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Dryococelus Australis ) A bacteriological disease investigation in a captive breeding colony of Lord Howe Island Stick insects (Dryococelus australis ) Christina Cheng ORCHID: 0000-0002-2615-6957 Submitted in fulfilment of the requirements of the degree in Master of Veterinary Science. April 2017 Faculty of Veterinary and Agricultural Sciences The University of Melbourne ABSTRACT The Lord Howe Island stick insect ( Dryococelus australis) is critically endangered in the wild. Once driven to near-extinction, the species now exists within just a few captive breeding programs that have been established to conserve the species, to increase population numbers through breeding, and to support the eventual reintroduction of the species into the wild. In the current study, the bacteria Serratia marcescens was investigated as a possible cause of mortality in a captive breeding colony of D. australis housed at Melbourne Zoo. S. marcescens was isolated from both dead insects and from the environment. The prevalence of the bacteria in dead insects was estimated, and the gross pathological and histopathological changes in infected insects were characterised. Strain types of S. marcescens were characterised using pulsed-field gel electrophoresis, and the strain type A1 was implicated as the most likely outbreak strain associated with insect deaths. Epidemiological risk factors for mortality were also investigated, and a novel PCR test was developed to facilitate rapid detection and identification of S. marcescens . The overall findings of this research suggest that S. marcescens is a potential opportunistic pathogen of D. australis , and is associated with haemocoelomitis and death. However, high virulence was not demonstrated in infected insects under experimental conditions. Recommendations for the future management of D. australis and the future reintroduction of the species to the wild are discussed in light of the findings from this research. i DECLARATION This is to certify that: (1) The thesis comprises only my original work towards the Master degree except where indicated in the Acknowledgments Section. (2) Due acknowledgment has been made in the text to all other material used. (3) The thesis is fewer than 50,000 words in length. ii ACKNOWLEDGEMENTS This thesis was completed as part of the course requirements for the Master of Veterinary Science degree (by research) at the University of Melbourne, in conjunction with the Veterinary Residency clinical program at Melbourne Zoo. The research and clinical components of this two-year combined program were weighted equally. I would like to take this opportunity to thank the many people who have made this research and clinical residency possible, and have given their support and guidance along the way. To my supervisors, Joanne Allen, Dr. Marc Marenda and Dr. Michael Lynch, thank you for all your endless wisdom, encouragement, and patience through some frustrating and testing times during my research, for steering me in the right directions, tirelessly reviewing my chapters, and pushing me to achieve beyond what I thought was possible. To my research chair, Professor Glenn Browning, thank you for your seemingly constant supply of ideas, optimism and somehow, time. To Dr Simon Firestone and Professor Mark Stevenson, thank you for lending me some much needed statistical and epidemiological guidance. To the Melbourne Zoo veterinarians, veterinary nurses, veterinary keepers and veterinary support officer, I am forever grateful for the opportunity that I had to work with and learn from each of you. If I have somehow gained just a hint of Dr. Helen McCracken’s wisdom and dedication to science, Dr. Kate Bodley’s incredible work ethic and meticulousness, Dr. Sarah Frith’s enthusiasm and fearlessness, and Dr. Michael Lynch’s professionalism and people skills, I will forever be a better veterinarian for it. To all the veterinary support staff, thank you for all your support and for allowing me to occupy much of the veterinary department post- mortem room, hallway, fridges and freezers with my vast collection of research samples. To the Melbourne Zoo invertebrate keepers, especially Rohan Cleave and Kate Pierce, thank you for sharing both your workspace and your wealth of knowledge in a species so close to your hearts. Special thanks to Rohan for your magnificent efforts in collating data for the epidemiological investigation in this study. And to all the other Melbourne Zoo animal keepers, for having trust in me to care for the zoo’s many wonderful creatures. Thank you to iii Zoos Victoria for making this research project possible, for funding my research stipend, and allowing me to be a part of the zoo family; and to both Zoos Victoria and Michael Magrath for funding the histopathological testing of insects in this study. To the pathologists at Gribbles Pathology, especially to Dr. Christine Bayley, thank you for all your pathology expertise, hard work in performing stick insect histopathology, and your continued enthusiasm throughout what has been a very steep learning curve in phasmid pathology. To the many other staff and students at the Asia Pacific Centre for Animal Health at the University of Melbourne who have been my sounding board for research ideas and troubleshooting in the laboratory, as well as great company over coffee, very late lunches, and extremely late laboratory sessions, thank you also. Finally, to my friends and family, thanks for your patience and encouragement through many long, challenging days and absences from your lives. I cannot thank you all nearly enough for your contributions and support on this journey! iv TABLE OF CONTENTS Abstract ......................................................................................................................................... i Declaration ................................................................................................................................... ii Acknowledgements ..................................................................................................................... iii List of figures ............................................................................................................................... ix List of tables ................................................................................................................................ xi List of abbreviations ................................................................................................................... xiii CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW .......................................................... 1 1.1. INTRODUCTION ..................................................................................................................... 1 1.2 THE LORD HOWE ISLAND STICK INSECT ................................................................................. 1 1.2.1 History of D. australis ...................................................................................................... 1 1.2.2 Conservation objectives and challenges ......................................................................... 3 1.2.3 Life history and biology of D. australis ............................................................................ 3 1.2.4 Climate conditions on Lord Howe Island ......................................................................... 4 1.3 BACTERIOLOGICAL DISEASE INVESTIGATION ......................................................................... 4 1.3.1 Historical disease in the colony ....................................................................................... 4 1.3.2 Preliminary disease investigation .................................................................................... 5 1.3.3 Serratia marcescens ........................................................................................................ 5 1.4 RESEARCH AIMS AND APPROACHES .................................................................................... 10 1.4.1 Overall aims ................................................................................................................... 10 1.4.2 Specific research aims and approaches ........................................................................ 10 CHAPTER 2. GENERAL METHODOLOGY AND MATERIALS ....................................................... 12 2.1 STUDY SUBJECTS AND LOCATION ........................................................................................ 12 2.2 HUSBANDRY OF THE D. AUSTRALIS COLONY ....................................................................... 12 2.3 EUTHANASIA ........................................................................................................................ 13 2.4 GENERAL BACTERIOLOGY..................................................................................................... 14 2.4.1 Culture media ................................................................................................................ 14 2.4.2 Isolation of S. marcescens ............................................................................................. 14 2.5 PULSED-FIELD GEL ELECTROPHORESIS (PFGE) ..................................................................... 15 2.5.1 Background .................................................................................................................... 15 2.5.2 Preparation of PFGE gel blocks and cell lysis ................................................................ 15 2.5.3 Restriction endonuclease digestion .............................................................................
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