An investigation into the role Toxoplasma gondii may play in the health of the southern brown bandicoot (Isoodon obesulus obesulus) and an assessment of environmental contamination with T. gondii. Amanda Jane Breidahl ORCID: 0000-0002-5947-3393 Masters of Veterinary Science March 2020 Melbourne Veterinary School Faculty of Veterinary and Agricultural Sciences The University of Melbourne Submitted in total fulfillment for the degree of Masters of Veterinary Science i Photograph on Title Page: Sculpture of a southern brown bandicoot placed in the Royal Botanic Gardens Cranbourne to raise awareness about the resident population of the nationally endangered southern brown bandicoots. Artists: Darrell Cordell, Vanessa Ellis, Trina Gaskell and Cliff Dolliver Sculpture formed from Kunzea ericoides ii Abstract The southern brown bandicoot (Isoodon obesulus obesulus), a small, ground-dwelling marsupial, is listed as ‘endangered’ under the Australian Environment Protection and Biodiversity Conservation Act 1999. While many factors contributing to population decline are understood, in particular predation and loss of high-quality, connected habitat, there is a lack of knowledge about other threatening processes, including disease, which has contributed to declines in other small mammal species. Toxoplasma gondii, a protozoan intracellular parasite, excreted into the environment by cats, has been shown to cause clinical disease, including death, in many small and medium sized captive and free-ranging marsupials, including the eastern barred bandicoot (Perameles gunnii). Little is known of its effect on southern brown bandicoot populations. This study aimed to investigate the significance of T. gondii to the health of southern brown bandicoot populations on the northern hinterland of Western Port, Victoria and methods of predicting probability of infection with environmental T. gondii. A series of necropsies was performed on 33 southern brown bandicoots collected opportunistically over a five-year period. The causes of death were identified as motor vehicle trauma (22); predation (4); ejected pouch young (2); drowning (1); pyometra (1); possible toxicity and stomach bloating through lentil ingestion (1) and unknown (2). Real- time qPCR was performed on tissues from 30 cases, all of which were negative for the presence of T. gondii DNA. A range of helminths and ectoparasites were collected and identified, most of which had been previously been reported in this species. However, a metastrongyloid helminth species, found by histopathology in the lungs, is reported for the first time in a southern brown bandicoot. To assess environmental contamination with T. gondii, two sites with different cat densities were compared. Seroprevalence of antibodies to T. gondii (n=24) using the Modified Agglutination Technique was performed on trapped southern brown bandicoots. iii No evidence of infection with T. gondii was found at either site. Molecular (qPCR) methods were used to measure T. gondii oocyst presence in soil samples (n= 594) and prevalence of T. gondii in tissues of rabbits (n=118) and mice (n=267). All tests were negative across both sites except for the presence of T. gondii in one rabbit (prevalence 0.85%). These results suggest that rabbits and mice may have the potential to be reliable sentinel species and inform conservation management of the probability of infection with T. gondii in small marsupials. The results from this study are consistent with the concept that opportunistic necropsy is a valuable strategy for passive disease and cause of death surveillance in native wildlife. No evidence was found that T. gondii was impacting the health of southern brown bandicoots, however, further longitudinal health surveys are necessary to determine the true prevalence of disease and causes of mortality. Further studies are recommended to confirm the effectiveness of mice and rabbits as potential sentinel species in a range of ecosystems, including those environments which have higher levels of contamination with T. gondii. iv Declaration by Author I certify that: i. this thesis comprises only my original work towards the degree of Masters of Veterinary Science, except where indicated in the preface and acknowledgement sections ii. due acknowledgement has been made in the text to all other material used iii. the thesis is fewer than 50,000 words in length, exclusive of maps, tables, bibliographies and appendices. Amanda Jane Breidahl v Preface Contribution of others: Honorary Professor Ian Beveridge (Faculty of Veterinary and Agricultural Science, The University of Melbourne) identified all of the internal parasites, both gross specimens and histopathology sections (Chapter 2); assisted in identification of some of the external parasites (Chapters 2 and 3); examined the blood smear slides for haemoparasites (Chapter 3); photographed the histopathology slides in Plates 2.1 and 2.2 (Chapter 2) and proof read the sections in the Thesis referring to parasites (Chapters 2 and 3). Dr David Spratt (Honorary Fellow in the Australian National Wildlife Collection, National Research Collections, Australia Commonwealth Scientific and Industrial Research Organisation) also examined the blood smear slides for the presence of haemoparasites (Chapter 3). Dr Jasmin Hufschmid (Faculty of Veterinary and Agricultural Science, The University of Melbourne) assisted in the identification of the external parasites (Chapters 2 and 3). Dr Pam Whiteley (Victorian Wildlife Health Surveillance, The University of Melbourne) performed the gross necropsy on seven bandicoots (408-16, 409-16, 410-16, 432-16, 433- 16, 434-16 and 435-16) (Chapter 2). Dr Janeen Samuel (retired veterinary pathologist) examined and interpreted all of the histopathology slides (Chapter 2). Pat Statham (Department of Primary Industries, Parks, Water and Environments, Mt Pleasant Animal Health Laboratory, Launceston, Tasmania) performed the Modified Agglutination Tests on eight southern brown bandicoot blood samples stored on Whatman Filter paper. Mr Barry Murphy (Murphy Landscape Consultancy) prepared the maps in Figures 1.2, 2.1, 2.2 and 3.1. Dr Duncan Sutherland (Phillip Island Nature Parks) prepared the maps in Figure 3.2. vi Mr Ian Caldwell supplied the majority of the rabbits. Publication Status: All chapters contain unpublished material that are not yet submitted for publication. Funding Sources: This research was funded by an Early Careers Grant awarded to Dr Jasmin Hufschmid (Faculty of Veterinary and Agricultural Science, The University of Melbourne). Zoos Victoria and Phillip Island Nature Parks provided some of the equipment and personnel used in the field work in Chapter 3. vii ACKNOWLEDGEMENTS Like children, this research project was conceived in naivety (blissfully unaware of what lay ahead), had a long gestation and now, after much labour, is finally delivered. As I now reflect on the experience, I am very grateful for the opportunities, skills and learning that it has encompassed. Importantly, I am extremely fortunate to have met so many wonderful, passionate people along the way, who care deeply about our precious wildlife. In particular, I would like to acknowledge and thank the many friends and colleagues from Wildlife Diseases Association Australasia, whose high standard of research, investigative and clinical work, fabulous conferences and commitment to Australian wildlife inspired me to take this step into the unknown. With good humour, my four fabulous supervisors have given me wonderful support and encouragement. It has been a great pleasure and wonderful opportunity to meet and work with each of you and I thank you for your support and friendships, which I hope will endure well beyond this project. Dr Jasmin Hufschmid has been instrumental in patiently teaching me the ways of research and guiding me through each step of the way. She has generously and cheerfully given innumerable hours over the duration of the project. Your commitment, care, patience, time, attention to detail and humour has been wonderful. Thank you. Professor Rebecca Traub has taught me all things molecular and protozoan. Your patience, knowledge, wisdom and support was invaluable, for which I am so very grateful. Thank you. Dr Michael Lynch, from Zoos Victoria, with his infectious enthusiasm and insight was a great support, particularly with envisioning the concept of eutherian sentinels, helping to equip the field work and providing a roster of zoo vets and nurses to help with the anaesthetics. Thank you. Dr Duncan Sutherland, from Phillip Island Nature Parks, provided the genesis of this project which arose from a direct bandicoot conservation management question. His quiet enthusiasm and love and knowledge of the natural world is inspiring. He generously supplied equipment and expertise for the fieldwork, helped with the GPS mapping and has provided wonderful insight and advice from an ecological perspective. Thank you. A special thank you to Dr Terry Coates, senior ecologist at Royal Botanic Gardens Cranbourne (RBGC). Terry gave permission for the field work to be conducted at the RBGC, viii supplied traps, advised trapping techniques, provided a room for our field clinic and collected and stored dead bandicoots and rabbits. In addition, Terry generously shared his vast knowledge and experience on the ecology of the southern brown bandicoot. Your warm, welcoming smile, friendship and unending practical help, even to the point of turning your office into a field veterinary clinic, was fabulous!