and the strain derived from the cDNA in which the protease cleavage site was modified showed dramatic increase in virulence (ICPI =
1.28).115
Although the sequence of the fusion protein cleavage site is a recognized virulence marker, the method for acquisition of that sequence is unknown. During 1990, a virulent
NDV (PMV-1/chicken/Ireland/34/90) isolated in Ireland showed a close antigenic relationship, demonstrated by MAbs, to a group of avirulent viruses. Nucleotide sequence analysis of a small region of the F gene of these viruses suggested that they may have arisen from the same gene pool. A few nucleotide changes resulted in differences in the deduced amino acid sequence (at residues 112, 115, and 117) giving 34/90 a typical virulent virus motif at the F cleavage site.37 Further analysis of a 309 nucleotide sequence of the F protein gene confirmed that the 34/90 isolate has a close genetic relationship to 15 an avirulent virus isolated from waterfowl, and was genetically quite distant from most other strains and isolates.38
A similar event but with more severe economic impact was observed in Australia during the 1998-2000 ND outbreaks affecting commercial poultry.53,77,138 The outbreaks were caused by a virulent virus that apparently evolved from a recognized Australian lentogenic strain. F and HN gene sequence comparison showed a strong relationship to sequences derived from an endemic low virulence virus rather than those of overseas viruses or wild bird isolates.53,138 It seems that the simple mutation of two nucleotides within an avirulent F0 cleavage site resulted in a highly virulent virus.53 It is more likely that mutations took place once the virus had been circulating in poultry. This is supported by the lack of isolation of virulent viruses from wild birds.12 The fact that virulent NDV strains might emerge from low virulence viruses by mutation points out possible future repercussions following the worldwide use of NDV live vaccines.12 However, the ND outbreaks caused by a mutated virus in Australia happened in the absence of NDV vaccination in that country.53,138
Sequential passages in chickens with infective spleen homogenates is a technique used for recovering of NDV infectivity and contagiousness.23,44 Experimentally, serial passages of NDV in chickens has resulted in virulence increase of isolates recovered from several avian species (mostly from pigeons).17,18,72,73,78 Virulence increase of some tested PPMV-1 isolates for chickens was accomplished when four passages were performed in 2-wk-old chickens by intramuscular injection of homogenized spleens from chickens at the previous passage. The increase in virulence was demonstrated by increase in the intravenous pathogenicity index (IVPI), comparing the results from pre- and post- 16 passaged viruses.17,18 IVPI increase was described after five passages in chickens of three out of eight tested PPMV-1 isolates from pigeons in Hungary.78 Pathogenicity enhancement was also limited to an IVPI increase for one out of three PPMV-1 isolates
(from USA and Canada) passaged in embryonated eggs.73 In another attempt to produce disease in chickens inoculated intranasally or with chicken-passaged USA PPMV-1 viruses, no clinical disease was observed in chickens after serial passages of the virus.112
It is known that both wild-type and laboratory cultured strains of NDV contain several subpopulations that are often distinguishable by their plaque morphology.57
Passage in MDBK cells may also aid in selecting for more virulent subpopulations of
NDV in a mixed culture.71 When cloned, these subpopulations may differ significantly from each other in their ability to infect and cause disease in bird species.57 Kissi &
Lomniczi 79 discussed possible mechanism(s) underlying the virulence increase observed after serial passages of PPMV-1 isolates in chickens. The authors suggested that the virulence increase might result from the selection of the most virulent virus clones among a heterogeneous population of genomes (“quasispecies”: a complex mixture of rapidly evolving and competing molecular variants of RNA virus genomes which occurs in most populations of RNA viruses)33. Upon passage in chickens, the most virulent virions of a heterogeneous population would overgrow the less virulent ones. As a result of selection, after a few passages, the most virulent virions would constitute the majority of the population. The authors also suggested that the heterogeneity of PPMV-1 strains fall into two categories: a) the strains that are probably homogeneous in virulence and do not contain subpopulations of higher virulence than the original strain will not change in 17 virulence upon passage in chickens; and b) the strains which undergo a substantial increase in virulence during passages in chickens are heterogeneous in virulence.79
Collins et al 39,40 evaluated the molecular basis for the virulence increase of PPMV-1 isolates. One isolate with low pathogenicity for chickens had a typical virulent motif at the F protein cleavage site (112G-R-Q-K-R-F117) that remained unaltered when virulence of the virus increased by bird to bird passage. The authors concluded that the wide variation in pathogenicity of PPMV-1 for chickens is not related to variation in the amino acid motif at the F protein cleavage site nor due to production of HN0 (HN precursor) which may also influence pathogenicity (see below).39 A similar study showed that the recorded differences in the IVPIs of passaged PPMV-1 viruses were unlikely to be associated with differences in the primary structure of the F0.40 The amino acid sequence of the F protein of some examined highly virulent PPMV-1 viruses showed that a double pair of amino acids in the F cleavage site is not necessary for the full expression of virulence. 39
Therefore, the exact minimum requirements for virulence based on the amino acid sequence at the F cleavage site remains unclear. Which particular factors - the importance of a basic amino acid at residue 112, the exact effect of interchanging lysine (K) and arginine (R) at various positions, and the real significance of a phenylalanine (F) at position 117 - are most important for virulence, remain to be determined.12
The main role of the extension of the HN protein in NDV virulence has to be more thoroughly evaluated. The HN protein may be produced in three different sizes (571, 577,
121 or 616 amino acids) depending on the position of stop codons in the gene. The HN0616 requires proteolytic cleavage for conversion to the biologically active form.102 The other 18
39 two forms, HN577 and HN571, appear to be already biologically active. Analyzed viruses such as strains Ulster 2C, D26, and QV4 that code for HN0 have been of extremely low virulence for chickens.121
C. Epidemiology
C. 1. Hosts
At least 236 avian species have a record of NDV isolation. Chickens are considered the most susceptible poultry species. Aquatic birds are considered the most resistant avian species.68 Generally, the NDVs isolated from them are of very low pathogenicity for domestic poultry.7 However, some low virulence isolates from migratory waterfowl in the USA differed in many respects from currently used commercial vaccine strains B1 and La Sota.118 Another exception was the occurrence of neurotropic velogenic ND in double-crested cormorants (Phalacrocorax auritus) in the USA 24,47,49 and Canada 81,82,142 during the 1990’s.
Captive (pet and exotic) birds are fully susceptible to NDV 46,68 and a high percentage of the isolates recovered from these birds are virulent for chickens.7 Virus shedding is usually brief in Galliformes and some song birds.68 In domestic geese, the NDV replication rate seems to be low and the excretion of NDV does either not exist or occurs only at very low levels and for a short period of time.28 Long-lasting shedding of NDV might be observed in Columbiformes (pigeons and doves) and Passeriformes with chronically infected kidneys.68 Virus shedding was detected until 21 days post- inoculation of highly virulent NDV in racing pigeons.45 Infected psittacine birds may become persistently infected (a carrier state) and can excrete virulent virus intermittently 19 for extremely long periods, sometimes exceeding one year.46 It is important to mention that NDV vaccination of poultry might prevent disease,111 but it does not prevent infection and excretion of the challenge virus.14 However, vaccination may reduce significantly the length of time virus is excreted. 14
Non-avian animal species, including humans, are suitable hosts for the replication of
NDV.7 The disease in humans is characterized by conjunctivitis due to infection of the conjunctival sac and is generally associated with accidents in laboratories during manipulation of NDV 7,10 and during vaccination procedures from either aerosolized virus or from hands contaminated during the reconstitution and dilution of vaccine.
C. 2. Transmission
Infection occurs either by inhalation or ingestion of the virus.7 NDV may be spread by any animal and any medium that results in contact directly or indirectly with susceptible poultry.12 Spreading from bird-to-bird depends on the availability of the virus in the infectious form.7 Birds with respiratory infection liberate large or small droplets containing virus and these virus-laden particles impinge upon mucous membranes, resulting in infection.10 Since large amounts of virus particles are excreted in feces, the ingestion of contaminated feces is probably the main method of spread, especially when respiratory disease is not present. The occurrence of vertical transmission of NDV is still controversial.10 Capua et al 35 reported a possible occurrence of NDV vertical transmission. In this case, a virulent strain was isolated from commercial embryonated eggs laid by clinically normal laying hens. 20
C. 3. Sources of virus and methods of spread
One of the most remarkable characteristics of ND is the ability of the disease to appear suddenly in a poultry population and spread quickly.7 The introduction of NDV into bird populations in an area or country has occurred by various means including:
7,8,10,12,68 a) Movement (migration, importation, smuggling, or transit) and introduction of
persistently-infected or virus-shedding live birds: a) feral birds (ND outbreaks in
cormorants in the USA 24 and Canada 81,82,142 during the 1990’s); b) pet and exotic
birds (California 1971-1973 ND epizootic in commercial poultry associated with
NDV of psittacine origin)132; c) game birds (ND outbreak in a game chicken flock in
California during 1998)42; d) racing pigeons (feral pigeons played a role as “vectors”
in the spread of NDV from racing pigeons to domestic fowl in England during the
mid 1980s)93, and e) commercial poultry (movement of live birds during the ND
panzootic of the early 1970s)7; b) Movement of people and equipment: considered the most important method of
secondary spread of NDV during the 1971-1973 California epizootic;132 c) Movement of poultry products: presence of virus in bone marrow and other parts of
the carcass; 7 d) Airborne spread: in recent years the significance of this virus transfer method has
been the subject of discussion.12 It was considered very important during the 1970-
1972 ND epizootic in Great Britain,7 but of minimal importance during the California
1971-1973 outbreaks with a similar virus;132 21 e) Non-avian species: potentially any animal might be involved in mechanical transfer
of NDV;7 f) Contaminated poultry feed: ND outbreaks in chickens in Great Britain after receiving
food contaminated with the variant pigeon-NDV;19,93 g) Water: captive kestrels (Falco tinnunculus) receiving contaminated rain water
gathered from a source frequented by feral pigeons infected with NDV;93 h) Vaccines: contaminated poultry vaccines in Denmark during 1997.65
D. Pathology, immunohistochemistry (IHC), and in situ hybridization (ISH)
The severity of the ND lesions does not always correspond to the degree of clinical signs.68,141 The occurrence and distribution of the lesions depend on the same factors described for the occurrence of clinical signs 10,11,68 including the host, the virus strain, and stress factors.
Specific gross or microscopic lesions are not observed in birds infected with NDV.10
However, the combined presence of gross lesions such as tracheal hemorrhage/necrosis associated with neck edema, hemorrhage/necrosis of the proventriculus (mostly at the opening of the mucosal glands), and necrosis or ulceration, or both, of the gut-associated lymphoid tissue (GALT) were considered highly indicative of VVNDV during the 1971-
1973 VVND outbreaks in California.96 In the USA, the occurrence of such lesions after intracloacal inoculation in adult chickens (intracloacal pathogenicity test) has been used to detect VVNDV as well as a criteria to differentiate VVNDV from NVNDV isolates especially those from imported birds in quarantine facilities.113 Microscopic lesions are also more severe with the VVNDV pathotype and are generally characterized by 22 extensive depletion and necrosis of lymphoid organs and aggregates (mostly in GALT).
Hemorrhages in the GI tract as well as myocardial necrosis and inflammation are common findings. Gliosis, neuronal degeneration, and mononuclear inflammatory infiltrates are often observed in the brain.31,56 Birds inoculated intraconjunctivally with
VVNDV have severe edema and mixed inflammatory infiltrates in the eyelids.31 The presence of intracytoplasmic eosinophilic inclusion bodies is rarely reported in birds infected with NDV. 42,56,128
Typical gross lesions are not usually seen in birds infected with the NVNDV pathotype.10,31 Histologically, the most remarkable findings are confined to the central nervous system;31,141 and the cerebellum is more commonly involved.141 The lesions are characterized by mononuclear perivascular cuffing (mostly lymphocytes and plasma cells), neuronal degeneration/necrosis (mostly of the Purkinje neurons), endothelial hypertrophy, and gliosis.31,83,141 Pneumonitis,141 lymphoid depletion, and myocarditis might also occur in birds infected with NVNDV isolates.31
PPMV-1 isolates from pigeons are generally classified as mesogenic viruses.10,73
Gross lesions might be absent or non-specific.25 However, generalized congestion, hepato- and splenomegaly, pancreatic necrosis, ulcerations of the ventriculus mucous membrane, and enteritis are often observed in some outbreaks in pigeons.145 The microscopic lesions described in those outbreaks were: interstitial nephritis, renal tubular necrosis,25 and lymphoplasmacytic infiltration within several organs including kidney, liver, intestines, and pancreas.25,145 The most consistent lesions were found in the brain and were characterized by perivascular cuffing, neuronal necrosis and chromatolysis, and gliosis.25,145 Necrosis of the spleen and pancreas might occur.145 With two other 23 mesogenic NDV isolates (Roakin and Anhinga), splenic lymphoid hyperplasia as well as myocardial degeneration/inflammation were also described.31
Minimal lesions, if present, are described in birds infected with lentogenic NDV isolates, affecting mostly the respiratory tract.31,55,58 Some isolates in Australia caused respiratory disease in broilers (called “late respiratory syndrome”) with detectable gross lesions (reddening of the trachea) and chronic non-suppurative tracheitis histologically.61
Tracheas of birds infected with lentogenic strains examined by scanning electron microscopy (EM) revealed hypertrophy of goblet cells and small patches of the deciliated epithelium scattered mainly around the opening of mucous glands.80 With the Australian lentogenic reference V4 strain, slight ulcerative tracheobronchitis and progressive lymphoproliferative hyperplasia in the respiratory system, GI tract, bursa and spleen were observed.5 Live lentogenic NDV vaccines such as B1, La Sota, and V4 might cause mild respiratory disease after immunization by aerosol.139
According to the virulence, NDV might cause necrosis of a variety of cells.31,86,87
More recently, another mechanism of cell injury, apoptosis, has been observed after NDV infection 84-86,88 or vaccination.117 NDV-induced apoptosis has been demonstrated in lymphocytes,86,87 macrophages,85 heterophils,86 cardiomyocytes,84,88 and cells of the brain.88
The use of IHC and ISH plays an important role in the diagnosis of NDV.31,62,83 There are several studies of the distribution of NDV (with APMV-1 but not with PPMV-1) in tissues of infected birds employing IHC 30-32,61,83,94,110 and ISH.30-32 In a broad pathogenesis study with NDV isolates representing all major pathotypes, Brown et al 31 detected considerably more widespread viral nucleic acid (mRNA) by ISH in tissues of 24 birds infected with VVNDV than in birds infected with the other pathotypes. Other important findings and conclusions of this study were: a) the widespread staining of macrophages mostly in the spleen and GALT of VVNDV-infected birds; b) the consistent detection of viral mRNA in the myocardium and air sacs of birds infected with most
NDV isolates (representing all pathotypes); and c) the route of inoculation may influence initial sites of viral replication.31
E. Current situation in the USA and around the world
Biological and molecular characterization of recent NDV isolates from chickens and turkeys showed no evidence that the isolates were more virulent than the NDV vaccine strains utilized to control ND in the USA.76 With the exception of the turkeys in North
Dakota that became infected with a virulent virus transmitted from a cormorant die-off during 1992, there have been no reported NDV isolates of mesogenic or velogenic pathotype from chickens and turkeys in the USA since the late 1970’s.72,74
The most recent outbreaks around the world have been due to viruses with properties of high or moderate virulence.69,106,137,143 There were outbreaks of ND in Ireland
(1990)37,38 and in Australia (1998-2000)38,53,77 attributed to viruses that apparently evolved from low virulence strains as discussed above. The reported occurrences of a mutation of a low virulence strain to high virulence are rare considering the opportunities that do exist from the widespread use of the lentogenic vaccines, and the continued exposure of poultry to low virulence field isolates.12,74,76
A phylogenetic study showed that there are two major phylogenetic groups of NDV isolates. One group includes viruses common worldwide, including viruses from 25 psittacines and isolates considered exotic to the USA and isolates from the recent ND outbreaks around the world. A separate clade includes viruses isolated in the USA during the 1940’s and 1950’s (including the vaccine strains B1 and La Sota) and current isolates from poultry that are closely related to the vaccine strains. Viruses isolated from psittacines during the 1970’s, the cause of the California outbreaks, are typical of viruses that are exotic to the USA or are like the viruses circulating in other parts of the world.124
The fact that the same strict control measures imposed on poultry are not imposed to pigeons has greatly aided the continued spread of pigeon-NDV.12 Despite evidence that the available vaccines are able to protect pigeons,9,29,129 their use is still variable.9
PPMV-1 has been repeatedly isolated from racing, show, and feral pigeons in many countries around the world 1,12,60,65,99,127,137 and is considered a pool of virus that is not only a continued threat to commercial poultry but also to indigenous feral birds.12
F. International regulation of ND and regulatory needs
Newcastle disease is one of two reportable avian diseases belonging to the OIE List A diseases along with HPAI. The occurrence of ND in an OIE member country is of major importance in the international trade of animals and animal products.107 According to the
OIE International Animal Health Code - 2001 (Chapter 2.1.15 – Newcastle disease,108
“Veterinary Administrations of ND free countries may prohibit importation or transit through their territory, from countries considered infected with ND, of the following commodities: 1) domestic and wild birds; 2) day-old birds; 3) hatching eggs; 4) semen of domestic and wild birds; 5) fresh meat of domestic and wild birds; 6) meat products of domestic and wild birds which have not been processed to ensure the destruction of ND 26 virus; and 7) products of animal origin (from birds) intended for use in animal feeding or for agricultural or industrial use.”
In the USA, as well as in many other countries, if an NDV isolate is suspected to be virulent for chickens, it should be submitted to a reference laboratory for assessment and its propagation must be only in a biosafety level 3 (BSL-3) laboratory.11
There is an international trend to separate NDV isolates into two virulence groups, the low virulence viruses (asymptomatic enteric and lentogens) and the virulent viruses
(mesogens and velogens) based especially on the ICPI as a differential test.74 The ND definition in Council Directive 92/66/EEC,41 for which control measures should be imposed when birds are affected in European Union (EU) countries is: “an infection of poultry caused by an avian strain of the paramyxovirus 1 with an ICPI in day-old chicks greater than 0.7.” This definition includes all known highly virulent (velogenic) and moderately virulent (mesogenic) viruses.9
The international trend mentioned above started with the EU definition of ND 41 and was codified by OIE during 1999, when the ICPI result became part of the new definition of ND.105 ND is now defined by OIE as “an infection of birds caused by a virus of avian paramyxovirus serotype 1 (APMV-1) that meets one of the following criteria for virulence: a) The virus has an intracerebral pathogenicity index (ICPI) in day-old chicks (Gallus
gallus) of 0.7 or greater; or: b) Multiple basic amino acids have been demonstrated in the virus (either directly or by
deduction) at the C-terminus of the F2 protein and phenylalanine at residue 117, 27
which is the N-terminus of the F1 protein. The term “multiple basic amino acids”
refers to at least three arginine or lysine residues between residues 113 and 116.
Failure to demonstrate the characteristic pattern of amino acid residues as described
above would require characterization of the isolated virus by an ICPI test.
In this definition, amino acid residues are numbered from the N-terminus of the amino acid sequence deduced from the nucleotide sequence of the F0 gene, 113-116 corresponds to residues –4 to –1 from the cleavage site.”105
With the implementation of the new OIE definition for ND and the ICPI becoming the international standard for assessing NDV virulence, the presence of moderately virulent isolates such as most PPMV-1 isolates (ICPI values greater than 1.0) in the USA
10,73 would become reportable to OIE 12,127 if there is epidemiological association of that infection with poultry.
Currently, the U. S. Code of Federal regulations refers to the presence of “exotic
Newcastle disease” (END) as any velogenic ND. EDN is defined as “an acute, rapidly spreading, and usually fatal viral disease of birds and poultry.” 36 It is still a critical issue for poultry exporting countries like the USA that the OIE International Animal Health
Code has not been revised to provide criteria for applying the new ND definition for the purposes of international trade .74
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VIRULENCE OF PIGEON-ORIGIN NEWCASTLE DISEASE VIRUS ISOLATES
FOR DOMESTIC CHICKENS1
1 Kommers, G.D., D.J. King, B.S. Seal, and C.C. Brown. 2001. Avian Diseases 45:906- 921. Reprinted with permission of the publisher. 53
SUMMARY
The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin- labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4- wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-
1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro- hemorrhagic lesions in the gastrointestinal tract. 54
Key words: chickens, in situ hybridization, immunohistochemistry, sequence analysis, pigeon paramyxovirus-1, avian paramyxovirus-1, Newcastle disease, virulence
Abbreviations: AAF = amnioallantoic fluid; APMV-1 = avian paramyxovirus-1;
BHI = brain-heart infusion broth; dpi = days postinfection; HA = hemagglutination; HI = hemagglutination-inhibition; ICPI = intracerebral pathogenicity index; IHC = immunohistochemistry; ISH = in situ hybridization; IVPI = intravenous pathogenicity index; Mab = monoclonal antibody; MDT = mean death time; N = nucleoprotein; NDV =
Newcastle disease virus; NVSL = National Veterinary Services Laboratories; PBS = phosphate-buffered saline; PPMV-1 = pigeon paramyxovirus-1; RT-PCR = reverse transcription-polymerase chain reaction; SEPRL = Southeast Poultry Research
Laboratory; SPF = specific-pathogen-free; VVNDV = velogenic viscerotropic Newcastle disease virus.
Avian paramyxovirus-1 (APMV-1) belongs to the family Paramyxoviridae, genus
Rubulavirus, and is synonymous with Newcastle disease virus (NDV). It infects approximately 236 species of pet and free-living birds in addition to domestic avian species (chicken, turkey, goose, duck, and pigeon) (27). Most pigeon paramyxovirus-1
(PPMV-1) isolates differ from other APMV-1 isolates by having unique monoclonal antibody binding profiles (7,28,33). Those antigenic differences and the difficulty in classifying PPMV-1 isolates by the standard NDV pathotyping scheme are the basis for their identification as NDV variants (2). PPMV-1 first emerged in the Middle East during the late 1970s (26), then spread throughout Europe (7) and now is found worldwide 55
(1,35,48). During 1984, PPMV-1 spread from feral pigeon populations into domestic chickens in Great Britain and caused more than 23 outbreaks among commercial chickens. Feedstuffs stored at Liverpool docks became contaminated with feces and carcasses of feral pigeons infected with PPMV-1 and were considered the source of the virus in most of those outbreaks (6,8).
Viruses confirmed as PPMV-1 were first isolated in the United States during 1984
(39), and these isolates differed from those pigeon isolates recovered in 1975 (19). Since then, numerous isolates have been recovered from both domestic and feral pigeons from all parts of the United States (11,39). During early 1998, severe disease attributed to
PPMV-1 was diagnosed in pigeon lofts in Texas and Georgia (45). There was some concern that spread to domestic poultry might occur. PPMV-1 isolates may cause minimal morbidity when chickens are inoculated intracloacally or intranasally (39).
However, when they were passaged through chickens by intramuscular inoculation these viruses increased in virulence on the basis of pathotyping test results after passage (4,5).
Consequently, there is a concern that a PPMV-1 isolate may infect poultry and circulate subclinically until it evolves to a more virulent form capable of causing disease among chickens.
The aim of this study was to perform sequential passage of pigeon NDV isolates in chickens and compare the initial virus inoculum and virus reisolated after passage by the classic NDV pathotyping tests (mean death time [MDT], intracerebral pathogenicity index [ICPI], and intravenous pathogenicity index [IVPI]). In addition, sequence analysis of the fusion protein cleavage site was completed, and chickens were inoculated with six 56 different pigeon-origin NDV isolates to assess clinical disease and pathogenesis through
14 days postinoculation.
MATERIALS AND METHODS
Viruses. Six different pigeon-origin isolates of NDV were examined: 1) Pigeon
TX (pigeon/U.S.(TX)/17498/98) from Texas outbreak, February 1998; 2) Pigeon GA
(pigeon/U.S.(GA)/21402/98) from Georgia outbreak, May 1998; 3) 84-44407
(pigeon/U.S./44407/84) early Northeastern United States isolate, 1984 (39); 4) Pigeon
84 (pigeon/U.S.(NY)/84) early United States isolate (New York), 1984 (28); 5) P1307
(pigeon/U.S.(CA)/1307/75) from import quarantine lots of fruit and crown pigeons, 1975
(19); and 6) P5658 (pigeon/U.S.(CA)/5658/75) from racing pigeons, 1975 (19).
All isolates except Pigeon 84 were provided by the USDA Animal and Plant
Health Inspection Service, National Veterinary Services Laboratories (NVSL).
Nucleotide sequence of the following NDV strains (42,43,44) was utilized for comparison in the phylogenetic analysis: chicken/U.S./LaSota/46, chicken/U.S./B1/48, chicken/Australia/QV4/66, chicken/U.S./GB/48, chicken/Australia/AustVict/32, pigeon/U.S.(MD)/3981/84, chicken/U.S./CA1083/71, turkey/U.S. (ND)/43084/92, and anhinga/U.S. (FL)/44083/93.
Eggs and chickens. The source of embryonated chicken eggs and chickens was the Southeast Poultry Research Laboratory (SEPRL) specific-pathogen-free (SPF) white leghorn flock. Embryonated eggs were inoculated for virus amplification of chicken- passaged virus, isolation, titration and MDT test. Chickens were inoculated for passage of 57 the virus, ICPI, IVPI, and pathogenesis tests. Birds were housed in negative-pressure isolators under BSL-3 agriculture conditions at SEPRL and provided feed and water ad libitum.
Virus passage in chickens. The method for virus passage in chickens was completed according to procedures described by Alexander and Parsons (5) for PPMV-1 isolates in Great Britain. Six groups of six 2-wk-old SPF white leghorns were inoculated with one of the six different pigeon-origin NDV isolates, and one group of six birds served as noninoculated controls. The initial chicken inoculation (passage 1) was 0.1 ml of undiluted amnioallantoic fluid (AAF) for each stock virus delivered intramuscularly
(i.m.; breast muscle). At 2 days postinfection (DPI), three chickens from each group were euthanatized, and their spleens were removed aseptically. To prepare inoculum for passage 2, frozen spleens harvested in passage 1 were quickly thawed, crushed, and homogenized. A 20% (w/v) homogenate in phosphate-buffered saline (PBS) without antibiotics was prepared by blending for 120 sec at high speed in a Stomacher 80 homogenizer (Brinkmann Instruments Inc., Westbury, NY). The spleen homogenate was clarified by centrifugation for 15 min at 2500 rpm (18,750 X g total), and the supernatant fluids were saved and used as inoculum for the next passage (0.1 ml/bird of undiluted supernatant of the homogenate i.m.). In addition, these supernatant fluids were inoculated into 9-to-10-day-old embryonated SPF eggs to confirm infectivity of passage inoculum and to prepare a virus stock for further testing of passaged virus. This procedure was repeated three times to make additional chicken passage for all isolates except with
P1307 and P5658 viruses which were passaged only one time. For passages 2, 3, and 4, the number of birds for spleen harvest was increased to four to expand the homogenate 58 volume (the number of birds per isolator was increased to seven). Embryo-propagated virus after passage was diluted as required for the ICPI, IVPI, MDT, and pathogenesis study.
On the first and fourth passages, three chickens inoculated with Pigeon TX,
Pigeon GA, 84-44407, and Pigeon 84 isolates were euthanatized at 4 dpi for collection of tissues for histopathology, immunohistochemistry (IHC), and in situ hybridization (ISH).
Chickens inoculated with P1307 and P5658 isolates (passage 1 only) died or were euthanatized at 2 DPI. Tissues for histopathology, IHC, and ISH were collected at that time. The following tissues were collected and fixed by immersion in 10% neutral buffered formalin for 48-56 hr: spleen, thymus, bursa, eyelid, proventriculus, pancreas, small intestine, cecal tonsils, large intestine, caudal thoracic air sac, trachea, lung, heart, liver, kidney, Harderian gland, and brain. Tissues were routinely processed into paraffin, and 3 µm tissue sections were sectioned for hematoxylin and eosin staining, IHC, and
ISH.
Pathogenesis in chickens. Chickens inoculated intraconjunctivally with postpassage virus were observed for morbidity and mortality in two separate experiments.
The first experiment in 4-wk-old SPF white leghorns included necropsy of dead infected birds as well as euthanasia of selected birds for necropsy at 2, 5, and 10 DPI (necropsy results to be reported separately). Six groups of 10 4-wk-old SPF white leghorns were inoculated with infective AAF propagated from the first (P1307 and P5658), third
(Pigeon 84), or fourth (Pigeon TX, Pigeon GA, and 84-44407) chicken passage. Infective
AAF was diluted to produce a dose of approximately 105.0 50% embryo infective dose 59
(EID50) (0.1 ml/bird). One group of 10 4-wk-old birds served as noninoculated controls.
Birds were observed for the occurrence of clinical signs or mortality for 14 days. In the second experiment, only Pigeon TX and Pigeon GA isolates were inoculated in 2-wk-old
SPF white leghorns, and the observations were limited to morbidity and mortality. Two groups per isolate of six 2-wk-old birds were inoculated intraconjunctivally with embryo
5.1 7.1 propagated fourth chicken-passage virus. The doses used were 10 and 10 EID50 with
4.5 6.5 Pigeon TX and 10 and 10 EID50 with Pigeon GA isolate. Birds were observed for the occurrence of clinical signs or mortality for 14 days.
Virus isolation and titration. Immediately prior to euthanasia, oral and cloacal swabs were obtained from each bird and placed in a tube containing 1.5 ml of brain-heart infusion broth (BHI) with antibiotics (2000 units/ml penicillin G, 200 µg/ml gentamicin sulfate, and 4 µg/ml amphotericin B; Sigma Chemical Co., St. Louis, MO). Swab fluids were centrifuged at 1000 X g for 20 min, and undiluted supernatant was inoculated into
9-to-10-day-old SPF embryonated chicken eggs and incubated for 7 days. Virus infectivity titers of inoculum during passage and pathogenesis experiments were calculated from the results of inoculation of 9- or 10-day-old embryonated eggs with serial 10-fold dilution in BHI containing antibiotics (100 units penicillin G/ml and 50 µg gentamicin sulfate/ml). NDV-infected dead or surviving embryos were identified by hemagglutination (HA) activity in AAF harvested from chilled eggs. NDV was confirmed in HA-positive samples by hemagglutination-inhibition (HI) test with NDV- specific antiserum or monoclonal antibodies (Mabs) (28). 60
MAbs and antiserum. Three MAbs with different NDV specificities were used for isolate differentiation by the HI test. The MAbs were obtained from the NVSL and included B79, 15C4 (33), and 161/617 (14). B79 reacts with all APMV-1 including most
PPMV-1 (33); 15C4 reacts with all APMV-1 except PPMV-1 (33); and 161/617 reacts only with PPMV-1 isolates within the APMV-1 group (14). The polyclonal chicken NDV antiserum was prepared at SEPRL by immunization of chickens with inactivated NDV-
La Sota.
HA and HI tests. The HA and HI tests were conducted by conventional microtiter methods (12). Four HA units of each of the virus stocks were used as test antigen in completing the HI test of MAbs (12,28).
Pathotyping tests. For MDT, a series of 10-fold dilutions of the original inoculum for virus passage of all six viruses and egg-amplified virus isolated from passage 4 of four viruses (Pigeon TX, Pigeon GA, 84-44407, and Pigeon 84) was made in sterile BHI. Subsequently, 0.1 ml of each dilution was inoculated into the allantoic cavity of five 9-to-10-day-old embryonated SPF chicken eggs. Inoculated eggs were incubated at 37 C and candled twice daily. The time of death of each embryo was recorded. The
MDT was determined as the mean time in hours for the minimum lethal dose to kill the embryos (3). For ICPI, AAFs from the original inoculum for virus passage of all six viruses and egg-amplified virus isolated from passage 4 of four viruses (Pigeon TX,
Pigeon GA, 84-44407, and Pigeon 84) were filtered through a 0.45µm filter prewet with
BHI. The HA titer of all filtrates was equal to or greater than 16. The filtrate was diluted 61
1:10 in PBS without antibiotics and 0.05 ml/bird was inoculated intracerebrally in 24-to-
40-hr-old SPF white leghorns. ICPI was performed and scored in the standard manner
(3). For IVPI, inoculum preparation was similar to that for ICPI. The dosage was 0.1 ml/bird intravenously in 4-week old SPF White Leghorn hatchmates. The IVPI test was performed and scored in the standard manner (3). Confirmation of infection of survivors at the termination of ICPI and IVPI tests was accomplished by testing for seroconversion as determined by HI.
Viral RNA extraction, oligonucleotide primers and reverse transcription- polymerase chain reaction (RT-PCR). Isolates of NDV were replicated in embryonated eggs (3), and RNA was extracted (13) directly from AAF as described
(42,43). Oligonucleotide RT-PCR primers were designed to amplify regions of the fusion protein gene, including the fusion protein cleavage site and the matrix protein gene region encoding the nuclear localization signal of the matrix protein (42,43). A single tube RT-PCR for genomic NDV RNA was completed as described (34), with
SuperscriptTM (Life Technologies, Gaithersburg, MD) (32) and AmplitaqTM (PE
Biosystems, Foster City, CA) polymerase (40). Amplification products were separated by gel electrophoresis in 1.0% agarose with Tris-borate buffer, stained with ethidium bromide and photographed during ultraviolet transillumination (42,43).
Direct nucleotide sequencing of RT-PCR products and phylogenetic analysis.
Amplification products were purified with MicroconTM (Amicon, Belford, MA) spin filters and spectrophotometrically quantified. Additionally, amplification products were 62 cloned with the TA cloning systemTM (36) according to the methods described by the manufacturer (Invitrogen, San Diego, CA). Direct double-stranded nucleotide sequencing (41) was completed with Taq polymerase (Applied Biosystems, Inc., Foster
City, CA) with the oligonucleotide primers used for RT-PCR, fluorescent-labeled dideoxynucleotides, and an automated nucleic acid sequencer (46). Nucleotide sequence editing, analysis, prediction of amino acid sequences and alignments were conducted with IntelliGenetics GeneWorks 2.5TM software (IntelliGenetics, Mountain View, CA).
Phylogenetic trees presented were constructed by the phylogenetic analysis using parsimony (PAUP; 47) software with a heuristic search and 1000 bootstrap replicates.
Nucleotide sequences for portions of the fusion protein and matrix protein genes from the RT-PCR reactions were performed before passage and submitted to GenBank as a single-sense strand contiguous sequence for each NDV isolate. Accession numbers assigned to each new isolate are AY008317 through AY008330.
Immunohistochemistry. To detect the nucleoprotein (N) of NDV a peptide antigen was used to produce antisera (22) with the sequence TAYETADESETRRIC.
This sequence represents residues 181 to 194 of the N protein (21) with a C addition for coupling. The peptide was coupled to keyhole limpet hemocyanin (KLH) (31) and used to immunize rabbits by standard procedures (Sigma-GenosysTM, The Woodlands, TX).
The immunoglobulin G fraction was purified by affinity-column chromatography (10) with the coupled peptide.
For IHC, paraffin tissue sections were sectioned at 3 µm. After deparaffinization, sections were rehydrated, and antigen sites were exposed by microwaving (10 min at full 63 power) in Vector antigen unmasking solution (Vector Laboratories, Burlingame, CA).
Sections were blocked (Universal Blocking Reagent; Biogenex, San Ramon, CA) and then incubated overnight at 4 C (or for 2 hr at 37 C) with primary anti-peptide (anti-N protein) antibody diluted 1:8000. After a brief wash, sections were incubated with biotinylated goat anti-rabbit antibody and then with avidin-biotin alkaline phosphatase
(Vector Laboratories). Substrate development was with Vector Red (Vector
Laboratories). Sections were counterstained with hematoxylin and coverslipped for a permanent record.
In situ hybridization. A negative-sense, digoxigenin-labeled riboprobe representing the matrix gene of Fontana (CA1083) NDV strain was used for ISH (12).
The procedures used to make the probe and for hybridization were as previously described (12). Briefly, the matrix gene of the Fontana strain was cloned into pCRII transcription vectors (Invitrogen, Carlsbad, CA). Anti-sense digoxigenin-labeled riboprobes were generated with RNA polymerase in the presence of labeled nucleotides.
Dot blot was used to verify the incorporation of digoxigenin. For hybridization, deparaffinized sections were rehydrated, digested with 30 µg/ml Proteinase K for 15 min at 37 C, and hybridized overnight at 42 C with approximately 20 ng of probe in prehybridization solution. After stringent washes, probe binding was visualized by the addition of anti-digoxigenin alkaline phosphatase and the chromogen/substrate nitroblue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate. Tissues were counterstained with hematoxylin and coverslipped. 64
RESULTS
MAb binding profiles. Mab binding profiles are summarized in Table 3.1.. Four isolates (Pigeon TX, Pigeon GA, 84-44407, and Pigeon 84) were inhibited by the pigeon- specific MAb 161/617 and classified as PPMV-1, and two isolates (P1307 and P5658) were not inhibited by the pigeon-specific MAb and then were designated as APMV-1.
Results were unchanged by virus passage in chickens.
Virus passage in chickens - inoculum titration, clinical disease, and virus re- isolation. The virus titer of the inoculum for passage one was higher than the titer of the homogenate of passage 3 spleens used as inoculum for passage 4. The passage 1 inoculum titers were 7.5, 6.9, 7.7, and 6.1 log10, and the passage 4 inoculum titers were
2.5, 1.9, 3.9, and 3.5 log10 for Pigeon TX, Pigeon GA, 84-44407, and Pigeon 84, respectively. Birds from each group inoculated with PPMV-1 isolates were clinically depressed during passage 1. Only birds infected with 84-44407 isolate had slight depression at passage 2, and a few birds of each group were depressed at passages 3 and
4. Several birds exhibited other neurologic signs (head shake or twitch and paralysis) at passages 1, 3, and 4. These clinical signs were seen mostly at 3-4 DPI in one or two birds out of the remaining three birds. No mortality was observed in chickens inoculated with
PPMV-1 isolates during the 4-day observation of any of the passages.
Birds inoculated with APMV-1 isolates (P1307 and P5658) developed clinical illness characterized by severe depression beginning at 2 DPI and died or were euthanatized in a moribund state. Because of the severity of the disease and high mortality observed with these two isolates only passage 1 was completed. 65
Virus isolation was positive from sampled spleens (2 DPI) and swabs (4 DPI) during passages 1-4 of the PPMV-1 isolates and at 2-3 DPI during passage 1 of the
APMV-1 isolates. Control chickens remained clinically normal and were negative by virus isolation throughout the passages.
Pathogenesis in chickens – clinical disease. The clinical observations in the 2- or 4-wk-old birds inoculated intraconjunctivally with postpassage inoculum are summarized in Table 3.2.. Clinical signs were not seen in all of the chickens inoculated with the PPMV-1 isolates (Pigeon TX, Pigeon GA, 84-44407, and Pigeon 84), but the disease was more severe and included some mortality in younger birds. The first signs were evident on days 5 and 6 postinoculation in 2-wk-old birds and days 7 and 8 in 4 wk olds. Depression and some nervous signs, tremors, paralysis, and birds down on their hocks were observed in sick birds. Severe disease resulted from inoculation with the
APMV-1 isolates, P1307 and P5658. Signs of conjunctivitis and periocular edema were observed by day 3 postinoculation, which preceded severe depression and mortality at day 5 post-inoculation. Results of the necropsy and histopathology at days 2, 5, and 10 postinoculation of chickens inoculated at 4 wk of age will be reported separately.
MDTs, ICPIs, and IVPIs. MDTs, ICPIs, and IVPIs obtained before and after passage in chickens are summarized in Table 3.3.. MDT decreased after passage in all four PPMV-1 isolates. Although the MDT of Pigeon GA decreased, it was still typical of a lentogenic (>90 hr) isolate before and after passage. Pigeon TX and Pigeon 84 had
MDTs typical of lentogens before passage and mesogens (60-90 hr) after passage in 66 chickens. The MDT of 84-44407 was typical of a mesogenic isolate before and after passage. The APMV-1 isolates, P1307 and P5658, were tested only before passage. Both viruses had a MDT typical of velogenic (<60 hr) isolates.
The ICPIs of all four PPMV-1 isolates increased after passage in chickens. Pigeon
TX and Pigeon 84 had ICPIs before and after passage typical of mesogenic (1.0-1.5) viruses. Pigeon GA had an ICPI (0.86) falling between lentogenic (0.2-05) and mesogenic (1.0-1.5) before passage and as a mesogenic isolate after passage. The isolate
84-44407 had an ICPI compatible with a mesogenic virus before passage and as a velogenic (1.5-2.0) isolate after passage in chickens. Both APMV-1 isolates, P1307 and
P5658, had ICPIs typical of velogenic viruses before passage.
The IVPIs of the PPMV-1 isolates also increased after passage. Pigeon TX,
Pigeon GA, and Pigeon 84 had IVPI values higher than classical mesogenic (0.0-0.5) and lower than velogenic (2.0-3.0) viruses before and after passage in chickens (Table 3.3.).
The IVPI of 84-44407 was similar to mesogenic viruses before passage and velogenic
(2.0-3.0) viruses after four passages in chickens. P1307 and P5658 had IVPI values of the velogenic pathotype before passage and were not tested further.
Gross pathology. With PPMV-1 isolates, the spleen was enlarged and congested in most of the birds from passage 1 and 4. With APMV-1 isolates, cecal hemorrhages were observed in two birds infected with P1307 and in one bird infected with P5658 at 2
DPI. Control birds did not have abnormal gross findings. 67
Histopathology, immunohistochemistry, and ISH. All four PPMV-1 isolates produced similar microscopic lesions that were more prominent during passage 1.
Lymphoid organs had mild to moderate abnormalities, with some lymphoid depletion, necrosis, and increased evidence of apoptosis. Lesions in lymphoid organs were more severe in birds inoculated with the isolate 84-44407. The most consistent positive lymphoid tissues by IHC and ISH were spleen and lymphoid aggregates in the gastrointestinal tract. Bursa and thymus had some positive cells in birds inoculated with the isolates 84-44407 and Pigeon 84. Within the myocardium, all isolates caused minimal to moderate disruption or necrosis of myofibers and mononuclear inflammatory infiltrates associated with the disruption (Fig. 3.1.A). With an anti-sense digoxigenin-labeled riboprobe, viral mRNA was detected in the heart, mostly in myofibers adjacent to areas of degeneration and inflammatory cellular infiltration (Fig. 3.1.B).
Perivascular cuffs, endothelial hyperplasia, and glial nodules were observed in the cerebrum and brain stem. In the cerebellum, there was necrosis or absence of Purkinje cells, and multifocal mononuclear inflammatory infiltrates, gliosis, and vacuolation were noted in the molecular layer (Fig. 3.2.A.). Viral mRNA was also detected by ISH as clusters of positively stained neurons within the cerebrum (Fig. 3.2.B.), brain stem, and cerebellum. For all four PPMV-1 isolates, staining by IHC and by ISH was more extensive in passage 1 than in passage 4.
Histopathologic changes in the birds inoculated with the APMV-1 isolates (P1307 and P5658) were severe and extensive. The spleen had severe multifocal to coalescent necrosis with fibrin replacement. Severe lymphoid depletion and multifocal necrosis were also observed in the bursa (Fig. 3.3.A.) and thymus. In the eyelid, there was epithelial 68 necrosis and necrosis of the lymphoid aggregates. Necrosis of the glandular epithelium was observed in the proventriculus. The cecal tonsils had necrosis and multifocal hemorrhages. Very strong positive stain was detected by IHC and ISH in all the affected lymphoid aggregates and organs (Fig. 3.3.B.). Individual cell necrosis was observed in the pancreas. Myofiber disruption or necrosis and mononuclear infiltrates were present in the myocardium. Several birds had infiltrates of plasma cells and hemorrhages in the air sacs. Brain lesions were present with both P1307 and P5658 isolates but were more severe in the former. They were characterized by multifocal necrosis in the granular layer, mild gliosis, and vacuolation in the molecular layer and white matter. In the brain, the most striking positive signal was detected by IHC and ISH in the granular layer of the cerebellum (Fig. 3.4.A.), in the Purkinje cells (Fig. 3.4.B.), and in neurons of the cerebrum. Although lesions were not observed in the trachea, lungs, and kidneys, viral N protein was detected by IHC in those organs.
No histopathologic abnormalities were detected in control chickens, and none of the control tissues had any positive staining with the IHC and ISH techniques.
Nucleotide and predicted amino acid sequence analysis. After alignment, contiguous nucleotide sequence information from the fusion protein and matrix protein gene amplification products was used to determine phylogenetic relationships among the
NDV isolates examined (Fig. 3.5.A.). All the pigeon NDV isolates were related to viruses with chicken/Australia/AV/32 as possible progenitor-type. This included recent
NDV isolates highly virulent for chickens previously considered exotic to North
America, such as chicken/U.S./CA1083(Fontana)/72 and cormorant/U.S./40068/92. 69
Fusion protein cleavage site sequences are presented in Fig. 3.5.B. Lentogenic vaccine isolates chicken/U.S./B1/48 and chicken/Australia/QV4/66 have the
109SGGGR[K]QGRLIG119 sequence at the fusion protein cleavage site, whereas the mesogenic and velogenic viruses have the sequence 109SGGRRQK[R]RFV[I]G119 containing the diagnostic pair of dibasic amino acids (RRQK[R]R) associated with the primary molecular determinant of virulence. Pigeon isolates P5658, P1307, and 84-44407 from the United States share a V for I substitution at position 118 that is present in the sequence from the turkey/U.S./43804/92 and the cormorant/U.S./40068/92 viruses but not in chicken/U.S/CA1083(Fontana)/72 virus fusion protein cleavage site sequence. The turkey/U.S./43804/92, cormorant/U.S./40068/92, and anhinga/U.S./44083/93 viruses also share an R for G substitution at position 110 with two of these pigeon isolates. Pigeon isolates 3981/84 (not part of this study), Pigeon 84, Pigeon GA, and Pigeon TX from the
United States share an S for T substitution at position 107 with chicken/U.S/CA1083(Fontana)/72.
DISCUSSION
The pathotyping of NDV isolates from pigeons by the classic velogenic, mesogenic, and lentogenic criteria has been more difficult than with most other NDV isolates. For example, many pigeon isolates have a MDT typical of lentogenic strains and an ICPI value of mesogenic strains even though the isolate caused severe disease and mortality in pigeons (2,3). Isolates from other bird species may not show their potential virulence for chickens in conventional pathogenicity tests unless the viruses were passaged several times in chickens (2,3). This makes the assessment of risk for poultry of 70 isolates from other species more difficult. In a previous study, passage of Pigeon 84 and
84-44407 in chickens inoculated by an eyedrop and intranasal route produced no virulence enhancement of either virus (28). In the present study those two isolates plus two recent isolates, Pigeon GA and Pigeon TX, and two isolates from pigeons in 1975,
P1307 and P5658 (19), were characterized before and after serial chicken passage by i.m. inoculation. The procedure of passage by i.m. inoculation increased the IVPI of several pigeon isolates in Great Britain to values of greater than 2.0, values comparable to isolates recovered from chickens during the outbreak in Great Britain in the mid-1980s
(5,8).
The HI assay with MAbs separated the six isolates of the present study into two groups. Pigeon GA, Pigeon TX, Pigeon 84, and 84-44407 were inhibited by the pigeon- specific MAb and identified as PPMV-1 isolates. P1307 and P5658 were not inhibited by the antibody and were called APMV-1 isolates. The pigeon-specific MAb 161/617 recognizes all but a few of the known PPMV-1 isolates (14,28). The identification of two antigenic groups among these pigeon isolates demonstrates that not all NDV isolates from pigeons are typical of the variant identified as PPMV-1.
The more severe clinical signs in birds inoculated with all four PPMV-1 isolates during passage 1 was probably caused by the difference in the inoculum titer during the passages. The inoculum titer for the first passage was equal to or greater than 2.6 log10 higher than the final passage inoculum titer, as might be expected with the sequential passage approach. Moreover, the termination of each passage at day 4 postinoculation was probably too early for clinical sign development, particularly when a lower dosage was given. The signs observed included only depression and some nervous signs. No 71 mortality occurred. In contrast, the APMV-1 isolates were highly virulent in the first passage. Within 2 days postinoculation, those isolates produced severe depression progressing to a moribund state and mortality or euthanasia. Because the APMV-1 isolates were initially of high virulence, no further passages were completed.
Chicken passage of the PPMV-1 isolates resulted in an increase in the ICPI and
IVPI values and a decrease in the MDT. The results are evidence of increased virulence in all the standard pathotyping tests. Although ICPI and IVPI values of the 84-44407 isolate increased to levels typical of velogenic strains, the MDT remained at a mesogenic value. Most values for the other PPMV-1 isolates remained in the range typical of the mesogenic pathotype. The results are similar to those in previous reports of increased virulence in pathotyping tests in which some but not all increases were to velogenic values (5).
Although these pathotyping indices did increase, in our study there was no clinico-pathologic evidence of overt pathogenicity increase of the PPMV-1 isolates for the inoculated chickens, either during the passages or in chickens inoculated by eyedrop with egg-amplified virus after passage. The inoculation route may have had some effect on onset of disease. Clinical signs appeared before day 4 in i.m. inoculated 2 wk olds but were not evident until day 5 or 6 in 2 wk olds and days 7 or 8 in 4 wk olds inoculated by eyedrop. Signs in birds inoculated with 84-4407, the virus with velogenic ICPI and IVPI indices, were no more severe than signs in birds inoculated with the other PPMV-1 isolates. More severe disease including mortality was evident in 2 wk olds inoculated by eyedrop with Pigeon TX than was observed when the same viruses were inoculated in 4 wk olds. Pigeon GA also produced more disease without mortality in 2 wk olds. No 72 apparent differences were noted between the two virus doses administered by eyedrop to
2 wk olds. Several of the neurologic signs described in pigeons naturally infected with
PPMV-1 isolates (9,11,39) were observed in the chickens in the present study. However, other clinical signs seen in infected pigeons such as polydipsia, watery green diarrhea, and mortality, except in the experiment in 2 wk olds (9,11,39), were not observed.
The APMV-1 isolates were highly virulent as indicated by the pathotyping results.
Clinical disease in chickens inoculated by eyedrop with the APMV-1 isolates was typical of viscerotropic velogenic Newcastle disease and all birds were dead or euthanatized by day 5.
All four PPMV-1 isolates caused microscopic lesions in tissues of the chickens inoculated for virus passage. Damage was more severe in the heart and brain, and these sites of viral tropism were confirmed by IHC along with ISH. Even in chickens that were not noticeably sick, virus was present in the heart and brain, causing a concern for possible low performance in the survivors because of residual myocardial or neural damage. There are descriptions of recovery from paralysis in pigeons experimentally infected with PPMV-1, but primarily among less affected birds (39). The brain lesions observed in this study were similar to those previously reported for pigeons (11,39).
Importantly, the lesions observed in the heart have not been described for pigeons naturally or experimentally infected (11,39).
The behavior of viruses P1307 and P5658 isolated from pigeons when experimentally inoculated into pigeons (19) and chickens was characteristic of velogenic viscerotropic NDV (VVNDV). Moreover, the IHC and ISH patterns in the present study were identical to other VVNDV strains previously examined (12). 73
The sequence analysis of the fusion protein cleavage site is a principal parameter in the characterization of NDV (APMV-1 or PPMV-1) isolates. The structure of the cleavage site is recognized as a correlate of virulence because it is extremely important for determining whether the virus is activated in a wide variety of tissues or in particular tissue types (23,24,37). Virulent viruses have multiple basic amino acids, arginine (R) or lysine (K), between the positions 112 and 116 (23,24) and a phenylalanine (F) at the position 117 (15). This is considered the primary molecular determinant of virulence
(23,24,37).
Classification of well-known NDV strains and more recent isolates on the basis of the fusion (15,16,18,30,42,43) and matrix (29,30,42,43,44) protein sequences has provided valuable phylogenetic information. Two major phylogenetic branches of NDV isolates have been identified previously by nucleotide sequence analysis of the fusion protein and matrix protein genes (42). Velogenic viruses obtained from exotic and other avian species since 1986 were found to be highly virulent as evident from the primarily viscerotropic form of disease and high ICPI values in inoculated chickens.
Phylogenetically, these viruses group in a clade with chicken/U.K./Herts/33 or chicken/Australia/AV/32 as the earliest reported isolate and include viruses isolated from various avian species (42,43). Several amino acid sequence differences surrounding the fusion protein cleavage site and matrix protein nuclear localization signal were detected that correlate with the phylogenetic data.
Viruses isolated from pigeons that were examined during our study all had a fusion protein cleavage site sequences that would place them in the virulent pathotype
(RRQKRF). Consequently, the fusion protein cleavage site sequence from pigeon 74 viruses reported herein are different from the sequence of viruses causing disease among poultry in the United Kingdom reported previously that had the sequence GRQKRF (17).
None of the viruses isolated from pigeons in the United States had the fusion protein cleavage site RRKKRF recently reported for variant PPMV-1 isolates in Germany (38).
The valine (V) for isoleucine (I) substitution found at position 118 just outside the fusion cleavage activation of PPMV-1 84-44407 was not observed in the PPMV-1 sequences reported by Collins et al. (17) or Oberdorfer and Werner (38).
Identification of several shared changes within the fusion protein and matrix proteins among virulent NDV isolates is consistent with the quasispecies nature of RNA viruses (25). These differences have occurred among virus isolates of different virulence types from a variety of birds with different geographic origins (17,38,42,43). This further indicates that multiple lineages of virulent NDV are circulating among domestic, pet and wild birds. This is particularly important because some exotic species may harbor velogenic NDV for extended periods of time (20). Consequently, highly virulent NDV isolates continue to circulate among birds other than chickens and threaten commercial poultry worldwide.
From this study, we determined that not all of the six pigeon-origin NDV isolates examined were the variant PPMV-1. Two of the viruses were APMV-1 of velogenic viscerotropic pathotype. The pathotyping test results with the four PPMV-1 isolates before passage in chickens would classify them as mesogenic viruses. Mesogenic indices were still obtained after four sequential passages of these viruses in chickens. An exception was the isolate (84-44407) that reached velogenic parameters of ICPI and IVPI
(but not of MDT). Additionally, it is important to note that the fusion protein cleavage 75 site amino acid sequence of all six studied pigeon-origin (PPMV-1 and APMV-1) isolates were compatible with virulent NDVs.
The fact that clinical disease was evident in intraconjunctivally inoculated chickens and the similarity of the viruses by nucleotide sequence analysis to more virulent viruses are evidences that the pigeon viruses are a potential hazard to chickens.
Every effort should be made to prevent infections of poultry with pigeon NDV.
ACKNOWLEDGMENTS
GDK is funded on a scholarship from Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Brazil. U.S. Poultry and Egg Association, grant 406 and USDA-ARS-CRIS project 6612-32000-021-00D funded the research. We acknowledge the technical support of Joyce Bennett, Phillip Curry, Melissa Scott, and
James Stanton.
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pp.184-196. 1988. 84
Table 3.1. Results of HI test of NDV isolates against NDV-specific monoclonal antibodies.
Monoclonal antibodyA
Isolate B79 15C4 161/617
Pigeon TX + - + Pigeon GA + - + 84-44407 + - + Pigeon 84 + - + P1307 + + - P5658 + + -
A + = antibody-inhibited HA; - = no HA inhibition. B79 reacts with all APMV-1 including most PPMV-1 (reference 33); 15C4 reacts with all APMV-1 except PPMV-1
(reference 33); 161/617 reacts only with PPMV-1 isolates within the APMV-1 group
(reference 14). 85
Table 3.2. Clinical disease and occurrence of mortality in 2- and 4-wk-old white leghorn chickens inoculated intraconjunctivally with chicken-passaged pigeon isolates. Isolate Clinical Clinical No. sick/ No. dead/ (age at signs signs total A total A inoculation) first evident Pigeon TX (4 wk) Depression: mild to severe 7 dpi 2/6 0/6
Pigeon TX (2 wk) Dose 10 7.1 Depression leading to paralysis 6 dpi 3/6 1/6 Dose 10 5.1 Depression leading to paralysis 5 dpi 4/6 2/6
Pigeon GA (4 wk) Down on hocks, tremors 7 dpi 1/6 0/6
Pigeon GA (2 wk) Dose 10 6.5 Depression leading to paralysis 6 dpi 3/6 0/6 Dose 10 4.5 Depression 6 dpi 3/6 0/6
84-44407 (4 wk) Depression 7 dpi 1/6 0/6
Pigeon 84 (4 wk) Depression, one down on hocks 8 dpi 2/6 0/6 B P1307 (4 wk) Conjunctivitis, periocular edema, 3 dpi 8/8 8/8 mortality or euthanasia by day 5 C P5658 (4 wk) Conjunctivitis, periocular edema, 3 dpi 8/8 8/8 mortality or euthanasia by day 5
A Total = number of birds per group of 2 wk olds and in 4 wk olds. It is the number of birds remaining in group when first clinical signs were observed. Of the birds inoculated at 4 wk old, sampling at 2 DPI had reduced the P1307 and P5658 groups from 10 chickens to 8. Sampling at 2 and 5 DPI had reduced the Pigeon TX, Pigeon GA, 84- 44407, and Pigeon 84 groups from 10 chickens to 6. B Three birds died at 5 DPI and five were euthanatized in extremis. C One bird died at 5 DPI and seven were euthanatized in extremis. 86
Table 3.3. Mean death time (MDT), intracerebral pathogenicity index (ICPI), and intravenous pathogenicity index (IVPI) results before and after virus passage in chickens.A
MDT ICPI IVPI (hr; pre-/ post- (pre-/ post- (pre-/ post- Isolate passage) passage) passage)
Pigeon TX 96 / 73 1.11 / 1.44 0.79 / 1.81 Pigeon GA 129 / 100 0.86 / 1.26 0.90 / 0.97 84-44407 81 / 75 1.39 / 1.61 0.56 / 2.25 Pigeon 84 108 / 84 1.33 / 1.39 0.51 / 0.70 P1307 48 / NDB 1.81 / ND 2.57 / ND P5658 58 / ND 1.76 / ND 2.32/ ND A Range of indices: velogenic (MDT = <60 hr; ICPI = 1.5 - 2.0; IVPI = 2.0 - 3.0), mesogenic (MDT = 60-90 hr; ICPI = 1.0 -1.5; IVPI = 0.0 - 0.5), lentogenic (MDT = >90 hr; ICPI = 0.2 - 0.5; IVPI = 0.0) (reference 3).
B ND = not done. 87
Fig. 3.1. Myocardial lesions induced by pigeon paramyxovirus-1 (PPMV-1) isolates at 4 days postinfection. (A) Heart, chicken infected with Pigeon GA isolate. Myofiber necrosis and multifocal infiltrates of lymphocytes, plasma cells, and macrophages are observed. Hematoxylin and eosin stain. Bar = 60 µm. (B) Heart, chicken infected with
Pigeon TX isolate. Positive myofibers for viral mRNA and mononuclear inflammatory infiltrate were detected in the myocardium. In situ hybridization. Mayer hematoxylin counterstain. Bar = 60 µm.
Fig. 3.2. Brain lesions in chickens induced by pigeon paramyxovirus-1 (PPMV-1) isolates at 4 days postinfection. (A) Cerebellum, chicken infected with Pigeon TX isolate.
Vacuolation, mononuclear inflammatory infiltrates, and gliosis are seen in the molecular layer. Purkinje cells are absent in the affected area. Hematoxylin and eosin stain. Bar =
120 µm. (B) Cerebrum, chicken infected with Pigeon 84 isolate. A cluster of positive neurons (dark staining) for viral mRNA is observed. In situ hybridization. Mayer hematoxylin counterstain. Bar = 40 µm.
Fig. 3.3. Bursal lesions in chickens induced by P1307 isolate at 2 days postinfection. (A)
Lymphoid depletion and necrosis of the lymphoid follicles are observed in this section.
Hematoxylin and eosin stain. Bar = 100 µm. (B) Abundant viral mRNA is detected in the cells of an affected lymphoid follicle. In situ hybridization. Mayer hematoxylin counterstain. Bar = 100 µm. 88
Fig. 3.4. Detection of viral nucleoprotein (N) or mRNA in the cerebellum of chickens inoculated with P1307 isolate at 2 days postinfection. (A) Clusters of neurons of the granular layer and Purkinje cells are positive for viral nucleoprotein.
Immunohistochemistry. Mayer hematoxylin counterstain. Bar = 150 µm. (B) Purkinje cells and cell processes are strongly positive for viral mRNA. In situ hybridization.
Mayer hematoxylin counterstain. Bar = 120 µm 89 90
Fig. 3.5. Phylogenetic relationships among Newcastle disease virus isolates reported and predicted amino acid sequence alignment of the fusion protein cleavage site sequences.
Isolates utilized in this study were pigeon/U.S.(TX)/17498/98 (Pigeon TX), pigeon/U.S.(GA)/21402/98 (Pigeon GA), pigeon/U.S./44407/84 (84-44407), pigeon/U.S(NY)/84 (Pigeon 84), pigeon/U.S.(CA)/1307/75 (P1307), and pigeon/U.S.(CA)/5658/75 (P5658). Nucleotide sequence of the following NDV strains
(42,43,44) was utilized for comparison in the phylogenetic analysis: chicken/U.S./LaSota/46, chicken/U.S./B1/48, chicken/Australia/QV4/66, chicken/U.S./GB/48, chicken/Australia/AustVict/32, pigeon/U.S.(MD)/3981/84, chicken/U.S./CA1083/71, turkey/U.S. (ND)/43084/92, and anhinga/U.S. (FL)/44083/93.
(A) An unrooted phylogram was generated by parsimony analysis after alignment of fusion and matrix protein gene contiguous nucleotide coding sequences. (B) Alignment of predicted amino acid sequences surrounding the fusion protein cleavage site. The fusion protein cleavage site sequence from position 112 to 116 is underlined. Virus identification was reduced to name or accession number and year as shown in Fig. 3.5.B. 91 CHAPTER 4
PATHOGENESIS OF SIX PIGEON-ORIGIN ISOLATES OF NEWCASTLE
DISEASE VIRUS FOR DOMESTIC CHICKENS1
1 Kommers, G.D., King, D.J., Seal, B.S., Carmichael, K.P. and Brown C. C. Veterinary Pathology 39:352-361, 2002 (in press). Reprinted with permission of the publisher. 93
ABSTRACT
The pathogenesis of six pigeon-origin isolates of Newcastle disease virus (NDV) was investigated in chickens. Four isolates were previously defined as the variant pigeon paramyxovirus-1 (PPMV-1), and two isolates were classified as avian paramyxovirus-1
(APMV-1). Birds inoculated with PPMV-1 isolates were euthanatized, and tissue samples were collected at 2, 5, and 10 days postinoculation (DPI). Birds inoculated with APMV-1 isolates died or were euthanatized, and tissue samples were collected at 2, 4, and 5 DPI.
Tissues were examined by histopathology, immunohistochemistry (IHC) for presence of
NDV nucleoprotein, and in situ hybridization (ISH) for presence of viral mRNA to the matrix gene. Spleen sections were stained by the terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) assay and by IHC using an anti-active caspase-3 antibody (IHC-Casp) to detect apoptotic cells. Brain sections of PPMV-1- infected birds were examined by IHC to detect T and B lymphocytes and glial fibrillary acidic protein (GFAP). Histologically, birds inoculated with PPMV-1 isolates had marked lesions in the heart and brain. Presence of viral nucleoprotein and viral mRNA in the affected tissues was confirmed by IHC and by ISH, respectively. Numerous reactive astrocytes were observed in brain sections stained for GFAP. Among all the isolates, the
IHC-Casp demonstrated that apoptosis was very prominent in the ellipsoid-associated cells of the spleen at 2 DPI. Results of the TUNEL assay indicated that apoptotic cells were prominent at 5 DPI and were more randomly distributed. The clinical signs and gross and histopathologic changes observed in the APMV-1-infected birds were characteristic of an extensive infection with highly virulent NDV evident by IHC. 94
Key words: Apoptosis; avian paramyxovirus 1; chickens; immunohistochemistry; in situ hybridization; Newcastle disease; pathogenesis; pigeon paramyxovirus 1; veterinary virology.
Newcastle disease (ND) is a globally distributed avian disease that can cause severe economic losses in commercial poultry. According to the Office International des
Epizooties, ND belongs to the reportable List A diseases (International Animal Health
Code, http://www.oie.int). Newcastle disease virus (NDV) is synonymous with avian paramyxovirus-1 (APMV-1), a nonsegmented, single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae, subfamily Paramyxovirinae, genus
Rubulavirus.3 Recent studies based on phylogenetic analysis have suggested that NDV should be assigned to a new genus within the subfamily Paramyxovirinae.15, 42
Pigeon paramyxovirus 1 (PPMV-1), a variant of NDV, is characterized by unique monoclonal antibody (Mab)-binding profiles.7, 20, 31 After its emergence in the Middle
East (late 1970s),32 pigeon NDV spread worldwide, 2, 32, 45 and dissemination from pigeon populations into commercial poultry has been documented in Great Britain 6 and
44 9, 20, 40 Austria. Newcastle disease virus has been recovered from pigeons in the USA with further characterization of the isolates as APMV-1 and PPMV-1 using Mab.20, 40 To date, there have been no reports of PPMV-1 spread with overt clinical disease among commercial poultry in the USA. However, because of the frequent isolation of PPMV-1 from pigeons across the USA, there is a need to assess the risk potential of these viruses causing disease in chickens. Previously, investigators demonstrated virulence increase based on NDV pathotyping tests after PPMV-1 isolates were serially passaged through 95 chickens, particularly using the intramuscular route of infection. 5, 6, 22, 23 Another concern regarding PPMV-1 is its ability to spread from infected pigeons to nearby chickens.4 In one study, the contact infection rate of two different PPMV-1 strains in 4-week-old specific-pathogen-free (SPF) chickens reached 100%.22
Further characterization of most PPMV-1 isolates demonstrated the presence of multiple basic amino acids at the fusion protein cleavage site and an intracerebral pathogenicity index equal to or greater than 0.7.12 Infections of birds with APMV-1 isolates typical of most PPMV-1 are now defined as Newcastle disease,38 a reportable disease that may result in the initiation of international trade restrictions depending on the species involved or the epidemiological association of birds with ND, or both. There is a concern that these PPMV-1 strains, if allowed to circulate among chickens, could evolve a greater disease-causing capability. The potential for such an occurrence has already been demonstrated for NDV. The 1998-2000 NDV outbreaks reported in Australia, 17, 21,
46 with clinical disease and mortality observed among commercial chickens, were caused by a virulent APMV-1 strain that evolved from a low virulence strain that was circulating among chickens apparently for a long time. Consequently, it is important to determine the real threat for domestic chickens represented by PPMV-1 isolates, which are usually characterized as NDV of mild to moderate virulence.
In this study, six pigeon-origin NDV isolates that were previously passaged in 2- week-old chickens 23 were inoculated in 4-week-old White Leghorns to assess clinical disease and pathogenesis through 14 days postinoculation. Infected birds were examined for gross and microscopic lesions, and tissue samples were evaluated using 96 immunohistochemistry (IHC), in situ hybridization (ISH), and two different assays for apoptosis.
MATERIALS AND METHODS
Eggs and chickens
The source of embryonated chicken eggs and chickens was the Southeast Poultry
Research Laboratory (SEPRL, ARS, USDA) SPF White Leghorn flock. Embryonated eggs were utilized for virus amplification of chicken-passaged virus. Chickens were inoculated for the pathogenesis study and housed in negative pressure isolators under
BSL-3 agriculture conditions at SEPRL and provided food and water ad libitum.11, 23
Viruses
Six pigeon-origin isolates of NDV were previously characterized by NDV Mab binding profiles using the hemagglutination-inhibition (HI) test. 20, 23 Four of these isolates were characterized as PPMV-1: Pigeon TX, from Texas outbreak, 1998; Pigeon
GA, from Georgia outbreak, 1998; 84-44407, early US PPMV-1 isolate, Northeastern,
1984; 40 and Pigeon 84, early US PPMV-1 isolate, New York, 1984. 20 The other two isolates were classified as APMV-1: P1307, from imported quarantine lots of fruit and crown pigeons, 1975 16 and P5658, from racing pigeons, 1975. 16 All isolates were provided by the USDA APHIS National Veterinary Services Laboratories with exception of Pigeon 84. 20 97
Pathogenesis study
The viruses utilized in this experiment were passaged in chickens as previously described.23 Six groups of six 2-week-old SPF White Leghorns were inoculated intramuscularly with six pigeon-origin NDV isolates. At 2 DPI, three or four chickens from each group were euthanatized and their spleens were removed aseptically and homogenized. The homogenate was inoculated intramuscularly into another group of 2- week-old SPF White Leghorns. This procedure was repeated for a total of four passages with the PPMV-1 isolates. Only one passage was performed with the two APMV-1 isolates because of the severity of the disease and high mortality observed during the first passage.
Infective amnioallantoic fluid from the first (P1307 and P5658), third (Pigeon 84, chosen because of higher titers than fourth passage virus) or fourth (Pigeon Tx, Pigeon
Ga, and 84-44407) chicken passages was prepared for inoculation into six groups of 10 4- week-old SPF White Leghorns. After dilution in brain-heart infusion broth,
5.0 approximately 10 EID50 was inoculated intraconjunctivally (0.1 ml/bird). One group of
10 4-week-old birds served as noninfected controls.
The disease was followed serially by examining tissues from birds infected with the PPMV-1 isolates that were euthanatized (2/day) at 2, 5, and 10 DPI.11, 23 The remaining four birds were observed clinically through 14 DPI, tested for the presence of
NDV antibodies using the HI,3 and euthanatized. Because of the severity of the clinical signs observed in birds inoculated with the APMV-1 isolates (P1307 and P5658), these birds were examined at 2, 4, and 5 DPI. Necropsies were performed immediately postmortem, and the following tissues were collected and fixed by immersion in 10% 98 neutral buffered formalin for approximately 48 hours: spleen, thymus, bursa, lower eyelid
(including conjunctiva and skin), Harderian gland, proventriculus, pancreas, small intestine, cecal tonsils, large intestine, caudal thoracic air sac, trachea, lung, heart, liver, kidney, and brain. All sampled tissues were routinely processed into paraffin, and 3 µm sections were cut for hematoxylin and eosin (HE) staining. Selected sections were processed for IHC,23 ISH,11 and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.37
Immunohistochemistry
The sampled tissues of all the infected birds were examined by IHC using the following generic protocol to detect viral nucleoprotein (N) and apoptotic cells. After deparaffinization, tissue sections were treated with 3% hydrogen peroxide and then subjected to antigen retrieval by microwaving for 10 minutes at full power in Vector antigen unmasking solution (Vector Laboratories, Burlingame, CA) followed by blocking
(only for IHC-N) with universal blocking reagent (Biogenex, San Ramon, CA) as recommended by the manufacturer. Tissues were incubated with the primary antibody overnight at 4 C or for 2 hr at 37 C. After washing, sections were incubated with biotinylated antibody against the species in which the primary antibody was made and then with either avidin-biotin alkaline phosphatase or elite-PO (Vector Laboratories).
Substrate was Vector Red or diaminobenzidine (DAB) (Vector Laboratories). Sections were counterstained lightly with hematoxylin and coverslipped for a permanent record.
To detect NDV nucleoprotein (IHC-N), the primary antibody was an anti-peptide antibody 23 made in rabbit and used at 1:8,000 dilution. Sections were incubated with 99 avidin-biotin alkaline phosphatase, and the substrate was Vector Red. Sections of the spleen were examined by IHC using a rabbit polyclonal anti-active Caspase-3 (IHC-Casp) antibody (Promega, Madison, WI) at 1:350 dilution. The enzyme was elite-PO and the substrate was DAB.
Selected sections of the brain of PPMV-1-infected birds were examined for the presence of T lymphocytes using anti-CD3 rabbit polyclonal antibody (Dako, Carpinteria,
CA), for the presence of B lymphocytes using the Mab BLA-36 (Biogenex), and for the presence of astrocytes expressing glial fibrillary acidic protein (GFAP) using an anti-
GFAP Mab (Biogenex). The following generic protocol was used with CD3 (IHC-CD3),
BLA-36 (IHC-BLA-36), and GFAP (IHC-GFAP) antibodies. Only the steps that differ from the previous protocol are mentioned. Deparaffinized sections were treated with 5% hydrogen peroxide. The antigen retrieval was performed using antigen retrieval Citra
Solution (Biogenex) incubated in a steamer for 25 minutes followed by blocking with 5% bovine serum albumin. Sections were incubated with the primary antibody was for 30 minutes at 37 C. Sections were then incubated with biotinylated antibody and with extra- avidinTM peroxidase conjugate (Sigma, St. Louis, MO). Substrate was DAB. The antibody dilutions for CD3, BLA-36, and GFAP were 1:100, 1:50, and 1:400, respectively.
In situ hybridization
Selected tissue sections of the PPMV-1-infected birds were stained with a negative-sense digoxigenin-labeled 850-base riboprobe representing the 5’ end of the matrix gene of NDV Fontana (CA 1083) strain as previously described.11 The matrix 100 gene from the Fontana strain was cloned into pCRII transcription vectors (Invitrogen,
Carlsbad, CA). Anti-sense digoxigenin-labeled riboprobes were generated using RNA polymerase in the presence of labeled nucleotides. For ISH, tissue sections were deparaffinized, rehydrated, and digested with 30 µg/ml Proteinase K for 15 minutes at 37
C. Hybridization was conducted overnight at 42 C with approximately 20 ng of probe in prehybridization solution. After stringent washes, anti-digoxigenin alkaline phosphatase was added to the sections. The development was with chromogen/substrate nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoylphosphate (BCIP). Tissues were counterstained lightly with hematoxylin and coverslipped.
TUNEL assay
The TUNEL assay37 (Boehringer Manheim, Indianapolis, IN) was utilized in addition to the IHC-Casp to detect apoptosis in sections of the spleen. The tissue sections were treated with 20 µg/ml Proteinase K for 15 minutes at 37 C. After washing, the sections were incubated with TUNEL reaction mixture for 1 hr at 37 C. Converter- alkaline phosphatase was added to the slides for 30 minutes at 37 C. The development was with chromogen/substrate NBT and BCIP. Tissues were counterstained lightly with hematoxylin and coverslipped with Permount.
The TUNEL and IHC-Casp methods were analyzed comparing the number of apoptotic cells detected at 2, 5, and 10 DPI in spleen sections of the infected versus control birds. Apoptotic cells were counted in 5 high power fields (400x), and the final result was based on the average between the fields. 101
RESULTS
Clinical findings
PPMV-1 isolates. Several chickens (10% - 20%) from each experimental group had mild periocular edema and bilateral conjunctivitis at 2 DPI. With the Pigeon TX isolate, two birds had slight depression by day 7, and one bird was very listless by day 8 postinoculation. At 10 DPI, all remaining Pigeon TX-infected chickens had mild to severe depression. With the Pigeon GA isolate, one bird was walking on its hocks and had head tremors, drowsiness, and incoordination by day 7 postinoculation. One bird infected with isolate 84-44407 was depressed at 7 DPI. Two birds inoculated with Pigeon
84 isolate had slight depression by day 8, and one bird was walking on its hocks by day 9 postinfection.
APMV-1 isolates. The clinical signs presented by chickens inoculated with the two pigeon-origin APMV-1 isolates (P1307 and P5658) were similar. However, birds inoculated with P1307 were more severely ill. With both APMV-1 isolates, all infected birds had severe bilateral conjunctivitis and periocular edema by 3 DPI. Only one bird infected with P1307 was depressed at that time. At 4 DPI, all APMV-1-infected chickens were severely depressed and inactive and had ruffled feathers. By 5 DPI, one P1307- infected bird was comatose and three were dead. Of the P5658-infected birds, one bird was dead and the remaining three were severely depressed and prostrate, and two of them had open-mouth breathing. The remaining birds were euthanatized in extremis at day 5 postinfection. No clinical signs were observed in the noninfected control birds. 102
Gross findings
PPMV-1 isolates. With all PPMV-1 isolates, the gross findings were similar.
Some infected birds had petechial hemorrhages and edema in the conjunctiva of the lower eyelid at 2 DPI. At 5 DPI, spleens were large and mottled, whereas one bird infected with
Pigeon 84 had petechial hemorrhages in the thymus. By 10 DPI, two Pigeon GA-infected birds had large mottled spleens, and one 84-44407-infected bird had petechiae in the eyelid.
APMV-1 isolates. In birds inoculated with both APMV-1 isolates, the macroscopic lesions were severe and widespread. At 2 DPI, the lesions were confined to the eyelids (edema and petechial hemorrhages) and spleen (large, friable, and mottled).
By 4 DPI, the grossly affected tissues were eyelids (edema and petechial hemorrhages),
Harderian gland (edema), spleen (small, pale or mottled), bursa (very small), and thymus
(small, edematous or with petechial hemorrhages). The gastrointestinal tract was empty in all infected birds and the carcasses were thin and dehydrated. Multifocal hemorrhages were observed in the mucosa of the proventriculus (P1307-infected birds) and small intestine and ceca (P5658-infected birds). At 5 DPI, gross lesions were observed in the eyelids (edema and petechial hemorrhages), spleen (small), bursa (very small), thymus
(edema and petechial hemorrhages), and kidneys (urate deposits). Multifocal hemorrhages were observed in the mucosa of the proventriculus (P1307- and P5658-infected chickens) and small intestine (only P5658-infected chickens). Noninfected control chickens did not have any gross lesions. 103
Histopathology, in situ hybridization, and immunohistochemistry
PPMV-1 isolates. The microscopic lesions observed in the infected birds were similar among all the PPMV-1 isolates. Viral nucleoprotein andl mRNA were detected in the affected tissues by IHC and by ISH, respectively. Mild conjunctivitis was observed at
2 DPI in birds inoculated with Pigeon TX, Pigeon GA, and 84-44407 isolates. Consistent among all the isolates was the presence of multifocal infiltrations of mononuclear inflammatory cells (lymphocytes, plasma cells, and macrophages) in the heart, sometimes associated with necrosis or disruption of cardiac myofibers or both, observed at 5 (Fig.
4.1.) and 10 DPI. Figure 4.2. illustrates the presence of viral mRNA in the myofibers at 5
DPI. Inflammatory cells, found in the affected areas of the heart, were sometimes also positive for viral mRNA. There was multifocal necrosis with fibrin deposits and apoptotic cells in the spleen of two birds infected with the 84-44407 isolate by 5 DPI. Lymphoid depletion, individual cell necrosis, and apoptotic cells were observed in the spleen, bursa, and thymus by 5 and 10 DPI, mostly with in birds inoculated with Pigeon GA and 84-
44407 isolates.
The most remarkable histologic finding with all PPMV-1 isolates was observed in the brain. At 2 DPI, a few cerebral neurons with central chromatolysis were observed in one bird inoculated with the 84-44407 isolate. At 5 DPI, mononuclear inflammatory infiltrations in the neuropil, endothelial cell hyperplasia, and central chromatolysis in the cerebral neurons characterized birds infected with 84-44407 isolate. Hypercellular areas, interpreted as gliosis and inflammatory reaction, were observed in the molecular layer of the cerebellum associated with Purkinje cell necrosis or loss or both. Vacuolation in the cerebellar white matter was observed in one Pigeon 84-infected bird at 5 DPI. 104
Brain lesions were severe by 10 DPI and similar among all PPMV-1-infected birds. Multifocal infiltrations of mononuclear inflammatory cells, mostly lymphocytes and plasma cells, were observed surrounding the small capillaries of the molecular layer of the cerebellum (Fig. 4.3.). Hypercellular areas, interpreted as multifocal gliosis, were associated with Purkinje cell loss in some birds at 10 DPI (Fig. 4.3., inset). Mild vacuolation was sometimes observed in the molecular layer, and focal lymphoplasmacytic meningitis was also observed in a few birds. Severe multifocal lymphoplasmacytic perivascular cuffs extending to the adjacent neuropil (Fig. 4.4.), perivascular edema, endothelial cell hyperplasia, central chromatolysis, neuronal necrosis with neuronophagia, multifocal gliosis, and vacuolation of the neuropil were observed in the cerebrum and brain stem of PPMV-1-infected chickens.
Numerous GFAP-positive astrocytes were observed in the cerebrum, cerebellum, and brain stem. These were more prominent surrounding areas of intense perivascular inflammatory response (Fig. 4.4., inset) and neuronal loss. A few GFAP-positive cells and cell processes were detected in the multifocal hypercellular areas of the cerebellar molecular layer, including the Bergmann-glia, at 10 DPI. The presence of GFAP-positive cells in the brain of the negative controls was within normal limits. A few lymphocytes were positively stained with IHC-CD3 (T cells) and IHC-BLA-36 (B cells) in the hypercellular areas of the cerebellar molecular layer at 5 DPI. By 10 DPI, many T and B lymphocytes were detected primarily surrounding the small capillaries and in the adjacent neuropil of the molecular layer. Numerous T and B lymphocytes were also observed in the perivascular cuffs and in the adjacent neuropil in other affected regions of the brain at
10 DPI. 105
Viral N protein was detected in Purkinje cells, other neurons, and glial cells at 5
DPI. One Pigeon 84-infected bird had large amounts of viral N protein and mRNA (Fig.
4.5.) in the cytoplasm of cerebral neurons at 10 DPI. The amount of N protein was quantitatively less in the affected tissues from birds inoculated with the PPMV-1 isolates than in those infected with the APMV-1 isolates. Staining was more often observed in sections of the heart and brain but less often in lymphoid organs at 5 DPI.
APMV-1 isolates. One P5658-infected bird had multifocal hemorrhages in the cecal tonsils at 2 DPI. Severe conjunctivitis, characterized by edema and lymphoplasmacytic infiltrates, was observed at 2, 4, and 5 DPI among the APMV-1- infected birds. At 4 and 5 DPI, severe lymphoid depletion and necrosis were observed in the lymphoid organs (thymus, bursa of Fabricius, and spleen). The presence of fibrin deposits was marked in the necrotic areas of the spleen. Similar lesions, sometimes associated with multifocal hemorrhages, were observed in the gut-associated lymphoid tissues (GALT), mostly in the cecal tonsils. Necrosis of the superficial epithelium and hemorrhages in the lamina propria were observed in the proventriculus and small intestine. Mild multifocal necrosis was observed in the pancreas and Harderian gland. In the heart, there was mild necrosis of the myofibers associated with mild multifocal infiltrates of macrophages and lymphocytes. Focal areas of necrosis and lymphoplasmacytic infiltrates were observed in the liver of a few birds. There was epithelial hyperplasia with necrosis and fibrin deposits in the trachea and severe pulmonary congestion in a few P1307-infected birds.
The brain lesions were similar but less frequent than those described for PPMV-1- infected chickens at 5 DPI and were more often observed in birds inoculated with the 106
P1307 isolate. At 2 DPI, only P1307-infected birds had very mild multifocal gliosis in the cerebellar molecular layer. Glial nodules and vacuolation were noticed in the cerebrum and brain stem at 4 and 5 DPI. By 5 DPI, one P1307-infected bird had intracytoplasmic eosinophilic inclusion bodies and distinct accumulations of eosinophilic granular material in neurons of the nucleus reticularis in the brain stem (Fig. 4.6.) and in Purkinje cells.
The affected neurons were strongly positive for N protein by IHC. The staining appeared as multiple granules in the cytoplasm (Fig. 4.6., inset) and the cell processes. No microscopic lesions were observed in the noninfected control chickens.
Large amounts of viral N protein were observed in all affected tissues. At 2 DPI,
N protein staining was observed infrequently in the conjunctiva of the lower eyelid (in epithelial and lymphoid cells), cecal tonsils (lymphoid cells), heart (myofibers), thymus
(more prominent in the medulla) and spleen (mostly in large macrophage-type cells). By 4 and 5 DPI, N protein-positive cells were more often seen in spleen (randomly distributed cells), brain (neurons of the cerebrum and brain stem, Purkinje cells, glial cells in the cerebellar molecular layer, and individual cells in the lymphoid aggregates of the choroid plexus), heart (mostly myofibers), thymus (widespread), bursa (widespread), proventriculus (superficial epithelial cells and lymphoid cells), and GALT (small intestine and ceca). Viral N protein was also detected in smaller amounts in epithelium of the conjunctiva and in associated lymphoid follicles, trachea (epithelial cells), lung (epithelial cells and bronchus-associated lymphoid tissue), liver (Kupffer cells), pancreas (lymphoid aggregates), kidney (tubular epithelial cells), feather follicles (epidermis of the follicle wall), and in the feather pulp. IHC-N was negative in all tissue sections of the noninfected control birds. 107
Apoptosis assays
Apoptotic cells were detected by two different assays, TUNEL and IHC-Casp, in spleen sections of chickens infected with the six pigeon-NDV isolates (PPMV-1 and
APMV-1) and of control chickens. At 2 DPI, the number of IHC-Casp-positive cells in the spleens of infected chickens was 3-5 times higher than in the spleens of control chickens. IHC-Casp-positive reaction was found mostly in the area of the ellipsoid- associated cells (EACs) and was characterized by dark brown staining of dendrite-shaped cells surrounding the penicilliform capillaries (Fig. 4.7.). This distribution pattern was very consistent among all the isolates. At 2 DPI, the number of randomly distributed apoptotic cells in TUNEL-stained spleen sections of infected birds was small (Fig. 4.7., inset) and equal or slightly higher than that detected in the noninfected controls. At 5 DPI, the number of TUNEL-positive cells increased 2-3 times (Fig. 4.8.), and the number of
IHC-Casp-positive cells decreased markedly (Fig. 4.8., inset) in spleen sections of birds inoculated with almost all isolates. At 10 DPI, no differences were observed in the number of positive cells (stained by IHC-Casp and TUNEL) found in the spleen sections of infected chickens versus the noninfected controls.
DISCUSSION
Six pigeon-origin isolates of NDV, previously passaged through 2-week-old chickens, 23 were inoculated intraconjunctivally into 4-week-old SPF White Leghorns.
Four isolates (Pigeon TX, Pigeon GA, 84-44407, and Pigeon 84) were previously classified by Mab binding profiles (using the HI test) as PPMV-1 and two isolates (P1307 and P5658) were defined as APMV-1. 20, 23 108
Although some birds presented with severe neurologic signs, overt clinical disease was not a major finding in chickens inoculated with PPMV-1 isolates, and no mortality occurred during the 14-day observation period. However, all four PPMV-1 isolates caused similar tissue damage in the infected chickens. Microscopic lesions were observed in the heart, brain, and lymphoid organs. The most remarkable and consistent histopathologic lesions were observed in the brain at 5 and more so by 10 DPI. The lesions were characterized by severe multifocal lymphoplasmacytic encephalitis with neuronal necrosis, perivascular edema, endothelial hyperplasia, gliosis, and less often neuropil vacuolation and meningitis. Multifocal perivascular lymphoplasmacytic inflammatory reaction and gliosis associated with areas of Purkinje cell loss were consistently observed in the cerebellar molecular layer. Sometimes vacuolation of the adjacent neuropil occurred. Of the 11 PPMV-1-infected birds with distinct brain lesions, only seven had overt neurologic signs.
Discordant temporal relationship between virus replication in the brain and occurrence of clinical disease has been reported with a neurotropic APMV-1 strain in chickens. In that study, 47 central nervous system disease developed several days after maximal virus replication had occurred in the brain. The occurrence of clinical disease, sometimes followed by death, was interpreted as a culmination of the brain damage associated with viral infection. 47 Although mortality was not observed in our PPMV-1- infected birds, the brain lesions probably would be severe enough to cause significant neurologic deficit and affect normal development of commercial chickens.
Significant lesions observed in the heart consisted of multifocal lymphoplasmacytic and histiocytic myocarditis associated with myofiber 109 necrosis/disruption. Similar lesions were also described in a previous passage study with the same PPMV-1 and APMV-1 isolates 23 and in several other studies with APMV-1 strains. 8, 27 Because NDV-infected chickens have altered electrocardiograms 35, 36 and because of the functional importance of the heart, bird performance, including weight gain and growth rate, could be reduced among commercial chickens infected with pigeon
NDV.
The use of IHC and ISH to detect APMV-1 infections in tissues of chickens has been reported several times. 11, 24, 33 However, to our knowledge, studies reporting the use of those methods to define tissue distribution of PPMV-1 infection in chickens are scarce.
23 In our study of PPMV-1-infected birds, viral replication was characterized by the presence of viral N and mRNA in the same sites of damage among the affected tissues.
Large amounts of virus were detected in the brain of one Pigeon 84-infected bird at 10 DPI. In comparison, in chickens infected with two APMV-1 neurotropic strains
(Texas GB and Turkey North Dakota) Brown et al 11 detected only very little viral mRNA by ISH at 10 DPI. In birds infected with another neurotropic APMV-1 strain (Missouri-
(H) Len 1950), NDV could not be recovered by virus isolation from pooled brain tissue after 9 DPI. 47 The abundant viral replication observed in the brain of PPMV-1-infected birds appears to be somewhat unusual compared with that of previously described
APMV-1 isolates which have a tropism for brain. 11, 47
In brain sections of PPMV-1-infected birds, GFAP-positive astrocytes were detected in remarkably high numbers, mostly surrounding the areas of perivascular inflammatory response and neuron degeneration/necrosis in cerebrum and brain stem.
GFAP-positive reaction was observed in the areas of gliosis adjacent to Purkinje cells at 110
10 DPI. Normally in chickens, the Bergmann-glia, a highly specialized astrocytic cell type found in the cerebellar molecular layer, is GFAP negative. 19 Reactive gliosis following a stab wound lesion was described in the cerebellar molecular layer in chickens. In that case, the Bergmann-glia showed a marked immunopositivity to GFAP around the lesion site.1 Similar GFAP staining was observed in the Bergmann-glial cells inside or adjacent to the multifocal cerebellar reactive gliosis of infected chickens in our study.
On HE-stained sections, it was not easy to identify all the cell types involved in the hypercellular areas observed in the cerebellar molecular layer mostly at 5 DPI. For this reason, brain sections were also stained for lymphocyte subset (CD3 and BLA-36 antibodies) to detect T- and B-lymphocytes, respectively. Only a few of the cells seen in the molecular layer were positive by IHC-CD3 and IHC-BLA-36 at 5 DPI. Both lymphocyte cell types were abundant in those areas at 10 DPI surrounding the walls of small capillaries and in the adjacent neuropil. T- and B-lymphocytes were also detected as cell components of the perivascular cuffs and inside of the adjacent neuropil, mostly in the affected cerebral sections.
With the two pigeon-origin APMV-1 isolates, severe clinical disease and mortality were observed in the inoculated chickens. Not all NDV isolates coming from pigeons are the variant PPMV-1. Pigeons infected with highly virulent NDV of the
APMV-1 type represent a very serious threat to commercial poultry flocks. P1307 and
P5658 were characterized as velogenic isolates in previous tests. 16 , 23 In the present study, both isolates produced gross and histopathologic lesions characteristic of highly virulent NDV, typical of the velogenic viscerotropic NDV pathotype 11, 18, 34, 39 Viral 111 replication, detected by IHC-N, in APMV-1-infected birds was abundant and much greater than in birds inoculated with the PPMV-1 isolates.
Intracytoplasmic inclusion bodies and distinct accumulations of eosinophilic granular material, as observed in the brain of one P1307-infected bird, are not often reported for NDV-infected birds. Case reports of ND include the presence of intranuclear and intracytoplasmic eosinophilic inclusion bodies in epithelial cells of esophageal glands and in neurons of the ganglia subjacent to the adrenal in infected pheasants and in hepatocytes of cuckoo doves.43 Transmission electron microscopy revealed intracytoplasmic inclusion bodies suggestive of paramyxovirus in endothelial cells in
14 blood vessel from the esophagus of an NDV-infected game chicken.
Several investigators have detailed many different techniques 26-28, 30 to detect
NDV-induced apoptosis. Most of the studies were performed in cell culture or in vivo with the APMV-1 Texas GB strain. Texas GB strain, a neurotropic virus, causes both necrosis and apoptosis of infected chicken peripheral blood lymphocytes, 30 apoptosis of chicken embryo fibroblasts, 26 and apoptosis of peripheral blood mononuclear cells 28 including virus-infected macrophages. 29 In contrast, during this study, apoptosis assays were confined to those that could be used in formalin-fixed paraffin-embedded tissues.
In the lymphoid organs and tissues examined in this study, the most striking results were obtained from APMV-1-infected birds where apoptosis was followed by extensive lymphocellular necrosis. Less prominent lesions were observed in lymphoid organs of the PPMV-1-infected birds that were characterized by apoptosis, depletion, and less often, by necrosis of lymphoid cells. The occurrence of apoptosis in lymphoid organs was further analyzed in sections of the spleen by two different approaches, the TUNEL 112 assay and the IHC-Casp staining. Among all the NDV isolates studied here, apoptotic cells were detected earlier (at 2 DPI) and in greater numbers mostly in the area of the
EACs of the spleen using the IHC-Casp staining as compared with the TUNEL assay. At
5 DPI, TUNEL staining was more extensive than IHC-Casp, and the apoptotic cells were randomly distributed in the spleen sections. These results could be explained by the fact that active caspase-3 is an enzyme that functions early effecting apoptosis, 13 and thus, its detection in a “preapoptotic” cell would occur before DNA breakdown, detected by the
TUNEL assay. 25, 37 The role of caspase-3 in apoptosis during NDV infection might be similar to that found in another study, where Sendai virus (another paramyxovirus) induced apoptosis through activation of caspase-3 and caspase-8.10 NDV infection followed by early NDV-induced apoptosis of the EACs in the spleen might have severe consequences for the bird immune response because EACs play such a crucial role in antigen processing and presentation. 41
We demonstrated that PPMV-1 causes severe lesions among infected chickens, mostly affecting the heart and brain. Pigeons must be considered seriously as a potential source of NDV infection and disease for commercial poultry flocks. Preventive NDV vaccination of domestic pigeons and strict biosecurity measures should be taken to reduce the risk of pigeon ND outbreaks in chickens.
ACKNOWLEDGEMENTS
G. D. Kommers was supported by a scholarship from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (Brasília, DF, Brazil). The work was funded by US Poultry and Egg Association, grant 406 and USDA-ARS-CRIS project 6612- 113
32000-021-00D. We acknowledge the excellent technical support of Phillip Curry and
Erica Behling-Kelly.
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Fig. 4.1. Heart; chicken inoculated with Pigeon TX isolate (PPMV-1), 5 DPI. The myofibers are necrotic or disrupted, and there is infiltration of lymphocytes, plasma cells, and macrophages. HE. Bar = 30 µm.
Fig. 4.2. Heart; chicken inoculated with Pigeon TX isolate (PPMV-1), 5 DPI. NDV mRNA (matrix gene) is detected in the myofibers (dark brown staining). ISH, hematoxylin counterstain. Bar = 30 µm.
Fig. 4.3. Cerebellum; chicken inoculated with Pigeon GA isolate (PPMV-1), 10 DPI.
Multifocal infiltrates of mononuclear inflammatory cells are surrounding the small capillaries of the molecular layer. HE. Bar = 250 µm. Inset: Purkinje cell loss and hypercellular area (gliosis and inflammatory reaction) in the molecular layer. HE. Bar =
150 µm.
Figure 4.4. Cerebrum; chicken inoculated with 84-44407 isolate (PPMV-1) at 10 DPI.
Severe multifocal lymphoplasmacytic perivascular cuffs are extensive to the adjacent neuropil. HE. Bar = 100 µm. Inset: Numerous GFAP-positive astrocytes (red stained) are present in the areas of intense perivascular inflammatory response. IHC, avidin-biotin- alkaline phosphatase, hematoxylin counterstain. Bar = 250 µm.
Figure 4.5. Cerebrum; chicken inoculated with Pigeon 84 isolate (PPMV-1), 10 DPI.
Abundant viral mRNA is present in the cytoplasm of neurons. Note intense inflammatory reaction in this area. ISH, hematoxylin counterstain. Bar = 200 µm. 122
Figure 4.6. Brain stem; chicken inoculated with P-1307 isolate (APMV-1), 5 DPI.
Intracytoplasmic eosinophilic inclusion bodies (arrow) and distinct accumulations of eosinophilic granular material are present in neurons of the nucleus reticularis.
Vacuolation of the neuropil is also visible. HE. Bar = 30 µm. Inset: Affected neuron of the nucleus reticularis is strongly positive for viral nucleoprotein (intracytoplasmic red granules). IHC, avidin-biotin-alkaline phosphatase, hematoxylin counterstain. Bar = 30
µm.
Figure 4.7. Spleen; chicken infected with Pigeon TX isolate (PPMV-1), 2 DPI. Large amounts of apoptotic ellipsoid-associated cells (EACs) are positive for active caspase-3 surrounding the penicilliform capillaries. IHC, avidin-biotin-peroxidase, hematoxylin counterstain. Bar = 230 µm. Inset: There are few randomly distributed TUNEL-positive apoptotic cells. TUNEL, hematoxylin counterstain. Bar = 100 µm.
Figure 4.8. Spleen; chicken infected with P-1307 isolate (APMV-1), 5 DPI. A large number of TUNEL-positive cells are evenly scattered throughout the spleen. TUNEL, hematoxylin counterstain. Bar = 230 µm. Inset: The number of active caspase 3-positive cells is markedly small. IHC, avidin-biotin-peroxidase, hematoxylin counterstain. Bar =
100 µm. 123 CHAPTER 5
VIRULENCE OF SIX HETEROGENEOUS-ORIGIN NEWCASTLE DISEASE
VIRUS ISOLATES BEFORE AND AFTER SEQUENTIAL PASSAGES IN
DOMESTIC CHICKENS1
1 Kommers, G.D., King, D.J., Seal, B.S. and Brown C.C. 2002. To be submitted to Avian Pathology. 125
ABSTRACT
Four serial passages of six Newcastle disease virus (NDV) isolates were performed in 2- week-old White Leghorns. The viruses were recovered from chickens (Ckn-LBM and
Ckn-Australia isolates), exotic (YN Parrot, Pheasant, and Dove isolates) and wild birds
(Anhinga isolate). Infected chickens were monitored clinically and euthanatized with tissues sampled for histopathology and immunohistochemistry. Pathotyping tests and sequence analysis of the fusion protein cleavage site were performed before and after passages. The moderately virulent Dove isolate became highly virulent with serial passage. The originally highly virulent Pheasant isolate had an increase in the intracerebral pathogenicity index (ICPI) and the intravenous pathogenicity index (IVPI) with passages in chickens. Virulence increase was not observed with Ckn-LBM, YN
Parrot, Ckn-Australia, or Anhinga isolates after four chicken passages. The results demonstrate the high risk for domestic chickens represented by some NDV-infected non- poultry species such as doves.
INTRODUCTION
Avian paramyxovirus-1 (APMV-1) is synonymous with Newcastle disease virus (NDV).
Viruses of the APMV-1 serotype vary in virulence from those that cause high mortality to those that cause asymptomatic infections. Isolates of NDV are non-segmented, single- stranded, negative-sense enveloped RNA viruses belonging to the family
Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus (Alexander 1997,
1998). Recent studies of phylogenetic relationships among the Paramyxoviridae are consistent with a proposal for expanding the taxonomic diversity within the 126
Paramyxoviridae to include a new genus for NDV (Leeuw & Peeters, 1999; Seal et al.,
2000; Westower & Hughes, 2001).
Virulence differences among NDV strains are determined by chicken and chicken embryo inoculation. Four pathotyping tests are employed to make that differentiation.
These include an intracerebral pathogenicity index (ICPI) in 1-day-old chicks from specific-pathogen-free (SPF) parents; an intravenous pathogenicity index (IVPI) in 6- week-old SPF chickens (Alexander, 1997, 1998); an intracloacal inoculation pathogenicity test in 6-to-8-week-old chickens (Alexander, 1997, 1998; Pearson et al.,
1975); and the mean death time (MDT) in 9-to-10-day-old embryonating eggs. Viruses are characterized as low (lentogens), moderate (mesogens), or high virulence (velogens) based on clinical signs and mortality in chickens along with time to embryo death postinoculation (Alexander, 1997, 1998).
The marked strain-dependent differences in tropism and virulence observed with
NDV depends upon the presence of cellular proteases required for the activation of the viral fusion (F) glycoprotein precursor (F0; Nagai & Klenk, 1977; Glickman et al., 1988;
Rott & Klenk, 1988; Gotoh et al., 1992; Nagai, 1995). The F0 of highly and moderately virulent NDV strains has dibasic amino acid cleavage motif (112R/K-R-Q-R/K-R-F117) allowing cleavage by proteases ubiquitously present in cells throughout the body, resulting in pantropic or systemic infection (Gotoh et al., 1992; Nagai, 1995; Alexander,
2001). Low virulence strains having monobasic cleavage motif (112G/E-K/R-Q-G/E-R-
L117) undergo F0 cleavage only in a few limited tissue types expressing specific trypsin- like protease(s), hence causing an infection more localized to particular organs as the respiratory and alimentary tracts (Gotoh et al., 1992; Nagai, 1995; Alexander, 2001). 127
The accurate virulence characterization of NDV isolates is extremely important because ND, an Office International des Epizooties (OIE) List A disease, requires reporting (Office International des Epizooties, 2000) that may result in international trade restrictions (Office International des Epizooties, 2002 a,b). A recent change in the
Newcastle disease (ND) definition was approved by the OIE. The new definition of ND, an OIE List A disease, relies on the ICPI and on determination of the F protein cleavage site amino acid sequence to differentiate virulent and low virulence APMV-1 isolates. An isolate with an ICPI equal to or greater than 0.7 or having an oligobasic cleavage motif at the F protein cleavage site will be classified as a virulent virus and its presence should be reported to the OIE (Office International des Epizooties, 1999).
Many bird species are susceptible to NDV and the manifestations of ND vary greatly in morbidity and mortality according to the virulence of the infecting NDV strain, the virus tropism for the body systems, and the avian species infected (Alexander 1997,
1998). Previous observations have suggested that isolates from non-poultry species may not show their potential virulence for domestic chickens in conventional pathogenicity tests, until passaged several times in chickens (Alexander 1997, 1998). The aim of this study was to determine by biological and molecular assays the effect of serial chicken passages on the virulence of six NDV isolates from chickens, exotic and wild birds.
MATERIALS AND METHODS
Viruses
Six NDV isolates were inoculated in chickens: 1) Ckn-LBM (chicken/U.S.(PA)/92-
31003/92), chicken isolate from a live bird market in Pennsylvania, 1992; 2) Yellow 128
Nape (YN) Parrot (parrot/U.S.(TX)/96-22027/96), isolate from a smuggled parrot in
Texas, 1996; 3) Ckn-Australia (chicken/Australia/98-09-14-1110/98), chicken isolate from Australia, 1998; 4) Anhinga (anhinga/U.S.(FL)/93-44083/93), isolate from an anhinga at a commercial marine park in Florida, 1993 (King & Seal, 1998, Brown et al.,
1999); 5) Pheasant (pheasant/U.S./F98-1208/98), isolated from an exotic pheasant, 1998
(Shivaprasad et al., 1999); and 6) Dove (dove/U.S./98-9248-10/98), isolated from an exotic dove in a quarantine station, 1998. The isolates Ckn-LBM, YN Parrot, Anhinga, and Dove were received from USDA, APHIS, National Veterinary Services Laboratories
(NVSL), Ames, Iowa. The Ckn-Australia isolate was received from Paul Selleck, CSIRO,
Australian Animal Health Laboratory at Geelong, Victoria, Australia.
The fusion (F) protein cleavage site amino acid sequences of the following NDV reference strains and isolates were utilized for comparison: 1) B1 (chicken/U.S./B1/48);
2) La Sota (chicken/ U.S./LaSota/46); 3) Kimber (chicken/U.S./Kimber/47); 4) Fontana
(chicken/U.S./CA1083/71); 5) Texas GB (chicken/U.S./GB/48) (Seal, 1996); and 6)
Pigeon GA (pigeon/U.S.(GA)/21042/98) (Kommers et al., 2001).
Eggs and chickens
The Southeast Poultry Research Laboratory (SEPRL) specific-pathogen-free (SPF) White
Leghorn flock was the source of embryonated eggs and chickens used in this experiment.
Embryonated eggs were inoculated for virus isolation and propagation. Chickens were inoculated for passage of the virus and pathotyping assays. Birds were housed in negative pressure isolators under BSL-3 agriculture conditions at SEPRL and provided feed and water ad libitum. 129
Virus passage in chickens
Groups of ten 2-week-old SPF White Leghorns were inoculated with one of the six NDV isolates for each passage and a group of ten birds was inoculated with diluent (phosphate- buffered saline [PBS]) as controls. The passage procedure (Figure 5.1.) was according to methods previously described for NDV (Alexander & Parsons, 1986, Kommers et al.,
2001). Briefly, chickens were inoculated with 0.1 ml of undiluted clarified infective amnioallantoic fluid (AAF) by the intramuscular (IM) route into the breast muscle. After
2 days postinoculation (DPI), six birds were euthanatized and the spleens were sampled aseptically and homogenized. A 20% weight/volume homogenate in PBS without antibiotics was prepared and used as inoculum for passage 2 (0.1 ml/bird of undiluted supernatant of the homogenate IM). This procedure was repeated 3 times (passages 2-4).
The supernatant fluids were also inoculated into 9- to 10-day-old embryonated SPF eggs to confirm infectivity of passage inoculum and to prepare a virus stock for characterization and pathotyping.
An alternate procedure for virus passage in chickens (Figure 5.2.) was used for the reasons described in the results with three isolates (Ckn-LBM, YN Parrot, and Ckn-
Australia). For passage 1 of this procedure, chickens were inoculated with 0.1 ml of undiluted infective AAF by eyedrop/intranasal (ED/IN) route (King, 1996). At 4 DPI, oral and cloacal swabs were collected separately into a tube of 1.5 ml brain-heart infusion broth (BHI) with antibiotics (2,000 units/ml penicillin G, 200 µg/ml gentamicin sulfate, and 4 µg/ml amphotericin B [Sigma Chemical Co. St. Louis, MO]) from six chickens per group and the birds were euthanatized for aseptic removal of spleens. Egg-amplified virus from splenic homogenate, cloacal swabs, or oral swabs was selected for ED/IN 130 inoculation of each subsequent passage through four passages. The virus positive sample selected for subsequent passage inoculum was the one that was the least likely to represent direct recovery of inoculum from the sampled passage. Preference was given to splenic virus positive, then cloaca and finally oral if other samples were not positive.
Virus amplified from spleens of YN Parrot-infected birds and from cloacal swabs of Ckn-
LBM- and Ckn-Australia-infected birds was used for subsequent passages.
At passages 1 and 4, three birds were euthanatized at 4 DPI. The following tissues were collected and fixed by immersion in 10% neutral buffered formalin for 48 to 56 hours: comb, spleen, thymus, bursa, eyelid (including conjunctiva and skin), Harderian gland, oropharynx, esophagus, ingluvium, proventriculus, pancreas, small intestine, cecal tonsils, large intestine, caudal thoracic air sac, trachea, lung, heart, liver, kidney, breast and thigh muscles, sciatic nerves, brain, and bone marrow (femur). The femur samples
(including bone marrow) were decalcified in 5% formic acid for 3-4 hours. All tissues were routinely processed into paraffin, and 3 µm tissue sections were prepared for hematoxylin and eosin (HE) staining and for immunohistochemistry (IHC).
Immunohistochemistry
An IHC protocol to detect the NDV nucleoprotein (N) was performed as previously described (Kommers et al., 2001). Briefly, paraffin tissue sections were deparaffinized, rehydrated, and antigen retrieval was by microwaving (10 min at full power) in Vector antigen unmasking solution (Vector Laboratories, Burlingame, CA). Sections were blocked (Universal Blocking Reagent; Biogenex, San Ramon, CA), and then incubated overnight at 4 C (or for 2 h at 37 C) with an anti-peptide (anti-N protein) antibody made 131 in rabbit and diluted at 1:8,000. Sections were incubated with biotinylated goat anti-rabbit antibody, and then with avidin-biotin-alkaline phosphatase (Vector Labs). Substrate development was with Vector Red (Vector Labs). Sections were counterstained lightly with hematoxylin and coverslipped for a permanent record.
Virus isolation and titration
Oral and cloacal swab fluids and splenic homogenate were centrifuged at 1,000 x g for 20 min. Undiluted supernatant was inoculated into 9- to 10-day-old SPF embryonated chicken eggs and incubated for 7 days. Virus infectivity titers were calculated from the results of inoculation of 9- to 10-day-old embryonated eggs with serial tenfold dilution in
BHI containing antibiotic (100 units penicillin G/ml and 50 µg gentamicin sulfate/ml).
NDV-infected dead or surviving embryos were identified by hemagglutination (HA) activity in AAF harvested from chilled eggs. NDV was confirmed in HA-positive samples by hemagglutination-inhibition (HI) test with NDV-specific antiserum or monoclonal antibodies (MAbs) (King, 1996).
Monoclonal antibodies and antiserum
Nine MAbs with different NDV specificities were used for isolate differentiation by the
HI test as previously described (Alexander et al., 1997). The MAbs obtained from NVSL included B79, 15C4, 10D11 (Lana et al., 1988), AVS (Srinivasappa et al., 1986), and
617/161 (Collins et al., 1989). B79 reacts with most APMV-1 including most PPMV-1;
15C4 reacts with most APMV-1 except PPMV-1; and 617/161 only reacts with PPMV-1 isolates within the APMV-1 group. The AVS antibody reacts with many lentogens, 132 including B1 and La Sota strains. The 10D11 antibody reacts with neurotropic velogens and mesogens like the Roakin strain. The MAbs prepared at SEPRL included P15D7,
P11C9, P3A11, and P10B8. These four antibodies identify additional antigenic diversity among APMV-1. The polyclonal chicken NDV antiserum was prepared at SEPRL by immunization of chickens with inactivated NDV-La Sota.
Hemagglutination and hemagglutination-inhibition tests
The HA and HI tests to identify NDV positive embryos and for antigenic characterization of virus stocks were conducted using conventional microtiter methods. Four HA units of each of the virus stocks was used as test antigen in completing the HI test of MAbs and polyclonal antiserum (King, 1996).
Pathotyping tests
The virulence of each NDV isolate before and after passage was evaluated by standard pathotyping tests (Alexander, 1998). These include the mean death time (MDT), the intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), and the intracloacal pathogenicity test. For MDT, a series of tenfold dilutions of the original inoculum for virus passage of all six viruses and egg-amplified virus isolated from passage 4 was made in sterile BHI. Subsequently, 0.1 ml of each dilution was inoculated into the allantoic cavity of five 9- to 10-day-old embryonated SPF chicken eggs.
Inoculated eggs were incubated at 37 C and candled twice daily. The time of death of each embryo was recorded. The MDT was determined as the mean time in hours for the minimum lethal dose to kill the embryos (Alexander, 1998). 133
For ICPI, pre- and post-passage infective AAF of all six viruses was filtered through a 0.45 µm filter pre-wet with BHI. The HA titer of all filtrates was equal to or greater than 16. The filtrate was diluted 1:10 in PBS without antibiotics and 0.05 ml/bird was inoculated intracerebrally in 24- to 40-hour-old SPF White Leghorns. The ICPI was performed and scored in the standard manner (Alexander, 1998). For IVPI, inoculum preparation was the same as that for ICPI. The dosage was 0.1 ml / bird intravenously in
6-week-old SPF White Leghorn hatchmates. The IVPI test was performed and scored in the standard manner (Alexander, 1998).
For the intracloacal test, four 6-week-old SPF White Leghorns were inoculated by swabbing the cloaca with a cotton swab saturated with 1:10 dilution of pre- or post- passage infectious AAF. Birds were observed daily for clinical signs during 10 days.
Dead birds were necropsied and lesions were scored as previously described for chickens inoculated intracloacally with viscerotropic velogenic NDV (VVNDV) (Pearson et al.,
1975). Gross lesions were rated as +4 to +1 in severity. The findings for each rating were:
+4 (edema of the subcutis in the head or neck region; prominent hemorrhage in the larynx or trachea; hemorrhage, necrosis or ulceration or both of the proventriculus through the cloaca); +3 (characteristic lesions in the upper respiratory and gastrointestinal [GI] tracts, as above); +2 (a prominent lesion in the cloaca and less extensive or fewer hemorrhages and necrosis elsewhere in the GI and respiratory tracts); and +1 (a recognizable lesion in the cloaca and a lesion elsewhere in the GI or respiratory tracts) (Pearson et al., 1975).
Confirmation of infection of survivors at the termination of ICPI, IVPI, and intracloacal tests was accomplished by testing for seroconversion as determined by HI. 134
Viral RNA extraction, oligonucleotide primers and reverse transcription-polymerase chain reaction (RT-PCR)
Isolates of NDV (before and after passage) were replicated in embryonated eggs
(Alexander, 1998) and RNA was extracted (Chomzcynski & Sacchi, 1987) directly from
AAF as described (Seal et al., 1995, 1998). Oligonucleotide RT-PCR primers were designed to amplify regions of the fusion protein gene, including the fusion protein cleavage site (Seal et al., 1995, 1998). A single tube RT-PCR for genomic NDV RNA was completed as described (Lewis et al., 1992), using SuperscriptTM (Kotewicz et al.,
1988; Life Technologies, Gaithersburg, MD) and AmplitaqTM (PE Biosystems, Foster
City, CA) polymerase (Saiki et al., 1985). Amplification products were separated by gel electrophoresis in 1.0% agarose using Tris-borate buffer, stained with ethidium bromide and photographed during UV transillumination (Seal et al., 1995, 1998).
Direct nucleotide sequencing of RT-PCR products
Amplification products were purified using MicroconTM (Amicon, Belford, MA) spin filters and spectrophotometrically quantified. Additionally, amplification products were cloned using the TA cloning systemTM (Mead et al., 1991) according to the methods described by the manufacturer (Invitrogen, San Diego, CA). Direct double-stranded nucleotide sequencing (Sanger et al., 1977) was completed using Taq polymerase
(Applied Biosystems, Inc., Foster City, CA) with the oligonucleotide primers used for
RT-PCR, fluorescent-labeled dideoxynucleotides, and an automated nucleic acid sequencer (Smith et al., 1986). Nucleotide sequence editing, analysis, prediction of 135 amino acid sequences at the F protein cleavage site and alignments were conducted using
IntelliGenetics GeneWorks 2.5TM software (IntelliGenetics, Mountain View, CA).
RESULTS
Virus passage in chickens
Direct bird-to-bird passage was successful with the Anhinga, Pheasant, and Dove isolates. Preliminary attempts to pass the Ckn-LBM, YN Parrot, and Ckn-Australia isolates by the intramuscular route resulted in virus recovery from spleen only with the
YN Parrot isolate. However, the splenic homogenate virus titer was too low to infect birds in the subsequent passage. Successful virus passage was accomplished with the alternate passage method (Figure 5.2.) by ED/IN inoculation of egg-amplified passaged viruses.
Clinical signs
Clinical signs were not observed during passages 1 to 4 with the Ckn-LBM, YN Parrot,
Ckn-Australia, or Anhinga isolates. With the Pheasant isolate from passage 1, one bird was depressed at 2 DPI. Depression and paralysis were observed at 3 DPI. At 4 DPI, four birds were dead. During passage 2, slight depression was observed at 2 DPI while at 3
DPI, severe depression and mortality occurred among infected chickens. All birds were depressed and a few were paralyzed or walking on hocks at 2 DPI in passage 3 and there was mortality at 3 and 4 DPI. At passage 4, all birds were less active and had ruffled feathers by 2 DPI. One bird was down on its hocks at 3 DPI while one bird died at 4 DPI. 136
With the Dove isolate in passage 1, one bird was depressed at 3 DPI and by 4
DPI, all birds were depressed and had ruffled feathers. Two birds had dark foci on the comb tips (interpreted to be foci of necrosis) and two other birds had periocular edema. A few chickens had periocular edema at 4 DPI during passage 2 and by passage 3, slight depression was observed at 3 DPI. At 4 DPI, the birds were depressed with ruffled feathers. During passage 4, a few birds had ruffled feathers at 2 DPI and all birds were depressed by 3 DPI. By 4 DPI, all infected chickens were very depressed and two birds had extensor rigidity and spastic leg paralysis.
Gross lesions
Gross lesions were observed only in birds infected with the Pheasant and Dove isolates.
In passage 1, birds infected with Pheasant isolate were dehydrated and had hemorrhages in the gastrointestinal (GI) tract, enlarged and mottled spleens, reticulated kidneys
(interpreted to be urate deposits), and necrosis of the comb tips. During passage 4, all infected chickens were dehydrated and had eyelid edema and GI tract hemorrhages. The lymphoid organs were very small and the lungs were edematous. Chickens infected with the Dove isolate were dehydrated and had eyelid edema and comb necrosis in passage 1.
During passage 4, the spleen was small and pale or mottled, the bursa was small while the thymus was small and sometimes surrounded by edema. Pulmonary edema was occasionally observed among chickens infected with the Dove isolate. 137
Histopathology
Microscopic lesions were observed only in birds inoculated with the Anhinga, Pheasant, and Dove NDV isolates. With the Anhinga isolate in passage 1, there was eyelid edema, multifocal myofiber disruption with infiltrates of lymphocytes and macrophages in the heart. Multifocal gliosis was observed in the molecular layer of the cerebellum. In passage 4, the cerebellum had multifocal gliosis with Purkinje cell loss. Epithelial necrosis was observed in the comb.
With the Pheasant isolate in passage 1, multifocal hemorrhages were observed in the cecal tonsils (Figure 5.3.) and there was severe diffuse necrosis of the lymphoid tissues (Figure 5.4.). Multifocal myofiber disruption and infiltrates of lymphocytes and macrophages were found in the heart. Multifocal gliosis was observed in the molecular layer of the cerebellum. Similar lesions were observed during passage 4 with the addition of lung edema and comb necrosis. Severe diffuse necrosis of the bone marrow (sampled only during passage 4) was a common finding.
With the Dove isolate in passage 1, there was moderate to severe lymphoid depletion in the lymphoid organs (Figures 5.5. and 5.7.) and mild individual cell necrosis was observed in the cecal tonsils. Mononuclear infiltrates were found in the heart. The cerebellum had mild multifocal gliosis, Purkinje cell loss, and vacuolation of the white matter. In passage 4, severe diffuse necrosis was a common finding in all lymphoid organs (Figure 5.6. and 5.8.), lymphoid aggregates, and in the bone marrow. Similar lesions as described in passage 1 were also observed in the heart and brain. Epithelial necrosis was observed in the comb. 138
Immunohistochemistry for NDV
Chickens inoculated with Ckn-LBM and YN Parrot isolates, positive staining for
NDV nucleoprotein was observed in the oropharyngeal salivary glands in passages 1 and
4. With the Ckn-Australia isolate, there were positive cells in the conjunctival epithelium
(Figure 5.9.) and in the oropharyngeal salivary glands (Figure 5.10.) from chickens in passages 1 and 4. With the Anhinga isolate in passage 1, positive staining was observed in the eyelids, heart, and brain of infected birds. In passage 4, the eyelids, heart, trachea, comb, and brain were IHC positive.
The most striking positive staining was observed in birds inoculated with the
Pheasant isolate. The N protein-positive organs detected by IHC during passage 1 were: eyelids, heart, lymphoid organs and lymphoid aggregates (Figure 5.11.), lungs, trachea, liver, kidney, and brain. The same organs were N protein-positive at passage 4 with the addition of the Harderian gland, pancreas, crop, and comb. With the Dove isolate, eyelids, bursa, thymus, heart, and brain were positive by IHC in passage 1 infected chickens. A greater number of organs were IHC-positive in passage 4 infected chickens including eyelid, heart, lymphoid organs (Figures 5.12. and 5.13.), lymphoid aggregates, bone marrow (Figure 5.14.), Harderian gland, oropharyngeal salivary glands, crop, esophagus, pancreas, lung, trachea, kidney, comb, and brain.
Monoclonal Antibodies
The MAb binding profile is summarized in Table 5.1. There were change(s) in the MAb binding pattern before and after passage with the YN Parrot and Anhinga isolates. The 139 changes with the YN Parrot isolate were observed with the MAbs 10D11 (HI-positive before and HI-negative after passage) and P10B8 (HI-negative before and HI-positive after passage). With the Anhinga isolate, one change was observed with the MAb P3A11
(HI-negative before and HI-positive after passage). The Dove isolate was HI-negative with MAb 617/161, specific for pigeon paramyxovirus-1 (PPMV-1) isolates, and had the same MAb binding profile as the Pheasant isolate.
MDT, ICPI, and IVPI
The results of the MDT, ICPI, IVPI, and intracloacal test performed before and after four passages in White Leghorn chickens are summarized in Table 5.2. There was some variation in the MDT results with the YN Parrot (MDT increased from 91 h to 104 h) and
Ckn-Australia (MDT decreased from 120 h to 113 h) isolates. Minor changes were observed in the ICPI with those isolates and no changes were observed in the IVPI results before and after passages. With the Anhinga isolate, the MDT increased, the ICPI slightly decreased, and the IVPI remained almost the same. However, important changes in the pathotyping tests results were observed with the Pheasant and Dove isolates. With the initially highly virulent Pheasant isolate, virulence increase was observed throughout all the pathotyping tests. Marked changes in virulence parameters were observed with the
Dove isolate, characterized by decrease in MDT and increase in ICPI and IVPI results.
Intracloacal test
Neither clinical disease nor mortality were observed in chickens inoculated intracloacally with Ckn-LBM, YN Parrot, Ckn-Australia, and Anhinga isolates before or after passages. 140
However, overt clinical disease and mortality were observed in birds inoculated with the
Pheasant (original and passaged viruses) and Dove (only with passaged virus) isolates
(Figure 5.15.). The clinical signs and mortality occurrence with these two isolates are summarized in Table 5.3. At necropsy, chickens inoculated with the original inoculum of the Pheasant isolate had +1 to +2 VVND lesions. The carcasses were thin and dehydrated, and the GI tract was void of contents. The principal lesions were in the eyelids (edema and petechial hemorrhages), proventriculus (petechiae and suffusions in the mucosa), cecum and cloaca (petechiation), lymphoid organs (very small), kidney
(urate deposits), and comb (multifocal necrosis). Birds inoculated with the passaged
Pheasant isolate had +4 lesions and with the passaged Dove virus the severity of the lesions ranged from +1 to +3. The lesions were characterized by hemorrhages
(petechiation or suffusions) in the eyelids (Figure 5.16.), larynx, proventriculus (Figure
5.17.), Peyer patches, cecal tonsils, and cloacal mucosa of infected chickens. Other affected tissues and lesions were observed in the neck (severe subcutaneous edema;
Figure 5.18.), spleen (enlarged and pale/mottled; Figure 5.19.), bursa (very small), thymus (edematous), liver (pale), and kidney (urate deposits; Figure 5.20.). Additionally, there were hemorrhages in the thigh muscles, in the mucosa of the junctions esophagus- ingluvium and proventriculus-ventriculus, blood clots in the ceca, and moderate hydropericardium in birds inoculated with the passaged Dove virus.
Nucleotide sequence
The amino acid sequence at the F protein cleavage site of each isolate is presented on Table 5.4. Before and after passages, the Ckn-LBM, YN Parrot, and Ckn-Australia 141 isolates had the F protein cleavage site sequence G-R/K-Q-G-R-L typical of low virulence viruses. Anhinga, Pheasant, and Dove isolates had the F protein cleavage site sequence R-R-Q-K-R-F typical of virulent viruses before and after passages. Pheasant and Dove isolates had identical F protein cleavage site amino acid motif, identical MAb binding pattern, and both caused lesions typical of the viscerotropic velogenic pathotype.
Nucleotide sequence analysis of the fusion gene did reveal that the Dove and Pheasant isolates were slight different (data not presented).
DISCUSSION
All six isolates were characterized by NDV polyclonal antibodies as viruses of the
APMV-1 serotype. Antigenic variation among these six APMV-1 isolates was evident in the results of the HI test with MAbs and was similar to the variation among NDV isolates described by others (Alexander et al., 1997; Aldous & Alexander, 2001). The Ckn-LBM isolate only reacted with the B79 MAb, and this binding pattern was previously reported with another chicken-origin isolate (ch/NY/13828/95) from a live bird market (King &
Seal, 1997). The Ckn-Australia was the only isolate that did not react with the B79 MAb.
The only previous NDV isolates not inhibited by B79 were from pigeons, isolates that were inhibited by the pigeon-specific antibody 617/161 (King, 1996). Two isolates, YN
Parrot and Anhinga, presented change(s) in the MAb binding pattern before and after passage. The YN Parrot isolate lost reactivity with the 10D11 MAb and gained reactivity with the P10B8 MAb after passage. The Anhinga isolate gained reactivity with the
P3A11 MAb after passage. These changes may have been a consequence of selection 142 from a mixed viral population during passage. There was no apparent change in other virus properties associated with the MAb binding changes.
The passage procedure by inoculation of infective spleen homogenates intramuscularly used previously in studies with PPMV-1 isolates (Alexander & Parsons,
1984, 1986; Kissi, 1988; King, 1996; Kommers et al., 2001) and recommended in the
NDV vaccine manual (Allan et al., 1978) was successful with only three isolates that were of moderate to greater virulence (Anhinga, Pheasant, and Dove). However, the other three viruses (Ckn-LBM, YN Parrot, and Ckn-Australia), of much low virulence, could not be effectively passaged by this method, as previously described with other low NDV isolates (Kissi, 1988). Although virus could be recovered from spleens of YN Parrot- infected birds, the splenic homogenate virus titer was still too low to infect birds in the subsequent passage. Therefore, with these three isolates, recovered virus from spleens or from cloacal swabs had to be grown in embryonated eggs (Kissi, 1988) to get enough titer to infect next passage birds. This change in the initial passage procedure along with the use of the ED/IN route of inoculation (King, 1996) resulted in successful virus passage. The infection of the birds was confirmed by virus isolation and by the seroconversion detected by the HI test.
These same three viruses (Ckn-LBM, YN Parrot, and Ckn-Australia) had only minor variation in the MDTs, ICPIs, and IVPIs after passage. None of these NDV isolates caused disease by intracloacal inoculation. All three isolates would be characterized as low virulence viruses based on the ICPI results before and after passage, according to the OIE criteria for NDV virulence (Office International des Epizooties,
1999). Additionally, all three isolates have the F protein cleavage site amino acid 143 sequence (residues 112 to 117) typical of low virulence viruses (Gotoh et al., 1992;
Nagai, 1995; Marin et al., 1996; Alexander, 2001).
With the Anhinga isolate, minor variation was observed in the pathotyping tests results before and after passages, but all results remained within the original pathotype.
The ICPI values before and after passage were typical of moderately virulent NDV
(Alexander, 1998). The F protein cleavage site was typical of virulent viruses and identical to the Fontana strain (Seal, 1996), responsible for the California VVND outbreaks in the early 1970’s (Utterback & Schwartz, 1973).
There was virulence increase with the initially highly virulent Pheasant isolate demonstrated by MDT decrease and by ICPI and IVPI increase. The lesions observed during the intracloacal test were equally severe and typical of the VVNDV pathotype before and after passages. The Pheasant and Dove isolates had the same F protein cleavage site amino acid sequence, which was identical to the NVNDV Texas GB strain
(Seal, 1996). Although there was a hypothesis that the Pheasant virus might be epidemiologically related to the Dove isolate (Shivaprasad et al., 1999), sequence analysis revealed that the Dove and Pheasant isolates were different viruses.
The most remarkable change after chicken passage was observed with the Dove isolate. The pathotyping indices obtained with the original virus were typical of moderately virulent NDV (Alexander, 1998). However, after four passages of the Dove isolate, there was virulence increase reaching values of highly virulent NDV (Alexander,
1998) based on MDT, ICPI, IVPI, and intracloacal test results. Several studies have demonstrated slight to moderate virulence increase after sequential passages of some
PPMV-1 isolates in chickens (Alexander & Parsons, 1984; 1986; Kissi, 1988; Kommers 144 et al., 2001). In those studies, the increase in virulence was characterized by changes in the pathotyping tests results, mostly the IVPI, but there were no significant changes in the pathogenicity of the viruses for chickens. The marked virulence and pathogenicity increase observed with the Dove isolate are not typically reported with NDV. This result agrees with the observation that some isolates from non-poultry species may not show their potential virulence for domestic chickens during conventional pathogenicity tests, until passaged several times in chickens (Alexander 1997, 1998).
Clinical signs, gross, or microscopic lesions were not observed with three viruses
(Ckn-LBM, YN Parrot, and Ckn-Australia) during passages 1-4. Viruses of similar low virulence as the Ckn-Australia isolate caused respiratory disease in broilers in Australia
(Hooper et al., 1999), with detectable chronic non-suppurative tracheitis. In those cases, some of the affected tracheal cells were positively stained by an indirect immunoperoxidase test for NDV antigen and virus isolation was made from the tracheas or conjunctivas (Hooper et al., 1999). In chickens infected with the Ckn-Australia as well as with Ckn-LBM and YN Parrot isolates, viral N protein was detected by IHC in the epithelial cells of the conjunctiva and of the oropharyngeal salivary glands in the absence of associated histologic lesions at the sampling time (4 DPI).
In chickens infected with the Anhinga isolate, microscopic lesions were detected mainly in the heart and brain. The number of N protein-positive organs detected by IHC at 4 DPI increased from passage 1 to passage 4. In a previous pathogenesis study with this isolate (original virus inoculum), Brown et al. (1999) observed viral mRNA by in situ hybridization only in the spleen (at 5 DPI) and heart (at 5 and 10 DPI). In our study, there was evidence of viral antigen in eyelids, heart, trachea, comb, and brain. 145
The severity of the lesions was similar in passages 1 and 4 with the Pheasant isolate. Viral N protein was detected by IHC in multiple organs throughout the body.
Similar multisystemic viral distribution has been described with other VVNDV isolates and reference strains (Brown et al., 1999). There was marked preferential viral tropism for the lymphoid organs and lymphoid aggregates associated with the GI tract than to nearby epithelial cells. Neurotropism was also observed in chickens infected with this isolate.
Chickens infected with the Dove isolate had severity of the lesions increase with passages. This isolate produced multisystemic distribution of viral N-protein detected by
IHC with abundant viral tropism for the lymphoid tissues as described for the Pheasant isolate. During this study, the serial passages in chickens resulted in the Dove isolate increasing its pathogenicity as demonstrated by the occurrence of much more severe lesions during passage 4. This was proven increased by the increased overt clinical disease, lesions, and mortality observed during the intracloacal test with the passaged virus. The selection of a more virulent clone among a “quasispecies” population of viral genomes (Hanson et al., 1988; Kissi, 1988; King, 1993, Morimoto et al., 1998), typical of
RNA viruses (Cann, 1997), is a possible mechanism to explain the virulence increase observed with the Dove isolate after passage in chickens.
Several studies have highlighted the importance of the F protein cleavage site as a major determinant of NDV virulence (Glickman et al., 1988; Gotoh et al., 1992; Collins et al., 1993; Nagai, 1995; Peeters et al., 1999). With the Dove isolate, there were no changes in the amino acid motif at the F cleavage site after passage even though virulence increased. When evaluating the molecular basis for PPMV-1 virulence increase, 146
Collins et al. (1994; 1996) concluded that the wide variation in pathogenicity of PPMV-1 isolates for chickens was not related to the variation in the amino acid motif at the F cleavage site nor due to the extension of the HN protein, which may also influence pathogenicity.
All these results suggest that other unknown virulence factors play a role in the marked virulence and pathogenicity diversity among NDV strains that have similar virulent amino acid motifs at the F protein cleavage site. The nature of the specific adaptation, selection, or mutation at other than F cleavage site that occurred with the
Dove isolate after passage remains undefined. The results reported here demonstrate the high risk for domestic chickens represented by some NDV-infected non-poultry species.
ACKNOWLEDGEMENTS
G.D. Kommers was supported by a scholarship from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The work was funded by
U.S. Poultry and Egg Association, Grant 448 and USDA-ARS-CRIS project 6612-32000-
021-00D. The authors acknowledge the excellent technical support of Phillip Curry,
Melissa Scott, and Dr. James Stanton.
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Phylogenetic Evolution, 21, 128-134. Table 5.1. HI test results of NDV isolates against polyclonal antiserum and NDV specific MAbs before (pre) and after (post) passage in chickens. Chicken serum MAbs
Isolates + control - control AVS 10D11 15C4 B79 P15D7 P11C9 P3A11 P10B8 617/161 Changes
Ckn-LBM (pre) + a - b - - - + - - - - -
c Ckn-LBM (post) + - - - - + - - - - - NC
YN Parrot (pre) + - + + + + + + + - -
d YN Parrot (post) + - + - + + + + + + - 2 C
Ckn-Austr (pre) + - + - + - + + + - -
Ckn-Austr (post) + - + - + - + + + - - NC
Anhinga (pre) + - - - + + + - - - -
d Anhinga (post) + - - - + + + - + - - 1 C
Pheasant (pre) + - - - + + + + + + -
Pheasant (post) + - - - + + + + + + - NC
Dove (pre) + - - - + + + + + + -
Dove (post) + - - - + + + + + + - NC a + = antibody-inhibited HA; b - = no HA inhibition; c NC = No changes; d C = Number of changes. AVS reacts with many lentogens including B1 and La Sota strains. 10D11 reacts with neurotropic velogens and mesogens like Roakin strain. 15C4 reacts with most APMV-1 except PPMV-1.
B79 reacts with most APMV-1 including most PPMV-1. P15D7, P11C9, P3A11, and P10B8 identify additional antigenic diversity among APMV-
1. The 617/161 MAbs reacts only with PPMV-1 within the APMV-1 group.
156 157
Table 5.2. Results of the MDT, ICPI, IVPI, and intracloacal test for pre- and post- passage NDV viruses
Isolates MDTa ICPI (0.0 – 2.0) IVPI (0.0 – 3.0) Intracloacal
(pre-/post-passage) (pre-/post-passage) (pre-/post-passage) (pre-/post-passage) Ckn-LBM >168 / >168 0.04 / 0.00 0.00 / 0.00 No dz / No dzb
YN Parrot 91 / 104 0.08 / 0.24 0.00 / 0.00 No dz / No dz
Ckn-Australia 120 / 113 0.00 / 0.05 0.00 / 0.00 No dz / No dz
Anhinga 68 / 76 1.39 / 1.31 0.04 / 0.05 No dz / No dz
Pheasant 62 / 51 1.65 / 1.88 2.31 / 2.58 VVND / VVNDc
Dove 72 / 66 1.20 / 1.60 0.24 / 2.35 No dz / VVND a Time in hours; b No dz, No disease; c VVND, Viscerotropic velogenic Newcastle disease. 158
Table 5.3. Clinical signs and mortality observed in birds inoculated intracloacally with Pheasant and Dove NDV isolates
Pheasant Dove a DPI Pre-passageb Post-passagec Post-passagec, d
1 NCSe slight depression (4/4) slight depression (4/4)
2 NCS depression (4/4) severe depression (4/4)
3 depression, ruffled feathers, severe depression (4/4) severe depression, periocular edema (1/4); ruffled feathers (4/4) less active (3/4)
4 depression (2/4); mortality (4/4) severe depression, less active (2/4) ruffled feathers (4/4)
5 Depresion, periocular severe depression (1/4) edema (4/4); mortality (3/4) comb necrosis (2/4); incoordination (1/4)
6 severe depresion, periocular mortality (1/4) edema (4/4); comb necrosis (1/4); torticollis (1/4)
7 mortality (2/4); severe depression, periocular edema (2/4); torticollis (1/4)
8 to severe depression, 10 periocular edema (2/4); torticollis (1/4) a DPI, Days postinoculation; b Original virus inoculum; c Four times passaged virus inoculum; d No clinical signs or mortality were observed with the pre-passaged Dove virus inoculum; e NCS, No clinical signs. 159
Table 5.4. Fusion protein cleavage site amino acid sequence of NDV isolates.
F cleavage site Virulence
Virus amino acid sequencea - OIE - Pathotypeb
Ckn-LBM 111GGKQGR-LIG119 Low virulence L
YN Parrot 111GGRQGR-LIG119 Low virulence L
Ckn-Australia 111GGRQGR-LIG119 Low virulence L
Anhinga 111GRRQKR-FVG119 Virulent M
Pheasant 111GRRQKR-FIG119 Virulent VV
Dove 111GRRQKR-FIG119 Virulent VV
B1c111GGRQGR-LIG119 Low virulence L
La Sota c111GGRQGR-LIG119 Low virulence L
Kimber c111GRRQKR-FIG119 Virulent M
Pigeon GAd111ERRQKR-FIG119 Virulent M
Texas GB c111GRRQKR-FIG119 Virulent NV
Fontana c111GRRQKR-FVG119 Virulent VV a G = Glycine; R = Arginine; Q = Glutamine; K = Lysine; F = Phenylalanine;
V = Valine; L = Leucine; I = Isoleucine; E = Glutamate (Garret & Grishan, 1999); b L = Lentogenic; M = Mesogenic; NV = Neurotropic velogenic; VV = Viscerotropic velogenic; c From Seal, 1996; d From Kommers et al, 2001 160
Figure 5.1. Serial chicken-passage method performed with the Anhinga, Pheasant, and
Dove NDV isolates.
Figure 5.2. Alternate serial chicken-passage method performed with the Ckn-LBM, YN
Parrot, and Ckn-Australia NDV isolates. 161
Figure 5.3. Chicken, 2 week-old, infected with Pheasant isolate at passage 1. Cecal tonsils. There is severe multifocal hemorrhage mostly at the luminal surface.
Hematoxylin and eosin. Bar = 300 µm.
Figure 5.4. Chicken, 2 week-old, infected with Pheasant isolate at passage 1. Cecal tonsils. Higher magnification of Figure 5.3. showing diffuse severe necrosis of the lymphoid cells. Hematoxylin and eosin. Bar = 60 µm.
Figure 5.5. Chicken, 2 week-old, infected with Dove isolate at passage 1. Bursa. Marked lymphoid depletion of the follicles. Hematoxylin and eosin. Bar = 100 µm.
Figure 5.6. Chicken, 2 week-old, infected with Dove isolate at passage 4. Bursa. Severe lymphoid necrosis of the follicles. Hematoxylin and eosin. Bar = 100 µm.
Figure 5.7. Chicken, 2 week-old, infected with Dove isolate at passage 1. Thymus.
Lymphoid depletion and mild multifocal necrosis affecting mostly the cortex.
Hematoxylin and eosin. Bar = 120 µm.
Figure 5.8. Chicken, 2 week-old, infected with Dove isolate at passage 4. Thymus.
Severe diffuse necrosis associated with interstitial and pericapsular edema. Hematoxylin and eosin. Bar = 100 µm. 162 163
Figure 5.9. Chicken, 2 week-old, infected with Ckn-Australia isolate at passage 4.
Eyelid. A few conjunctival epithelial cells are positive (red-stained) for NDV N protein.
IHC, avidin-biotin-alkaline phosphatase, hematoxylin counterstain. Bar = 60 µm.
Figure 5.10. Chicken, 2 week-old, infected with Ckn-Australia isolate at passage 4.
Salivary glands. N protein-positive epithelial cells (red-stained) are observed. IHC, avidin-biotin-alkaline phosphatase, hematoxylin counterstain. Bar = 60 µm.
Figure 5.11. Chicken, 2 week-old, infected with Pheasant isolate at passage 4. Cecal tonsils. Numerous N protein-positive cells (red-stained) are observed. IHC, avidin-biotin- alkaline phosphatase, hematoxylin counterstain. Bar = 120 µm.
Figure 5.12. Chicken, 2 week-old, infected with Dove isolate at passage 4. Bursa. N protein-positive cells (brown-stained) are observed mostly in the cortex of the follicles.
IHC, avidin-biotin-peroxidase, hematoxylin counterstain. Bar = 60 µm.
Figure 5.13. Chicken, 2 week-old, infected with Dove isolate at passage 4. Spleen.
Numerous N protein-positive cells (brown-stained) are observed. IHC, avidin-biotin- peroxidase, hematoxylin counterstain. Bar = 60 µm.
Figure 5.14. Chicken, 2 week-old, infected with Dove isolate at passage 4. Bone marrow (femur). N protein-positive cells (red-stained) are randomly distributed, IHC, avidin-biotin-alkaline phosphatase, hematoxylin counterstain. Bar = 60 µm. 164 165
Figure 5.15. Intracloacal pathogenicity test in 6-week-old chicken with the Dove isolate.
Bird with severe depression at 2 DPI.
Figure 5.16. Intracloacal pathogenicity test in 6-week-old chicken with the Dove isolate.
There is focal hemorrhage in the sclera and edema and hemorrhage in the eyelid at 5
DPI.
Figure 5.17. Intracloacal pathogenicity test in 6-week-old chicken with the Pheasant isolate. Multifocal hemorrhages in the mucosa of the proventriculus at 4 DPI. Bar = 0.5 cm.
Figure 5.18. Intracloacal pathogenicity test in 6-week-old chicken with the Dove isolate.
Marked subcutaneous neck edema at 5 DPI.
Figure 5.19. Intracloacal pathogenicity test in 6-week-old chicken with the Pheasant isolate. The spleen is enlarged, pale, and mottled (necrosis) at 4 DPI. Bar = 0.5 cm.
Figure 5.20. Intracloacal pathogenicity test in 6-week-old chicken with the Pheasant isolate. Kidneys with urate deposits secondary to severe dehydration at 4 DPI. 166 CHAPTER 6
PATHOGENESIS OF CHICKEN-PASSAGED NEWCASTLE DISEASE VIRUSES
ISOLATED FROM CHICKENS, WILD, AND EXOTIC BIRDS1
1 Kommers, G.D., King, D.J., Seal, B.S. and Brown C.C. 2002. To be submitted to Avian
Diseases. 168
ABSTRACT
The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens
(Ckn-LBM and Ckn-Australia isolates), wild (Anhinga isolate) and exotic (YN Parrot,
Pheasant, and Dove isolate) birds was examined after the isolates had been passaged four times in domestic chickens. Groups of ten four-week-old specific-pathogen-free (SPF)
White Leghorn chickens were inoculated intraconjunctivally with each one of the chicken-passaged isolates. The infected birds were observed for clinical disease and were euthanatized and sampled at selected times from 12 hours to 14 days postinoculation or at the occurrence of mortality. Tissues were examined by histopathology, by immunohistochemistry (IHC) for presence of NDV nucleoprotein (N), by in situ hybridization (ISH) for viral mRNA, and were double labeled for apoptosis and viral N protein. Birds inoculated with the Pheasant and Dove isolates were compared to the reference velogenic viscerotropic Fontana (CA 1083) strain during an early pathogenesis study (from 12 hours to 2 days postinfection [DPI]). Among the six chicken-passaged isolates, birds infected with Pheasant and Dove isolates had the most severe clinical disease, which was characterized by marked depression and mortality at 4 and 5 DPI.
Severe diffuse necrosis of the lymphoid organs and lymphoid aggregates was the main histologic finding in birds inoculated with those two isolates. Although birds inoculated with the Anhinga isolate did not show clinical signs, microscopically there were encephalitis, myocarditis, and tracheitis. Birds inoculated with Ckn-LBM, YN Parrot, and
Ckn-Australia did not become clinically ill, had only mild histologic lesions, and viral N protein detection was restricted to the inoculation site. In chickens infected with the most virulent viruses (Pheasant and Dove isolates), viral N protein and mRNA were detected 169 by IHC and ISH, respectively, among most of the affected organs especially at 2, 4, and 5
DPI. Double-labeled sections of the lymphoid organs revealed the presence of apoptotic cells containing intracytoplasmic viral N protein. The results of this study demonstrate that moderate to highly virulent NDV isolates from wild and exotic birds represent a serious threat for commercial poultry flocks.
Key words: Apoptosis; avian paramyxovirus-1; chickens; double labeling; immunohistochemistry; in situ hybridization; Newcastle disease; pathogenesis; veterinary virology.
INTRODUCTION
Newcastle disease (ND) is one of the most important avian viral diseases because of its economic impact on the poultry industry. Newcastle disease virus (NDV) is synonymous with avian paramyxovirus type 1 (APMV-1; [1,2]). It has been classified in the order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae, genus
Rubulavirus (1,2,21). However, it has been proposed that NDV should have its own genus within the family Paramyxoviridae (22,28,33).
The clinical signs, gross or microscopic lesions observed in birds infected with NDV are not specific for ND. The clinical disease might range from subclinical infection to
100% mortality in a short period of time. Many factors related to the host (species, age, and immune status), virus strain (pathotype, dosage and route of infection), and environmental or social stress can influence the severity and the course of the disease as well as the occurrence and distribution of the lesions (1,2,9). 170
In this study, six NDV isolates recovered from chickens, exotic and wild birds that were previously passaged in 2-week-old White Leghorn (WL) chickens (14) were inoculated in 4-week-old WLs to assess clinical disease and pathogenesis after intraconjunctival inoculation. Infected birds were examined for gross and microscopic lesions. Tissue samples were also evaluated by immunohistochemistry (IHC), in situ hybridization (ISH), and were double labeled for viral antigen (ICH) and for apoptosis
(TUNEL and IHC/caspase-3 assays).
MATERIALS AND METHODS
Eggs and chickens
The source of embryonated chicken eggs and chickens was the Southeast Poultry
Research Laboratory (SEPRL, ARS, USDA) specific-pathogen-free (SPF) White
Leghorn (WL) flock. Embryonated eggs were utilized for virus amplification of chicken- passaged virus. Chickens were inoculated for the pathogenesis study and housed in negative pressure isolators under BSL-3 agriculture conditions at SEPRL and provided feed and water ad libitum (3,12).
Viruses
Six chicken-passaged isolates of NDV were previously characterized by the OIE criteria for virulence as low virulence viruses and virulent viruses (14). The low virulence isolates were: 1) Ckn-LBM (chicken/U.S.(PA)/92-31003/92; intracerebral pathogenicity index (ICPI) = 0.00), a chicken isolate from a live bird market in Pennsylvania, 1992; 2)
Yellow Nape (YN) Parrot (parrot/U.S.(TX)/96-22027/96; ICPI = 0.24), an isolate from a 171 smuggled bird in Texas, 1996; 3) Ckn-Australia (chicken/Australia/98-09-14-1110/98;
ICPI = 0.05), a chicken isolate from Australia, 1998.
The virulent isolates were: 1) Anhinga (anhinga/U.S.(FL)/93-44083/93; ICPI = 1.31), isolate from an anhinga at a commercial marine park in Florida, 1993; 2) Pheasant
(pheasant/U.S./F98-1208/98; ICPI = 1.88), isolated from an exotic pheasant, 1998; (29); and 3) Dove (dove/U.S./98-9248-10/98; ICPI = 1.60), isolated from an exotic dove in quarantine station, 1998. The isolates Ckn-LBM, YN Parrot, Anhinga, and Dove were provided by the USDA, APHIS, National Veterinary Services Laboratories (NVSL),
Ames – IA. The Ckn-Australia isolate was received from Paul Selleck, CSIRO,
Australian Animal Health Laboratory at Geelong, Victoria, Australia.
Pathogenesis experiment
The viruses studied here were passaged four times in chickens as previously described (14). Briefly, three viruses (Anhinga, Pheasant, and Dove) were passaged in 2- week-old SPF WLs by intramuscular inoculation of infective spleen homogenates recovered from the previous passage. The other three viruses (Ckn-LBM, YN Parrot, and
Ckn-Australia) were passaged in 2-week-old SPF WLs using a modified passage procedure. The inoculum was egg-amplified virus recovered from spleens or cloacal swabs from the previous passage and administered by the eyedrop/intranasal (ED/IN) route.
Infective amnioallantoic fluid from the fourth chicken passage was prepared for inoculation into six groups of 10 4-week-old SPF WLs. After dilution in brain-heart
5.0 infusion broth, approximately 10 EID50 was inoculated intraconjunctivally (0.1 ml per 172 bird). One group of 10 4-week-old birds served as noninfected controls. With all six isolates, the disease was followed serially by examining tissues from birds euthanatized
(2/day) at 2, 5, 10, and 14 days postinoculation (DPI) or at the occurrence of mortality.
With the Pheasant and Dove isolates, an early pathogenesis study was performed by intraconjunctival inoculation (0.1 ml per bird) of two groups of 8 4-week-old chickens. Birds were euthanatized (2/day) at 12, 24, and 36 hours postinoculation (HPI) and at 2 DPI. One group of 8 4-week-old birds was infected with the reference velogenic viscerotropic Fontana strain (chicken/U.S./CA1083(Fontana)/71; ICPI = 1.80; [11]) for comparison. One group of 8 4-week-old birds served as noninfected controls.
Necropsies were performed immediately postmortem and the following tissues were collected and fixed by immersion in 10% neutral buffered formalin for approximately 48-52 hours: spleen, thymus, bursa, lower eyelid (including conjunctiva and skin), Harderian gland, turbinates, esophagus, ingluvium, proventriculus, pancreas, small intestine, cecal tonsils, large intestine, caudal thoracic air sac, breast and thigh muscles, comb, trachea, lung, heart, liver, kidney, brain, turbinates (including nasal mucosa), and bone marrow (femur). The sections of femur and turbinates were decalcified in 5% formic acid for 3-4 hours. All sampled tissues were routinely processed into paraffin, and 3 µm sections were cut for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), (ISH), and apoptosis assays.
Virus isolation, titration, and hemagglutination-inhibition (HI) test
Immediately prior to euthanasia, oral and cloacal swabs were obtained from each bird and placed in a tube containing 1.5 ml of brain-heart infusion broth (BHI) with 173 antibiotics (2000 units/ml penicillin G, 200 µg/ml gentamicin sulfate, and 4 µg/ml amphotericin B; Sigma Chemical Co., St. Louis, MO). Swab fluids were centrifuged at
1000 X g for 20 min, and undiluted supernatant was inoculated into 9-to-10-day-old SPF embryonated chicken eggs and incubated for 7 days. Virus infectivity titers of inoculum during the experiments were calculated from the results of inoculation of 9- or 10-day-old embryonated eggs with serial 10-fold dilution in BHI containing antibiotics (100 units penicillin G/ml and 50 µg gentamicin sulfate/ml). NDV-infected dead or surviving embryos were identified by hemagglutination (HA) activity in AAF harvested from chilled eggs. NDV was confirmed in HA-positive samples by the HI test with NDV- specific antiserum (10). At 10 and 14 DPI, birds were bled immediately prior to euthanasia. The HI test to detect seroconversion was conducted by conventional microtiter methods (10).
Immunohistochemistry
All sampled tissues were examined by IHC using the following protocol to detect viral nucleoprotein (IHC/NP). After deparaffinization, tissue sections were subjected to antigen retrieval by microwaving for 10 minutes at full power in Vector antigen unmasking solution (Vector Laboratories, Burlingame, CA) followed by blocking with universal blocking reagent (Biogenex, San Ramon, CA) as recommended by the manufacturer. Incubation with the primary antibody, an anti-peptide antibody made in rabbit (12) and used at 1:8,000 dilution, was overnight at 4 C or for 2 hr at 37 C. After washing, sections were incubated with biotinylated antibody against the species in which the primary antibody was made and then with avidin-biotin alkaline phosphatase (Vector 174
Laboratories). Substrate was Vector Red (Vector Laboratories). Sections were counterstained lightly with hematoxylin and coverslipped with Permount for a permanent record.
Double labeling for detection of apoptosis and viral protein
The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; [24]) assay (Boehringer Manheim, Indianapolis, IN) was utilized to detect apoptosis in sections of lymphoid organs (spleen, bursa, and thymus) of Fontana-,
Pheasant-, and Dove-infected birds. The tissue sections were microwaved for 10 minutes at full power in Vector antigen unmasking solution (Vector Laboratories). After washing, the sections were incubated with TUNEL reaction mixture for 1 hr at 37 C. Converter-AP was added to the slides for 30 min at 37 C. The development was with chromogen/substrate nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3- indoylphosphate (BCIP). After performing the TUNEL assay, the tissue sections were washed and stained by the IHC/NP protocol as described above, starting at the blocking step. Sections were counterstained lightly with hematoxylin and coverslipped for a permanent record.
Sections of the spleen were also double labeled using two sequential rounds of
IHC to detect apoptotic cells (IHC/caspase-3) and viral N protein (IHC/NP). In the first round of IHC, deparaffinized spleen sections were treated with 3% hydrogen peroxide and then subjected to antigen retrieval followed by blocking as described in the IHC/NP protocol. Incubation with the primary antibody, a rabbit polyclonal anti-active caspase-3 antibody (Promega, Madison, WI) at 1:350 dilution (13), was for 2 hours at 37 C. After 175 washing, sections were incubated with biotinylated antibody against the species in which the primary antibody was made and then with elite-PO (Vector Laboratories). Substrate was diaminobenzidine (DAB) (Vector Laboratories). After performing the IHC/caspase-
3 protocol, tissue section were washed and a second round of IHC was performed using the IHC/NP protocol as described above, starting at the incubation with the primary antibody. Sections were counterstained lightly with hematoxylin and coverslipped with
Permount for a permanent record.
In situ hybridization
Selected tissue sections of birds infected with three of the virulent viruses
(Pheasant, Dove, and Fontana) were stained with a negative-sense digoxigenin-labeled
850-base riboprobe representing the 5’ end of the matrix gene of NDV Fontana (CA
1083) strain as previously described (3,12,13). The matrix gene from the Fontana strain was cloned into pCRII transcription vectors (Invitrogen, Carlsbad, CA). Anti-sense digoxigenin-labeled riboprobes were generated using RNA polymerase in the presence of labeled nucleotides. For in situ hybridization, tissue sections were deparaffinized, rehydrated, and digested with 30 µg/ml Proteinase K for 15 minutes at 37 C.
Hybridization was conducted overnight at 42 C with approximately 20 ng of probe in pre- hybridization solution. After stringent washes, anti-digoxigenin alkaline phosphatase was added to the sections. The development was with chromogen/substrate NBT and BCIP.
Tissues were counterstained lightly with hematoxylin and coverslipped. 176
RESULTS
Clinical signs
Low virulence viruses - Neither clinical signs nor mortality were observed in chickens inoculated with the Ckn-LBM, YN Parrot, and Ckn-Australia isolates. Birds inoculated with these three isolates were euthanatized and sampled as scheduled (at 2, 5,
10, and 14 DPI). The infection of all inoculated birds was confirmed by virus isolation or by seroconversion (HI test).
Virulent viruses – Neither clinical signs nor mortality were observed in the
Anhinga-infected birds and they were euthanatized and sampled as scheduled (at 2, 5, 10, and 14 DPI). In the early pathogenesis study, clinical signs were not observed in birds infected with Pheasant and Dove isolates during the first 36 HPI. A few Fontana-infected birds had ruffled feathers at 36 HPI and some birds had ruffled feathers and hunched posture at 2 DPI. With the Pheasant isolate, at 2 DPI all birds were slightly depressed and a few had head twitching. At 3 DPI, all birds were depressed, with diarrhea, and four birds had reddened and swollen conjunctiva. All the remaining birds were found dead at
4 DPI. With the Dove isolate, all birds were slightly depressed with ruffled feathers at 3
DPI. At 4 DPI, all birds were very depressed. Five birds were found dead at 5 DPI. One remaining bird was very depressed. Clinical signs were not observed in the noninfected controls. The infection of all infected birds was confirmed by virus isolation or by seroconversion. 177
Gross Findings
Low virulence viruses - At 2 DPI, the infected birds had slightly enlarged spleens
(Ckn-LBM isolate), eyelid petechiation (YN Parrot isolate), and very large dark spleens
(Ckn-Australia isolate). At 5 DPI, there were slight reddening of the eyelids (Ckn-
Australia isolate), mild pulmonary edema (Ckn-LBM, YN Parrot, and Ckn-Australia isolates) and slight petechiation in the thymus (YN Parrot isolates). At 10 DPI and 14
DPI, a few petechiae was observed in the eyelids of all infected birds.
Virulent viruses – At 2 DPI, Anhinga-infected chickens had petechiae in the eyelids, multifocal hemorrhages on the thigh muscles, petechiae in the thymus, and the spleens were small. At 5 DPI, there were slightly enlarged spleens, pulmonary edema, and hemorrhages on the thigh muscles. Edema and petechiae in the eyelids and crusty dark spots on the comb tips were observed at 10 DPI. At 14 DPI, a few birds had petechiae in the eyelids.
Slight reddening and a few petechiae were observed at 12 HPI in birds infected with
Pheasant, Dove, and Fontana isolates. At 24 HPI, there were foci of reddening at the cecal tonsils (Pheasant and Fontana isolates) and very fluid intestinal contents (Fontana isolate). At 36 HPI, there were petechiae in the eyelids (Pheasant isolate), dark red foci at the serosal surface of the cecal tonsils (Pheasant, Dove, and Fontana isolates), very fluid intestinal contents (Dove and Fontana isolates), and large spleens (Pheasant isolate).
At 2 DPI, there were petechiae and edema in the eyelids, large mottled spleens
(Fontana-, Dove-, and Pheasant- infected birds), fluid intestinal contents (Fontana- infected birds), white foci on the serosal surface of the cecal tonsils (Pheasant- infected birds), and bone marrow pallor (Dove-infected birds). 178
Moderate to severe widespread gross lesions were observed in the chickens infected with both Pheasant (4 DPI) and Dove (4 and 5 DPI) isolates. The gross findings were eyelid edema and reddening, dehydration, muscle wasting, subcutaneous neck edema, pale/mottled spleens, small/edematous thymus, very small bursa, diffusely pale bone marrow, and edematous pancreas. Petechial hemorrhages, mostly at the opening of the mucosal glands, was a very common finding in the proventriculus. Other findings were the gastrointestinal (GI) tract devoid of contents, white foci in the serosa of the ceca, petechiation in the cloacal mucosa, and reticulate kidneys (interpreted to be urate deposits).
Histopathology
Low virulence viruses – Microscopic lesions were first detected at 10 and 14 DPI in Ckn-
LBM-infected birds consisting of very mild pulmonary edema. In YN Parrot-infected birds, mild to moderate lymphoplasmacytic infiltrates were observed in the eyelids at 2,
5, and 10 DPI. There were mild lymphoplasmacytic infiltrates in the lamina propria of the trachea and mild to severe lymphoplasmacytic infiltrates with fewer macrophages in the caudal thoracic air sacs at 10 (Fig. 6.1) and 14 DPI. Australia-infected birds had lymphoplasmacytic conjunctivitis (2, 5, and 10 DPI). In the trachea, there were mucous gland hyperplasia (5, 10, 14 DPI) and epithelial cell hyperplasia (14 DPI). Infiltrates of lymphocytes, plasma cells, and fewer macrophages were observed in the lamina propria
(5 and 14 DPI), sometimes extending into the epithelium (10 DPI).
Virulent viruses – With the Anhinga isolate, there was mild lymphoplasmacytic encephalitis at 2 and 5 DPI. Severe brain lesions were detected at 10 DPI consisting of 179
Purkinje cell necrosis, neuron necrosis with neuronophagia, gliosis, and perivascular cuffing in the cerebellar molecular layer (Fig. 6.2), cerebrum, and brain stem. There was multifocal myocardial necrosis associated with mononuclear infiltrates at 5, 10, and 14
DPI. Mild lymphoplasmacytic tracheitis was observed at 5 and 10 DPI. Epithelial degeneration, necrosis, and heterophilic inflammation was observed in the comb at 5 and
10 DPI.
Microscopic lesions were not observed in birds inoculated with the Fontana strain at 12 HPI. At 24 HPI, there was mild edema and hemorrhage in the nasal mucosa and mild foci of hemorrhage in the cecal tonsils. At 36 HPI, increased apoptosis was observed in the conjunctiva-associated lymphoid tissue (CALT) and in the cortex of the thymus and bursal follicles. There were multifocal hemorrhages and slight lymphoid depletion in the cecal tonsils, pulmonary congestion and edema, and lymphoplasmacytic infiltrates in the nasal mucosa, larynx, and air sacs. At 2 DPI, there was severe eyelid edema and conjunctivitis, apoptosis and small foci of necrosis in the splenic ellipsoids, mild lymphoid depletion and necrosis in the gut-associated lymphoid tissues (GALT) and cecal tonsils, and foci of necrosis in the bone marrow.
Microscopic lesions were first observed at 36 HPI in Pheasant-infected birds and were characterized by edema, hemorrhagic foci, and lymphoplasmacytic infiltrates in the eyelids, mucus accumulation in the nasal cavity, and mild lymphoid depletion in the cecal tonsils. At 2 DPI, there was increased apoptosis, depletion, and lymphocellular necrosis in the CALT, conjunctival epithelial necrosis and mixed inflammatory reaction. Increased apoptosis and lymphoid depletion were observed in the bursal follicles (cortex), thymus
(cortex), and pancreas (lymphoid aggregates). Apoptosis and lymphocellular necrosis 180 were more prominent in the spleen (ellipsoids; Fig. 6.3), thymus (medulla), small and large intestines (GALT), and cecal tonsils. There was moderate hyperplasia of the mucous glands with mucous accumulation in the nasal cavity. Epithelial necrosis and lymphoplasmacytic infiltrates with fewer heterophils were observed in the larynx. Mild lymphoplasmacytic tracheitis, focal gliosis in the cerebellar molecular layer, and necrosis of the bone marrow lymphoid series were also observed.
At 4 DPI, the most severe lesions were diffuse necrosis of all the lymphoid tissues, associated with fibrin deposits and heterophils. There was severe conjunctivitis, occasional necrosis of the proventricular glands, and multifocal hemorrhages in the lamina propria of the proventriculus and of the intestines. Severe multifocal necrosis affecting both myeloid and lymphoid series was observed in the bone marrow (Fig. 6.4).
Less frequent lesions were individual cell necrosis in the pancreas, piecemeal necrosis and mononuclear infiltrates in hepatic portal areas, cardiac myofiber disruption/necrosis and lymphocytic infiltrates, focal gliosis in the cerebellar molecular layer, and small areas of epithelial necrosis and pustules in the comb.
Microscopic lesions were first observed at 24 HPI with the Dove isolate. There was mild conjunctivitis, small foci of hemorrhage in the nasal mucosa, and pulmonary congestion. At 36 HPI, there were small foci of necrosis and apoptotic cells in the splenic ellipsoids. Apoptotic cells were also observed in the cortex of the thymus and bursal follicles, GALT, and cecal tonsils. There was lymphoplasmacytic conjunctivitis, laryngitis, and tracheitis. At 2 DPI, there was increased apoptosis in the spleen (germinal centers and ellipsoids), bursa (cortex follicular), thymus (cortex), and cecal tonsils 181
(germinal centers). Lymphoplasmacytic conjunctivitis, tracheitis, and rhinitis (with epithelial necrosis) were also observed.
At 4 and 5 DPI, there were widespread lesions and the most remarkable ones were found in the spleen (severe diffuse necrosis), bursa (lymphoid depletion and necrosis of follicles; Fig. 6.5), thymus (severe diffuse necrosis; Fig. 6.6), GALT and cecal tonsils (diffuse necrosis), comb (epithelial necrosis and pustules), and bone marrow
(severe diffuse necrosis). Necrosis of osteoclasts was occasionally observed in sections of femur. Microscopic lesions were not found in the noninfected controls.
IHC/NP
Low virulence viruses – There were a few IHC/NP-positive epithelial cells in the conjunctiva at 2 (with YN Parrot and Ckn-Australia isolates) and 5 DPI (with Ckn-LBM,
YN Parrot, and Ckn-Australia isolates; Fig. 6.7).
Virulent viruses – With the Anhinga isolate, IHC/NP positive staining was only detected at 5 DPI in the cardiac myofibers and in the comb epithelium. Detectable amounts of viral N protein were not observed in the sampled tissues at 12, 24 (with
Fontana, Dove, and Pheasant isolates), and 36 HPI (with Pheasant isolate). With the
Fontana strain, one bird had a few IHC-positive cells in the lymphoid aggregates of the eyelids and of the nasal mucosa at 36 HPI. A few weakly positive cells were observed in the nasal epithelium in one Dove-infected bird at 36 HPI. In chickens infected with
Pheasant and Dove isolates, the most striking staining was observed at 2 (Fig. 6.8), 4, and
5 DPI, where there was extensive staining in multiple organs. The lymphoid organs and lymphoid aggregates (mostly GALT) were the tissues with the largest amounts of viral N 182 protein at 4 and 5 DPI. The IHC results for 2 (Pheasant, Dove, and Fontana isolates), 4
(Pheasant, and Dove isolates), and 5 DPI (Dove isolate) are summarized in Table 6.1.
The tissues of the noninfected controls were negative by the IHC/NP assay.
Double labeling
The double-labeled sections of the lymphoid organs of Fontana-, Pheasant-, and
Dove-infected birds stained with the combined TUNEL-IHC/NP assays revealed similar results for all three isolates. In sections of the spleen, slightly to moderately increased numbers of apoptotic cells (TUNEL-positive) were observed mostly surrounding the penicilliform capillaries in the ellipsoids at 2 DPI. Most of the TUNEL-positive cells were negative for viral N protein at that time, but where located in the neighborhood of
IHC/NP-positive cells (Fig. 6.9). A few double-labeled cells, characterized by black- stained nuclei (TUNEL-positive) and red-stained cytoplasm (IHC/NP-positive), were seen mostly in the ellipsoids at 2 DPI (Fig. 6.10). At 4 and 5 DPI, numerous widespread splenic cells were either positive by IHC/NP or by the TUNEL reaction. Double-labeled cells were more often seen and were randomly distributed at that time. In the noninfected controls, rare randomly distributed TUNEL-positive cells were observed in the spleen sections and all cells were negative for viral N protein.
In sections of the bursa at 2 DPI, slightly increased numbers of TUNEL-positive cells were observed mostly in the cortex of the follicles. Scarce N-protein-positive cells were observed mostly in the medulla at this time. In later infection (4 and 5 DPI), the number of apoptotic cells was remarkably increased especially in some follicles that were also strongly positive for viral N protein than in others (Fig. 6.11). Double-labeled cells 183 were occasionally observed. In the noninfected controls, a small number of TUNEL- positive cells were detected mostly in the cortex follicular and all cells were negative for viral N protein.
In sections of the thymus at 2 DPI, slightly increased numbers of TUNEL-positive cells were observed in the cortex. Viral N protein was detected in a few medullary cells at
2 DPI. There were apoptotic cells in the vicinity of the N protein-positive cells. In some birds, the presence of TUNEL-positive and N protein-positive cells was more striking in some thymic lobes than in others after 2 DPI. Scattered TUNEL-positive cells were observed mostly in the cortex of noninfected controls. All cells were negative for viral N protein in the controls.
With the combined IHC/caspase-3 and IHC/NP in spleen sections, increased number of apoptotic cells were observed in the ellipsoids at 2 DPI and were located close to N protein-positive cells (red-stained cytoplasm). The morphology (dendrite-shaped) and location of several IHC/caspase-3-positive cells were compatible with the ellipsoid- associated cells (EACs). Double-labeled cells were hardly seen by this method. Caspase-
3-positive cells were seen in small numbers and randomly distributed in the spleen sections of the noninfected controls. Viral N protein was not detected in the controls.
ISH
The selected tissue sections of birds infected Fontana, Pheasant, and Dove isolates stained by ISH confirmed the distribution of the viral infection detected by IHC/NP.
More striking positive staining was observed at 2, 4 (Fig. 6.12), and 5 DPI. The tissues of the noninfected controls were negative by the ISH assay. 184
DISCUSSION
Birds infected with three chicken-passaged low virulence viruses (Ckn-LBM, YN
Parrot, and Ckn-Australia) did not develop clinical disease after intraconjunctival inoculation. Histologic lesions were observed in the inoculation site (conjunctiva) and in organs of the respiratory system. More prominent lesions (tracheitis and airsacculitis) were observed late after infection (at 10 and 14 DPI). The detection of viral nucleoprotein
(N) in affected organs was restricted to the inoculation site. It is possible that the virus load in the infected cells of other affected tissues was too low to be detected by IHC.
Previous studies have demonstrated minimal positive staining detected by ISH with two low virulence reference strains (B1 and QV4; [3]).
Among the virulent viruses, the Anhinga isolate showed moderate virulence for chickens after intraconjunctival inoculation. Clinical signs were not observed. However, microscopically there was encephalitis, myocardial necrosis/inflammation, tracheitis, and comb necrosis. By IHC, viral N protein could only be detected in the heart and comb.
The absence of detectable viral N protein in the brain might be because the most remarkable brain lesions occurred late (at 10 and 14 DPI) when NDV is rarely detected
(3,34). Brown et al (3), working with the original Anhinga isolate inoculated intraconjunctivally in chickens, detected viral mRNA by ISH only in the spleen (5DPI) and heart (5 and 10 DPI). However, during a passage study (14) with this virus, viral N protein was detected in the eyelids, heart, trachea, comb, and brain of chickens inoculated intramuscularly. The route of inoculation might have influenced the sites of viral replication as previously suggested (3). 185
The chicken-passaged Pheasant and Dove isolates were highly virulent for chickens inoculated by the intraconjunctival route. The early pathogenesis of these two viruses for chickens was compared to the highly virulent Fontana strain, a psittacine- origin virus which caused the California viscerotropic velogenic ND outbreaks in chickens during the early 1970’s (32). For this study, the infected birds were euthanatized and sampled at 12 hours interval until 2 DPI. The Fontana-infected birds started presenting clinical signs earlier (36 DPI) than the Pheasant- and Dove-infected birds (2 and 3 DPI, respectively). With all three highly virulent viruses, mild gross lesions were confined to the inoculation site (slight reddening in the eyelids) at 12 HPI. Microscopic lesions were first observed at 24 HPI consisting mostly of circulatory changes
(congestion, edema, and hemorrhages) in the eyelids and cecal tonsils. Apoptosis, depletion, and small foci of necrosis affecting lymphoid organs and aggregates were first detected at 36 HPI in birds infected with all three highly virulent viruses.
Viral N protein and mRNA were first detected (at 36 HPI) in large mononuclear cells in the conjunctiva-associated lymphoid tissue (CALT) and in lymphoid aggregates of the nasal turbinates of Fontana- and Dove-infected birds. It seems that the next areas infected after intraconjunctival inoculation of these highly virulent NDV isolates were the lymphoid aggregates of the nasal turbinates. This result might suggest the involvement of macrophage-type cells in the replication and dissemination of NDV. Brown et al (3) reported evidence of extensive viral replication within macrophages with subsequent spread to many organs (mostly lymphoid tissues) in birds infected with the viscerotropic velogenic pathotype. An in vitro study reported that chicken macrophages support the 186 growth and replication of NDV. Intact virus particles were also observed by electron microscopy in macrophages exhibiting features of apoptosis (17).
Based on the number of organs positive for viral N protein or mRNA, detected by
IHC and ISH respectively, the Pheasant isolate spread more rapidly throughout body tissues than the Fontana and Dove isolates at 2 DPI. After 2 DPI, birds infected with
Pheasant and Dove isolates were severely ill and mortality occurred at 4 and 5 DPI, respectively. At this time, disseminated gross and microscopic lesions were observed mostly affecting the lymphoid tissues as previously described with other highly virulent
NDV isolates (3,12,15,25). The tropism for lymphoid organs and aggregates in this study was confirmed by the presence of large numbers of cells positive for viral N protein and viral mRNA, detected by IHC and ISH respectively. The presence of large amounts of viral antigen and mRNA in areas where the vast majority of the cells are lymphocytes suggests that these cells are also permissive to viral replication in vivo, as reported in vitro (18).
A prominent finding in our study was the presence of large amounts of IHC/NP- positive and mRNA-positive cells in the bone marrow (femur) of birds infected with the
Pheasant (4 DPI) and Dove (5 DPI) isolates. However, the presence of infected cells in the skeletal muscles examined (breast and thigh muscles) was negligible. These findings are important when considering trade of fresh poultry meat from countries where ND has been observed in commercial poultry.
Although the ability of NDV to induce apoptosis and lysis of several types of neoplastic cells has been the focus of many cancer therapy studies (4,5,6,23,30), there is a relatively limited number of studies reporting the importance of NDV-induced apoptosis 187 in avian infected cells or tissues (16-20,13,27). In this experiment, an increased number of apoptotic cells was observed microscopically (HE stained sections) in the splenic ellipsoids mostly at 2 DPI. The splenic ellipsoids and especially the ellipsoid-associated cells (EACs; antigen-presenting cells) are the first localization of several different kinds of antigens, including infectious agents, in the spleen of chickens (7). The apoptosis assays (TUNEL and IHC/caspase-3) confirmed the increased number of apoptotic cells in the ellipsoids at 2 DPI comparing to the noninfected controls. Several IHC/caspase-3- positive apoptotic cells had the morphology of the EACs, as previously described with other APMV-1 and PPMV-1 isolates (13). Viral N protein was also detected in the cytoplasm of cells surrounding the penicilliform capillaries (center of the ellipsoids).
Most of the apoptotic cells were negative for viral N protein at 2 DPI and were located in the neighborhood of N protein-positive cells. Double-labeled cells were better visualized using the combined TUNEL-IHC/NP than IHC/caspase3-NP and were more often seen at
4 and 5 DPI.
These results along with the increased numbers of apoptotic cells, especially in the bursal follicles or thymic lobes where N protein was more abundantly detected, confirm that apoptosis is an important mechanism in lymphoid depletion during NDV infection. Similar results were reported with other avian viral diseases including highly pathogenic avian influenza (HPAI; [26]) and infectious bursal disease virus (IBDV) in chickens (8,31). NDV-induced apoptosis seems to be caused by either direct or indirect effect of viral infection. The simultaneous positive staining for apoptosis and viral N protein demonstrated in this study clearly illustrates the direct effect of the NDV infection causing apoptosis of the infected cells. The indirect effect is possibly related to 188 the release of cytokines (enrolled in the induction of apoptosis) by the infected cells, as previously suggested for NDV (30) and for other avian viral agents (8,26,31).
ACKNOWLEDGEMENTS
G.D. Kommers was supported by a scholarship from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The work was funded by
U.S. Poultry and Egg Association, Grant 448 and USDA-ARS-CRIS project 6612-32000-
021-00D. The authors acknowledge the excellent technical support of Phillip Curry,
Melissa Scott, and Dr. James Stanton.
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B Affected tissues Pheasant Dove Fontana 2 DPI 4 DPI 2 DPI 4 DPI 5 DPI 2 DPI Comb - + - + + - C Eyelid + +++ + ++ ++ + Harderian gland + - - - + - Bursa + +++ - + ++ - Cecal tonsils ++ +++ + +++ +++ + Spleen + +++ + ++ ++ + Thymus + +++ + +++ +++ + Bone marrow + +++ + +++ +++ - Tongue - - - - + - Salivary gland + + - + + - Crop + ++ - + + - Esophagus - - - - + - Proventriculus + ++ + ++ ++ - Intestines ++ ++ + +++ +++ - Pancreas - - - - + - Kidney + ++ - + +++ - D C C Nasal mucosa - NS + NS NS + Larynx ++ +++ + ++ - - Trachea - + - + + - Air sac - - - - + - Lung + ++ - + ++ - Heart - + - - + - Brain + + - + + - A - = negative for NDV nucleoprotein (N); + = small amounts of viral N detected; ++ = moderate amounts of viral N detected; +++ = large amounts of viral N detected.; B = all the infected birds were dead by 4 DPI; C Also positive at 36 HPI; D NS = Not sampled.
195 196
Figure 6.1. There are multifocal lymphoplasmacytic infiltrates with fewer macrophages in the air sac of a chicken infected with the YN Parrot isolate at 10 days postinfection.
Hematoxylin and eosin. Bar = 60 µm.
Figure 6.2. There are multifocal perivascular cuffing and gliosis in the cerebellar molecular layer of a chicken infected with the anhinga isolate at 10 days postinfection.
Hematoxylin and eosin. Bar = 100 µm.
Figure 6.3. Apoptotic cells and lymphoid cell depletion are observed in the splenic ellipsoids of a chicken infected with the Pheasant isolate at 2 days postinfection.
Hematoxylin and eosin. Bar = 40 µm.
Figure 6.4. There is severe necrosis affecting both myeloid and lymphoid series in the bone marrow (femur) of a chicken infected with the Pheasant isolate at 4 days postinfection. Hematoxylin and eosin. Bar = 60 µm.
Figure 6.5. Lymphoid depletion and lymphocellular necrosis are observed in the bursa of a chicken infected with the Dove isolate at 4 days postinfection. Hematoxylin and eosin.
Bar = 90 µm.
Figure 6.6. There is severe diffuse necrosis of the thymus of a chicken infected with the
Dove isolate at 5 days postinfection. Hematoxylin and eosin. Bar = 90 µm. 197 198
Figure 6.7. Detection of viral nucleoprotein in the cytoplasm (arrows) of conjunctival epithelial cells of a chicken infected with Ckn-Australia isolate at 5 days postinfection.
Immunohistochemistry, avidin-biotin-peroxidase, hematoxylin counterstain. Bar = 60
µm.
Figure 6.8. Viral nucleoprotein-positive cells (red-stained) are observed in the splenic ellipsoids of a chicken infected with the Dove isolate at 2 days postinfection.
Immunohistochemistry, avidin-biotin-alkaline phosphatase, hematoxylin counterstain.
Bar = 40 µm.
Figure 6.9. TUNEL-positive cells (dark-stained nuclei) and nucleoprotein-positive cells are observed in the splenic ellipsoids of a chicken infected with the Pheasant isolate at 2 days postinfection. Double labeling, TUNEL assay and immunohistochemistry (avidin- biotin-alkaline phosphatase), hematoxylin counterstain. Bar = 40 µm.
Figure 6.10. Double-labeled cell with a red-stained cytoplasm (nucleoprotein-positive; arrowhead) and an apoptotic dark-stained nucleus (TUNEL-positive; arrows) in the splenic ellipsoids of a chicken infected with the Pheasant isolate at 2 days postinfection.
Double labeling, TUNEL assay and immunohistochemistry (avidin-biotin-alkaline phosphatase), hematoxylin counterstain. Bar = 10 µm. 199
Figure 6.11. There is a large number of apoptotic cells (dark-stained nuclei) in a bursal follicle where viral nucleoprotein-positive cells (red-stained cytoplasm) are also observed. Chicken infected with the Dove isolate at 4 days postinfection. Double labeling, TUNEL assay and immunohistochemistry (avidin-biotin-alkaline phosphatase), hematoxylin counterstain. Bar = 90 µm.
Figure 6.12. Numerous cells (dark-stained) are positive for viral mRNA in the bone marrow (femur) of a chicken infected with the Pheasant isolate at 4 days postinfection. In situ hybridization, hematoxylin counterstain. Bar = 60 µm. 200 CHAPTER 7
CONCLUSIONS
Several biological and molecular assays were performed to investigate the effect of serial passages in domestic chickens and the potential threat represented by twelve NDV isolates recovered from pigeons (six isolates), chickens (two isolates), wild (one isolate), and exotic (three isolates) birds.
With the pigeon-origin NDV isolates, a monoclonal antibody (MAb) panel revealed that four of them (Pigeon TX, Pigeon GA, 84-44407, and Pigeon 84) were the variant pigeon paramyxovirus-1 (PPMV-1) and two of them (P1307 and P5658) were avian paramyxovirus-1 (APMV-1) isolates. Pathotyping tests results performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. However, the PPMV-1 were still of moderate virulence (mesogenic pathotype) for chickens after passages. The fusion protein cleavage site amino acid sequences of all six pigeon-origin (PPMV-1 and APMV-
1) isolates were typical of virulent NDVs. Although the pathotyping indices indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The two pigeon-origin 202
APMV-1 isolates when inoculated into chickens caused severe lesions, characteristic of the viscerotropic velogenic NDV (VVNDV) pathotype.
All six heterogeneous origin-isolates were characterized as viruses of the APMV-1 serotype. Antigenic variation among these six APMV-1 isolates was evident in the results of the HI test with MAbs. The passage procedure by inoculation of infective spleen homogenates intramuscularly was successful only with three isolates – those of moderate to greater virulence (Anhinga, Pheasant, and Dove). However, the other three viruses
(Ckn-LBM, YN Parrot, and Ckn-Australia), of much lower virulence, could not be effectively passaged by this method. Therefore, with these three isolates, recovered virus from spleens or from cloacal swabs had to be grown in embryonated eggs to get enough titer to infect next passage birds. This change in the initial passage procedure along with the use of the eyedrop/intranasal route of inoculation resulted in successful virus passage.
Three viruses (Ckn-LBM, YN Parrot, and Ckn-Australia) had only minor variation in the pathotyping indices after passage. All three isolates would be characterized as low virulence viruses based on the Office International des Epizooties (OIE) criteria for virulence (ICPI results and the F protein cleavage site amino acid sequence).
With the Anhinga isolate, minor variation was observed in the pathotyping indices before and after passages, but all results remained within the original mesogenic pathotype. The ICPI values before and after passage were typical of moderately virulent
NDV and the F protein cleavage site was typical of virulent viruses.
There was virulence increase with the initially highly virulent Pheasant isolate demonstrated by the pathotyping indices before and after passage. The lesions observed 203 during the intracloacal test were typical of the VVNDV pathotype before and after passages. The F protein cleavage site amino acid sequence was typical of virulent viruses.
The most remarkable change after chicken passage was observed with the Dove isolate. The pathotyping indices obtained with the original virus were typical of moderately virulent NDV. However, after four passages, there was virulence increase reaching values of highly virulent NDV. There were no changes in the amino acid motif at the F cleavage site after passage. The marked virulence and pathogenicity increase observed with the Dove isolate are not often reported with NDV.
The nature of the specific adaptation, selection, or mutation at other than F cleavage site that occurred with the Dove isolate after passage remains undefined. All these results suggest that other unknown virulence factors play a role in the marked virulence and pathogenicity diversity among NDV strains that have similar virulent amino acid motifs at the F protein cleavage site.
The pathogenesis studies performed with all twelve NDV isolates revealed important aspects in the pathology of NDV. The highly virulent viruses, including P1307,
P5658, Pheasant, and Dove isolates, showed marked tropism for lymphoid organs and lymphoid aggregates. The largest amounts of viral nucleoprotein and viral mRNA were detected in the lymphoid tissues by IHC and ISH, respectively. Apoptosis and necrosis were the major viral-induced mechanisms for lymphocellular depletion. Moderately virulent viruses, including all four PPMV-1 isolates and the Anhinga isolate, showed marked neurotropism and degenerative/inflammatory lesions were consistently found in the heart of the infected birds. The low virulence viruses (Ckn-LBM, YN Parrot, and 204
Ckn-Australia) caused mild lesions that were limited to the site of inoculation and to organs of the respiratory system as described with most lentogenic viruses.
As stated in the introduction, the difference in the Newcastle disease (ND) definition between the OIE and United States Code of Federal Regulations (US-CFR) raises several questions. The results of this investigation may provide some answers for these questions but not all of them.
For the question – What is the threat to poultry represented by isolates that are classified as virulent by OIE but are not by the US-CFR? – the answer is not simple. All moderately virulent viruses, including the PPMV-1 isolates and the Anhinga isolate, would be classified by OIE as virulent viruses. However, the disease caused by these viruses would not fit the US-CFR definition of ND. Although mortality was not a major finding in the PPMV-1-infected or Anhinga-infected birds inoculated intraconjunctivally, the brain lesions observed probably would be severe enough to cause significant neurologic deficit and affect normal development of commercial chickens. Additionally, the lesions observed in the heart probably would affect bird performance, including weight gain and growth rate among commercial chickens infected with pigeon NDV.
For the second question – Could passage of these isolates in chickens result in increased virulence that justifies using a more severe standard than the US-CFR? – the answer would be yes mostly for isolates like the Dove isolate. For still undetermined reasons this isolate had virulence increase after passage in chickens reaching pathotyping indices of VVNDV isolates. The disease caused by this virus before passage in chickens would not fit the US-CFR criteria for virulence. However, the disease caused by the Dove 205 isolate after passage would fit the definition of “exotic Newcastle disease” stated in the
US-CFR.
The answer for the third question – What is the risk of trade sanctions if the USA has to adopt the OIE standards? – depends on several factors, especially in the establishment of epidemiological association between the source of the isolated virus and commercial poultry flocks. It is still a critical issue for poultry exporting countries like the USA that the OIE International Animal Health Code has not been revised to provide criteria for applying the new ND definition for the purposes of international trade.
In conclusion, the hypothesis of virulence increase after serial passages in chickens was confirmed only with some of the NDV isolates examined. The fact that clinical disease was evident in intraconjunctivally inoculated chickens and the similarity of the
PPMV-1 viruses by nucleotide sequence analysis to more virulent viruses is evidence that the pigeon viruses are a potential hazard to chickens. Pigeons must be considered as a potential source of NDV infection and disease for commercial poultry flocks. The results reported here with the highly virulent viruses demonstrate the high risk for domestic chickens represented by some NDV-infected non-poultry species.