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Importance of a Siderophore in the Pathogenesis and Virulence of Photobacterium Damsela Subsp Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1996 Importance of a Siderophore in the Pathogenesis and Virulence of Photobacterium Damsela Subsp. Piscicida in Hybrid Striped Bass (Morone Saxatili X Morone Chrysops). John Phillip Hawke Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Hawke, John Phillip, "Importance of a Siderophore in the Pathogenesis and Virulence of Photobacterium Damsela Subsp. Piscicida in Hybrid Striped Bass (Morone Saxatili X Morone Chrysops)." (1996). LSU Historical Dissertations and Theses. 6189. https://digitalcommons.lsu.edu/gradschool_disstheses/6189 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor M 48106-1346 USA 313/761-4700 800/521-0600 IMPORTANCE OF A SIDEROPHORE IN THE PATHOGENESIS AND VIRULENCE OF PHOTOBACTERIUM DAMSELA SUBSP. PISCICIDA IN HYBRID STRIPED BASS (MORONE SAXATILIS X MORONE CHRYSOPS) A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Veterinary Medical Sciences through the Department of Veterinary Microbiology and Parasitology by John P. Hawke B.S., Auburn University, 1972 M.S., Auburn University, 1974 May 1996 UMI Number: 9637776 UMI Microform 9637776 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 DEDICATION To the memory of my mother, Evelyn Hawke and to Mae and Earle Cole who taught me to love and respect the estuarine environment ACKNOWLEDGMENTS I wish to extend my gratitude and appreciation to my graduate advisor, Dr. Ronald Thune. His continuous support and encouragement made it possible to remain steadfast in my pursuit of the Ph.D degree until the end. I greatly appreciate all the opportunities he has provided for me. I would also like to thank Dr. Johannes Storz and Dr. David Huxsoll for their support of our fish disease research program. I particularly want to thank Dr. Richard Cooper whose support and ideas continued to motivate me. I am also grateful to the other members of my graduate committee, Dr. David Horohov, Dr. William Todd, Dr. Wayne Taylor and Dr. Richard Goyer for their guidance and helpful suggestions. In the beginning of the project Dr. Joe Newton was very helpful in forming a plan for the pathogenesis studies. More recently, Dr. A1 Camus provided valuable suggestions concerning pathological techniques and Mae Lopez cut beautiful sections. Dr. Lisa Stanley-Collins always had helpful comments and suggestions in many areas. Over the years, there have been many people in the departments of Veterinary Microbiology and Parasitology, and Veterinary Science who have helped me with various parts of this dissertation project. I would particularly like to thank Denise Fernandez and Christy Limbers in Dr. Thune7s lab and Karen Cooley, Brandye Sawyer and John Buchanan in Dr. Cooper's lab. For help in designing fish iii challenge systems I am indebted to Mike Pena and John Dobias. I would also like to recognize Mark Lawrence and Michelle Moore for helpful discussions concerning my research. Most importantly, I would like to thank my wife, Becky, without whose love and support and a gentle push every now and then, I would never have been able to complete the dissertation. To my children Autumn, Rachel and Phillip, thanks for being patient and supportive. I also appreciate the understanding and support of my dad, Jack Hawke, who has endured difficult times very gracefully during the course of this project. Also, my thanks go to all my family in Birmingham and to my other mom and dad, Peggy and Vernon Bullock whose patience and generosity have been very important to me. TABLE OF CONTENTS DEDICATION ......................................... ii ACKNOWLEGMENTS ..................................... iii LIST OF TABLES .................................... ix LIST OF FIGURES .............. ..................... X ABSTRACT ......................................... xii INTRODUCTION ....................................... 1 CHAPTER I. LITERATURE REVIEW ........................ 7 Hybrid striped bass ............................. 7 Striped bass spawning and production ........ 7 Hybrid striped bass production and culture ... 8 Fish pasteurellosis ............................. 10 Geographic distribution .................... 10 Epizootics in the United States ........ 10 Epizootics in Japan ................... 11 Epizootics in Europe and the Mediterreanean ........................ 12 Erroneous identification .............. 13 Species susceptibility ..................... 13 Taxonomy, morphology and biochemical characteristics ............................ 15 Pasteurella piscicida ................. 15 Photobacterium damsela subsp.piscicida... 17 Structural components important in virulence.. 22 Capsule ............................... 22 Fimbria ............................... 22 Flagella ..................... ,....... 22 Lipopolysaccharides ................... 22 Resistance to bactericidal effects of s e r u m ................................ 24 Outer membrane proteins ............... 25 Non-structural components important in virulence ................................. 26 Plasmids .............................. 26 R-plasmids and antibiotic susceptibility. 28 Siderophores .......................... 3 0 Siderophores produced by fish pathogens . 31 Vibrio anguillarum ............... 31 Aeromonas hydrophila ............. 32 P. damsela subsp. piscicida ....... 3 3 Alternative mechanisms of iron acquisition........................... 34 Toxins and proteolytic enzymes ........ 35 Antigenic homogeneity ...................... 37 Clinical signs ............................. 38 v Acute disease ........... 38 Chronic disease ....................... 38 Histopathology ............................. 39 Viability in aquatic environments and transmission ........................... 41 Pathogenicity .............................. 42 Pathogenesis ............................... 45 Vaccination ................................ 45 Bacterial transformation ........................ 47 Competent cells ..................... 48 CaCl2 transformation 48 Electroporation ............................ 49 Transpositional mutagenesis ..................... 49 Minitransposon TnlO derivative 103 .......... 52 Plasmid vehicle pNK2859 . .......... 52 Plasmid vehicle pEIS ....................... 52 Study objectives ................................ 53 CHAPTER II. MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF STRAINS OF PHOTOBACTERIUM DAMSELA SUBSP. PISCICIDA ISOLATED FROM DISEASED HYBRID STRIPED BASS CULTURED IN LOUISIANA ...................... 56 Introduction .................................... 56 Materials and methods ........................... 59 Isolation of the etiological agent .......... 59 Bacterial strains .......................... 59 Phenotypic analysis ........................ 59 Rapid identification by ELISA .............. 60 Plasmid content ............................ 62 Antimicrobial susceptibility ............... 62 Enzymic activity ........................... 63 Determination of midpoint denaturation temperature and G/C ratio ................. 63 Growth of cultures .................... 63 DNA extraction ........................ 64 DNA base composition .................. 64 Results .......................... 65 Isolation of the etiological agent ......... 65 Phenotypic and biochemical characteristics of Louisiana isolates ...................... 66 Comparison of enzymatic activities ......... 69 Plasmid content ............................ 69 Antimicrobic
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