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Iridovirus Infections of Captive and Free-Ranging Chelonians in the United States

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Iridovirus Infections of Captive and Free-Ranging Chelonians in the United States IRIDOVIRUS INFECTIONS OF CAPTIVE AND FREE-RANGING CHELONIANS IN THE UNITED STATES By APRIL JOY JOHNSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006 Copyright 2006 by April Joy Johnson ACKNOWLEDGMENTS Funding for this work was provided in part by the University of Florida College of Veterinary Medicine Batchelor Foundation, grant No. D04Z0-11 from the Morris Animal Foundation, and a grant from the Disney Conservation Fund. I also thank the University of Florida, College of Veterinary Medicine for awarding me an Alumni Fellowship for tuition and stipend over the first three years. I would like to thank the many people who helped collect and submit turtle and tortoise samples including Dr. William Belzer, Dr. Terry Norton, Jeffrey Spratt, Valorie Titus, Susan Seibert and Ben Atkinson. Also, I would like to thank all the pathologists who contributed to this work including Dr. Allan Pessier, Dr. Nancy Stedman, Dr. Robert Wagner and Dr. Jason Brooks. I thank Drs. Jerry Stanley and Kathy Goodblood for allowing me access to chelonian and amphibian populations at Buttermilk Hill Nature Sanctuary and to Drs. Mick Robinson and Ken Dodd for providing necropsy reports on archived cases. I would also like to thank the Mycoplasma Research Laboratory at the University of Florida including Dr. Mary Brown, Dr. Lori Wendland and Dina Demcovitz for providing access to their wild gopher tortoise plasma bank. I thank Yvonne Cates at the Zoological Society of San Diego for excellent histology support and Lynda Schneider from the University of Florida Electron Microscopy Core Laboratory. I also thank Dr. Harvey Ramirez, Rachelle Wright and Kevin Chadbourne with Animal Care Services for excellent care of the turtles and frogs. iii Several people went above and beyond normal expectations in assisting with this research in various ways. I would especially like to thank Dr. Allan Pessier for all his time, patience and assistance with necropsies and histopathology as well as making me a better scientific writer. I thank Dr. Jim Wellehan for all his advice and assistance with molecular and laboratory techniques. I also am indebted to our laboratory technicians, Sylvia Tucker and April Childress. They both provided invaluable laboratory support and became great friends throughout my time at the University of Florida. I thank my parents for their support and encouragement, without which I would never have made it to this point in my career. They have always believed in me and, although they might not always understand my unique interests, have never discouraged me from pursuing my goals. For that I will always be thankful. And last, but far from least, I am extremely grateful to my supervisory committee members: Dr. Elliott Jacobson, Dr. David Bloom, Dr. Jack Gaskin, Dr. Jorge Hernandez and Dr. Gail Scherba. I thank them for the advice, support and encouragement they have given me since the beginning. I thank them for helping me develop my skills and for their confidence in my abilities. I have learned so much from them and have thoroughly enjoyed this opportunity to work with them. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT...................................................................................................................... xii CHAPTER 1 INTRODUCTION ........................................................................................................1 2 RANAVIRUS INFECTION OF FREE-RANGING AND CAPTIVE BOX TURTLES AND TORTOISES IN THE UNITED STATES.....................................11 Introduction.................................................................................................................11 Materials and Methods ...............................................................................................12 Animals................................................................................................................12 Necropsy and Histopathology .............................................................................16 Nucleotide Amplification, Sequencing, and Sequence Analysis ........................16 Virus Isolation .....................................................................................................17 Transmission Electron Microscopy.....................................................................18 Restriction Enzyme Analysis ..............................................................................18 Results.........................................................................................................................19 Necropsy and Histopathology .............................................................................19 PCR and Sequence Analysis ...............................................................................20 Virus Isolation .....................................................................................................21 Transmission Electron Microscopy.....................................................................21 Restriction Enzyme Analysis ..............................................................................21 Discussion...................................................................................................................22 3 DEVELOPMENT AND USE OF AN INDIRECT ENZYME LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF IRIDOVIRUS EXPOSURE IN GOPHER TORTOISES (Gopherus polyphemus) ...........................34 Introduction.................................................................................................................34 Materials and Methods ...............................................................................................36 Virus ....................................................................................................................36 v Antigen Preparation.............................................................................................37 Positive and Negative Reference Plasma ............................................................38 ELISA Procedure.................................................................................................39 Experimentally Inoculated Turtles ......................................................................40 Reproducibility....................................................................................................41 Protein Expression and Immunoblotting.............................................................42 Wild Gopher Tortoises Samples with Unknown Exposure.................................43 Results.........................................................................................................................43 Antigen Preparation.............................................................................................43 ELISA Parameters...............................................................................................44 Experimentally Inoculated Turtles ......................................................................44 Reproducibility....................................................................................................45 Protein Expression and Immunoblotting.............................................................45 Wild Gopher Tortoises Samples with Unknown Exposure.................................46 Discussion...................................................................................................................46 4 EXPERIMENTAL TRANSMISSION OF A RANAVIRUS IN WESTERN ORNATE BOX TURTLES (Terrapene ornata ornata) AND RED-EARED SLIDERS (Trachemys scripta elegans)......................................................................65 Introduction.................................................................................................................65 Materials and Methods ...............................................................................................66 Experimental Animals and Husbandry................................................................66 Pre-inoculation Sample Collection......................................................................67 DNA Preparation, Polymerase Chain Reaction and Nucleotide Sequencing......67 ELISA..................................................................................................................68 Virus Preparation.................................................................................................70 Transmission Studies...........................................................................................71 Study 1..........................................................................................................71 Study 2 ..........................................................................................................73 Results.........................................................................................................................74 Experimental Animals and Pre-inoculation Sampling ........................................74 Transmission Studies...........................................................................................74
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