CHARACTERISATION OF EXPERIMENTALLY INDUCED AND SPONTANEOUSLY OCCURRING DISEASE WITHIN CAPTIVE BRED DASYURIDS Scott Andrew Lindsay A thesis submitted in fulfillment of requirements for the postgraduate degree of Masters of Veterinary Science Faculty of Veterinary Science University of Sydney March 2014 STATEMENT OF ORIGINALITY Apart from assistance acknowledged, this thesis represents the unaided work of the author. The text of this thesis contains no material previously published or written unless due reference to this material is made. This work has neither been presented nor is currently being presented for any other degree. Scott Lindsay 30 March 2014. i SUMMARY Neosporosis is a disease of worldwide distribution resulting from infection by the obligate intracellular apicomplexan protozoan parasite Neospora caninum, which is a major cause of infectious bovine abortion and a significant economic burden to the cattle industry. Definitive hosts are canid and an extensive range of identified susceptible intermediate hosts now includes native Australian species. Pilot experiments demonstrated the high disease susceptibility and the unexpected observation of rapid and prolific cyst formation in the fat-tailed dunnart (Sminthopsis crassicaudata) following inoculation with N. caninum. These findings contrast those in the immunocompetent rodent models and have enormous implications for the role of the dunnart as an animal model to study the molecular host-parasite interactions contributing to cyst formation. An immunohistochemical investigation of the dunnart host cellular response to inoculation with N. caninum was undertaken to determine if a detectable alteration contributes to cyst formation, compared with the eutherian models. Selective cell labelling was observed using novel antibodies developed against Tasmanian devil proteins (CD4, CD8, IgG and IgM) as well as appropriate labelling with additional antibodies targeting T cells (CD3), B cells (CD79b, PAX5), granulocytes, and the monocyte-macrophage family (MAC387). Toluidine blue labelling of mast cells complemented results. Effective labelling was not obtained with CD79a, interleukin-4, interferon-γ, or MHCII antibodies. Descriptions of pathology such as the site and extent of tissue necrosis in S. crassicaudata resemble those reported in immunocompromised rodent models. The exception is the lack of major involvement of the brain and associated neurological signs. Immunohistopathologic findings suggest the dunnart host cell response resembles that described in eutherians – strongly neutrophilic with fewer macrophages focused on sites of tissue necrosis, with a predominantly T cell nature to any lymphoid response inclusive of CD4 positive T helper cells. Intracellular parasite replication is frequently observed in the absence of a detectable host cellular response. More sensitive alternative techniques such as real time polymerase chain reaction using effective cytokine labelling is recommended to further define the host response to neosporosis. Active surveillance of disease to further define the animal model involved collecting cadavers over 28 months from the dunnart colony maintained at the University of Sydney. An additional 66 S. crassicaudata and S. macroura animals were examined. Of the 28.3% of animals diagnosed with a disorder of growth (n=15), 80.0% were diagnosed with a malignancy (n=12), and the most frequently diagnosed neoplasm was squamous cell carcinoma. Lesions were seen on the rostral mandible (2), pouch (2), distal limb (1), and tail (1) and five of six of these tumours were diagnosed in S. macroura. The single squamous cell carcinoma diagnosed in the pouch of an S. crassicaudata showed a distinctive verrucous growth pattern, prompting consideration of a viral causation. Immunohistochemical investigation of carcinomatous proliferations using multiple bovine papillomavirus markers and an antibody targeting the p16 cell protein found no supportive evidence for a viral association. Additional colony pathology included single or multicentric nodular pyogranulomatous or suppurative disease (botryomycosis) – sometimes a comorbidity of carcinoma. In S. crassicaudata eight cases of gastric dilatation were also identified – with and without trichobezoar formation. Collection and examination of specimens from the colony is ongoing. ii ACKNOWLEDGEMENTS The completion of this thesis would not have been possible without the contributions of the following individuals and groups. Firstly, I’m indebted to my supervisor, Mark Krockenberger, for his guidance and encouragement with this project. His ongoing professional and personal support is greatly appreciated. I’m also grateful for the advice provided by my co-supervisor, Damien Higgins, relating to immunological and immunohistochemical aspects of this thesis, and to Jan Slapeta with regard to its parasitological components. Jan’s use of the dasyurid animal model for investigation of experimental neosporosis provided the material basis for the immunohistochemical investigations in Chapter Two. Patricia Martin has always been enthusiastic with her assistance in identifying infectious agents of disease in the dasyurid colony, including liaising with The Reference Laboratory at the Centre for Infectious Diseases and Microbiology, Westmead, Sydney. Paul Canfield has provided invaluable guidance in interpreting histopathology in the small marsupials. Dane Hayes from the Department of Primary Industries, Parks, Water and Environment Tasmania, generously provided the novel Tasmanian devil antibodies used in Chapter Two and their use was made possible through an introduction and logistical support from Kathy Belov and Katrina Morris. Without Bronwyn McAllen’s in depth understanding of captive dasyurid health and well being, and her willingness to provide cadavers for examination, Chapter One of this thesis would not have been achievable. Likewise, Chapter Three was made possible through the encouragement of Mark Bennett from Murdoch University, Western Australia, to seek out a viral cause for carcinomatous disease identified in the dunnart colony. His laboratory generously funded, and through Michael Slaven conducted the papillomavirus immunohistochemistry. Ongoing technical support and advice in the Veterinary Pathology Diagnostic Service (VPDS) laboratory from Elaine Chew, Karen Barnes and Huy Tran is greatly appreciated. Most importantly, no project such as this is achievable without the support and understanding of family. This project was partially funded by the generosity of a grant from The Dr William Richards Awards in Veterinary Pathology with remaining laboratory expenses incurred by VPDS. Thanks to all. iii TABLE OF CONTENTS STATEMENT OF ORIGINALITY .................................................................................................................................. I SUMMARY .............................................................................................................................................................. II ACKNOWLEDGEMENTS.......................................................................................................................................... III TABLE OF CONTENTS ............................................................................................................................................. IV LIST OF FIGURES...................................................................................................................................................... V LIST OF TABLES....................................................................................................................................................... VI GENERAL INTRODUCTION....................................................................................................................................... 1 CHAPTER ONE: THE PATHOLOGY OF A CAPTIVE-BRED DUNNART COLONY (S. CRASSICAUDATA AND S. MACROURA)........................................................................................................................................................ 3 CHAPTER TWO: IMMUNOHISTOCHEMICAL CHARACTERISATION OF THE HOST CELLULAR IMMUNE RESPONSE FOLLOWING INOCULATION WITH N. CANINUM (S. CRASSICAUDATA)................................................................. 39 CHAPTER THREE: IMMUNOHISTOCHEMICAL INVESTIGATION OF PAPILLOMAVIRUS ASSOCIATION WITH CARCINOMATOUS PROLIFERATIONS IN A CAPTIVE-BRED DUNNART COLONY (S. CRASSICAUDATA AND S. MACROURA).................................................................................................................................................... 127 GENERAL DISCUSSION ........................................................................................................................................ 144 APPENDICES........................................................................................................................................................ 148 REFERENCES........................................................................................................................................................ 189 iv LIST OF FIGURES Figure 1.1. Animal housing and colony management.................................................................................................................19 Figure 1.2. Gross and histological features of a cutaneous sarcoma in animal 64........................................................31 Figure 1.3. Gross and microscopic appearance of gastric trichobezoar and pancreatic nodular