Health and disease status in a threatened marsupial, the quokka (Setonix brachyurus) Pedro Martínez-Pérez Veterinarian (Hons), MVS Conservation Medicine This thesis was submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Wildlife Veterinary Studies, Murdoch University School of Veterinary and Life Sciences Murdoch University Murdoch, Western Australia January 2016 Declaration I declare that this thesis is my own account of my research and contains as its main content, work which has not been previously submitted for a degree at any tertiary educational institution. _______________________________________________ Pedro A. Martínez-Pérez I Abstract Between 1901 and 1931, there were at least six anecdotal records of disease outbreaks in mainland quokkas (Setonix brachyurus) that were associated with mass. This time period pre-dates the arrival of the red fox (Vulpes vulpes). Despite these outbreaks, little or no research has been carried out to establish health and disease baseline data of the fragmented and scattered, extant populations. Epidemiological data was determined for a range of potential pathogens, and established physiological reference intervals of apparently healthy, wild quokkas on Rottnest Island and mainland locations. There were significant differences between Rottnest Island and mainland quokkas. Rottnest Island animals had haemograms with mark evidence of oxidative injury and bone marrow response consistent with a regenerative normocytic hypochromic anaemia. Except alkaline phosphatase (ALP), all blood chemistry analytes where higher in mainland animals, with particular emphasis on creatine kinase (CK), alanine amino transferase (ALT), aspartate amino transferase (AST) and vitamin E. Some other key findings include a widespread presence of a novel herpesvirus (MaHV-6), the recovery of Cryptococcus neoformans var. grubii from quokkas in highly altered ecosystems on Rottnest Island, and new Salmonella spp. serovars in Rottnest Island quokkas. Atypical lymphocytes resembling those in proliferative disorders of the lymphoid and haematopoietic tissues in other species were observed in blood smears of animals on Rottnest Island but not on the mainland. The presence of potentially-pathogenic organisms is likely to increase synergistic effects of ongoing and future threats (e.g. habitat clearing, climate change), and could increase quokka extinction risk. Disease surveillance would make a valuable contribution to Recovery Plans for the quokka, enabling preparedness for a rapid response if clinical disease is to happen, and to manage populations in a more integrated way. II Acknowledgments This dream would have not been possible without the support and encouragement of many people. First and foremost, my most sincere gratitude and appreciation to my supervisors, A/Prof. Trish Fleming (principal supervisor) and Dr Tim Hyndman (co-supervisor) from Murdoch University, for their tireless and relentless help. To both of you, thanks for your abounding commitment to see me through this long intellectual journey and for the many hours you spent reading and commenting my work. Trish, I know it was never easy, and your effort has been immense. From taking on a student you didn’t know, who’s project had no funding, but who stubbornly (some will call it passionate) decided to see it through, to supporting a project that was not within your immediate area of expertise. Thanks for staying with me and my project, despite the many challenges it presented. Tim, although you came into this journey halfway, your support and knowledge has been key in the final outcome. Thanks for your unceasing effort to get me think about my science, to get me re- examined my own thoughts. Your advice was always filled with encouragement and a smile. Will I split the infinitive again? I cannot promise you that much my friend, but rest assure that Star Trek’s opening sequence “to boldly go where no man has gone before” will stay engraved in my mind. I would not be here seeing this journey ending without you two. What lies ahead only God knows, but I am taking what you have given me as a lighthouse that will guide my future career. To Prof. Phil Nichols from Murdoch University, an unmeasurable gratitude for standing by my side, in the good ones and the bad ones. Our short and sometimes long catch-ups were always filled with encouragement and companionship. To you too, thanks for seeing me through this cause called a PhD. To Prof. Colin Wilks at the Faculty of Veterinary and Agricultural Science of The University of Melbourne who, together with Prof. Joanne Devlin’s team, carried out the serology studies for Macropodid herpesvirus 1 and Macropodid herpesvirus 2, my gratitude. Colin, thanks for your valuable input in the gammaherpesvirus manuscript, and for your encouraging and supportive company. This project would have not been the same without you. I thank Murdoch University, DPaW, the Australian and New Zealand Trustees and the Rottnest Island Authority for their financial support, without which this project could not have gone ahead. To the staff of the Veterinary Clinical Pathology (Murdoch University) III thanks for carrying out most of the haematology and blood chemistry analyses. To Dr Kirsty Townsend (Murdoch University), my greatest appreciation for your support in the area of microbiology. Your teachings and friendship were a fundamental part in this project. To Dr Mark Bennett (Murdoch University), thanks for your guidance and expertise in the area of clinical pathology, and for the time you invested looking down the microscope making sure I was doing the right thing while assessing blood smears. Also, although involved only at the beginning of my project, I am thankful to Dr Cree Monaghan (Perth Zoo) for supporting my idea and providing valuable feedback in the area of Conservation Medicine. I am also grateful to Dr Mark Krockenberger (Faculty of Veterinary Science at the University of Sydney) and Dr Nicky Buller at the Department of Agriculture and Food WA (DAFWA) for their involvement and support in some of the molecular and microbiological aspects of the fungal work in this project. Nicky, your knowledge and diligence is greatly appreciated. To Pat Statham at the Animal Health Laboratory, Department of Industries, Parks, Water and Environment (DPIPWE) in Tasmania, thanks for carrying out the serology for T. gondii. Gratitude goes to many others that helped me at various stages of my PhD, Prof. Una Ryan, Andrea Paparini, Josephine Ng, Rongchang, Carlo and Sabrina, Halina and her lovely monsters, Roberta Bencini (The University of Western Australia), Paul de Tores (Department of Environment and Conservation) and many more. I would like to also extend my gratitude to Dr Shannon Dundas (Murdoch University) and Karlene Bain from the Department of Parks and Wildlife (DPaW) in Walpole, for allowing me to merge my project with theirs. I would like to acknowledge your dedication and effort for allowing me to work with the animals you were trapping in the mainland. Thanks to you two, I was able to work with the wonderful quokka. Karlene, thanks for your hospitality and for the adventurous trips through the majestic forests of the south west of Western Australia. Likewise, I thank Helen Shortland-Jones (Rottnest Island Authority), for assisting and organising all the logistics needed to work with quokkas on Rottnest Island (e.g. accommodation, transportation, maps), and for assisting in animal trapping and handling. It was a pleasure work with you all. To Dr Simone Vitale and Dr Rebecca Vaughan at Perth Zoo as well as the rest of the personnel at the veterinary hospital, my gratitude for granting me access to some of the captive individuals and hospital records, as well as for assisting in sample collection. Thanks to Dr Karrie Rose at the Australian Registry of Wildlife Health (ARWH) for sharing her knowledge and given me access to confidential pathology records in the ARWH. Dr Larry Vogelnest (Taronga Zoo) and Dr Rupert Woods (Wildlife Health Australia), thanks for your encouraging words. To my uni mates, Elvina, Mel, Narelle, Wan Ho, Tina, Linda, and to all other colleagues, PhD students or not, with whom I shared time, thanks for being there. Lauren, thanks for the IV good and the bad times we shared at our office over at Murdoch, and to Andy Demos, Lauren’s partner, it was an absolute pleasure meeting you, this work is partly dedicated to your memory. Wherever you are Lauren, I wish you well my dearest friend. To Jessica Lee my best friend and her family. Cacatua, who would have thought that two weeks in Costa Rica -weeks of blisters and barb wire injuries- would have evolved into the friendship we have today. Thanks for our long scientific-religious debates [you’ve said this before ☺], for your hard work while trapping quokkas, taking samples and reading maps. Thanks for your infectious and rejuvenating laugh, and for all the life lessons we have learned. But above all, my neverending gratitude for giving me the greatest gift of all. To your family, uncle Jimmy, Julie, Sammy and Daniel, and Davis too, thanks for being there to lift me up in my darkest and most difficult times during my PhD. Without you all, I would have not gone far. To my family. Mom and Dad, thanks for your encouragement, patience, understanding and endless support. Thanks for the great lengths you have gone to support and help my family, to care for them and see them grow. Davicho, thanks for your regular communications and for looking after our parents, and caring for my family. Diana, I can only imagine how difficult it was to raise our son in my absence and find yourself alone with such task. My heart has an eternal debt with you for keeping me alive in our son’s mind. Andrés my beloved and only son, we shall be together once more, and live a thousand-fold those years that we missed, I love you.