CHARACTERISATION OF NATIVE TRYPANOSOMES AND OTHER PROTOZOANS IN THE AUSTRALIAN MARSUPIALS THE QUOKKA (SETONIX BRACHYURUS) AND THE GILBERT’S POTOROO (POTOROUS GILBERTII) Thesis presented by Jill Austen Bachelor of Science with First Class Honours in Biomedical Science for the degree of Doctor of Philosophy School of Veterinary and Life Sciences Murdoch University 2015 i DECLARATION I declare that this thesis is my own account of my research and contains as its main content, work that has not previously been submitted for a degree at any tertiary institution. Jill Austen ii ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude and appreciation to my supervisors: Professor Una Ryan, Associate Professor Simon Reid, Dr Tony Friend and Dr William Ditcham. Una you introduced me into the wonderful world of parasites over a decade ago and since then my fascination for microscopic organisms has grown experientially. Your exceptional knowledge, guidance, belief in my research and kindness have never failed to impress me and I hope that one day I may strive to walk in your footsteps. If earth angels do exist I truly believe you are one. Simon, or should I say trypanosome guru. Thank you so much for initially introducing me to your parasite of choice ‘Trypanosomes”. These blood borne parasites have won my heart and curiosity and trypanosomes are now my preferred parasite of choice. I will always remember your brain storming sessions and exceptional advice and for that I am truly grateful. Tony, thank you so much for your continuous support, wildlife expertise and early morning walks through the bush. This PhD would not have been possible without your collaboration and dedication and our adventures on Rottnest Island will never be forgotten. William, thank you so much for your exceptional laboratory intellect and for always being my go to man for when I had endless questions. You always managed to enlighten me with your novel ideas and boost my self-esteem in the process and for that I am truly grateful. I would like to acknowledge the support and friendship extended by the staff and students of the Vector and Waterborne Pathogens Group and staff at Murdoch University. Particular thanks go to Professor Peter Irwin (with his famous quote ‘You need more evidence’), Frances Brigg, Gordon Thompson, Peter Fallon, Dr Kirsty Townsend, Russell Hobbs, Aileen Elliot, Dr Scott Edwards, Dr Rongchang Yang, Garry iii Allen, Ryne Charsley, Gavin D’Mello, Michael Slaven and Gerard Spoelstra for their skills and assistance in facets of this study. Thank you to my fellow colleagues who have now become close personal friends, particular thanks goes to Tegan McNab and Linda Davies for their help on Rottnest Island, my office buddies, Andrea Ducki, Ahmed (aka the King,), and Emilija Filipovska-Naumovska for all the laughs over the years as well as Bong Sze How, Ryan Jefferies, Joanne McCoubrie, Annika Estcourt, Mark O’Dea, and Amanda Barbosa for making my PhD student days memories to treasure. Family and friends without your continuous love and support I would not have been able to have accomplished so much and fulfilled my dream of studying Australian trypanosomes. I would like to thank my mum (Margaret Smith) and dad (Chris Meinema) for always believing in me and encouraging me to live my dreams and to never give up. I have learnt that a successful PhD relies mostly on the support you get from home. I am therefore truly indebted to my husband Jeff who has continuously supported me throughout my studies and has been the sole carer of our three gorgeous boys on the weekends. Jeff you have sacrificed so much, therefore I would like to dedicate this thesis to you and our three beautiful boys Jacob, Dylan and Brodie. iv SYMBOLS AND ABBREVIATIONS Symbols ~ approximately = equals > greater than < less than - minus % percent x times ± plus-minus sign Abbreviations µL Microlitre ACP Alternative complement pathway AIDS Acquired immunodeficiency syndrome AMP Ampicillin ANOVA Analysis of variance ApoLi Apolipoprotein Li BI Bald Island BIIT Blood incubation infectivity test B Breadth measurment bp Base pair C° Degrees Celsius CI Confidence interval CM Cunninghams liquid medium CO2 Carbon dioxide CO1 Cytochrome c oxidase subunit 1 CRISPs Cysteine-rich secretory proteins DA Diminazene aceturate DAPI 4’6-diamidino-2-phenylindole DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate v EDTA Ethylenediamine tetra acetic acid Eg or e.g. Exempli gratia – for example et al. et alia: and others. EIDs Emerging infectious diseases FAM 6-carboxy fluorescein, acronym FCS Foetal calf serum FISH Fluorescent in situ hybridisation FITC Fluorescein isothiocyanate FF Free flagellum measurement GAPDH glyceraldehyde-3-phosphate dehydrogenase g relative centrifugal force HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HDL High density lipoprotein HPR Human haptoglobin-related protein HSS Human serum sensitive HuMSEM Human modified sloppy Evan’s medium HoMSEM Horse modified sloppy Evan’s medium H2O Water i.e. id est – in other words ICN Pharmaceutical company IgG Immunoglobulin G IPTG Isopropyl β-D-1-thiogalactopyranoside IU International units k Kinetoplast K+ Potassium kb Kilobase kDNA Kinetoplast deoxyribonucleic acid Km Kilometers KN Kinetoplast to nucleus measurement L Liter LB Luria-Bertani L8C4 Anti-paraflagellar rod antibody M Molar concentration MAC Membrane attack complex mg Milligram MgCl2 Magnesium chloride min Minute ML Maximum likelihood vi mL Millilitre mM Millimolar mm Millimetre MP Maximum parsimony mtDNA Mitochondrial deoxyribonucleic acid MSEM Modified sloppy Evan’s medium n Number NA Nucleus to anterior measurement Na+ Sodium NCBI National Center for Biotechnology Information ng Nanograms NHS Normal human serum NSW New South Wales p Probability of an event due to chance alone PBS Phosphate-buffered-saline PCR Polymerase chain reaction PCV Packed cell volume pers. comm. Personal communication PH Negative logarithm of hydrogen ion concentration PI Post inoculation PK Posterior to the kinetoplast measurement pmol Picomoles PN Posterior to nucleus measurement Qld Queensland rDNA Ribosomal deoxyribonucleic acid RNA Ribonucleic acid RI Rottnest Island RPM Revolutions per minute SD Standard deviation SE Standard error SEM Scanning electron microscopy SOC Super Optimal Broth sp. Unknown species (singular) sp. n Novel species spp. Several species SPSS Statistics Package for Social Studies SRA Serum resistance associated gene 18S rRNA 18S ribosomal RNA vii Taq Thermus aquaticus deoxyribonucleic acid polymerase TEM Transmission electron microscopy TgsGP T. b. gambiense specific gene TL Total length TLFs Trypanosome lytic factors TPB Two Peoples Bay USA United States of America UV Ultra violet light VSG Variable surface glycoprotein WA Western Australia WHO World Health Organisation YT Yeast extract and tryptone X-GAL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside viii ABSTRACT Trypanosomes are blood-borne parasites that can cause severe disease in both humans and animals, resulting in very large economic losses worldwide. In contrast to the wealth of information on pathogenic species such as Trypanosoma cruzi and Trypanosoma brucei, little is known of the pathogenicity, prevalence and life-cycles of trypanosomes in native Australian mammals. The aim of this thesis was to characterise trypanosomes and other protozoans from the critically endangered Gilbert’s potoroo (Potorous gilbertii) and the quokka (Setonix brachyurus) from Western Australia using morphological and molecular analysis. A novel Trypanosoma species, Trypanosoma copemani was identified in Gilbert’s potoroos and quokkas using molecular and morphological analysis. Further molecular characterisation of T. copemani in quokkas at both the 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loci identified multiple T. copemani genotypes revealing that the parasite is genetically variable. Given the phylogenetic grouping of T. copemani as a stercorarian (requiring faecal transmission by vector), the fact that ticks are common ectoparasites of Gilbert’s potoroos and quokkas and the previous identification of trypanosomes in ticks, ticks were examined as potential vectors of T. copemani. Motile trypanosomes were detected in both the haemolymph and midgut sections of Ixodes australiensis ticks removed from quokkas and Gilbert’s potoroos and stained trypanosomes were detected within a faecal smear. Morphologically, the tick trypanosomes resembled in vitro forms of T. copemani, representing epimastigotes and slender trypomastigote stages, with dividing stages detected within the midgut region. Molecular analysis of the tick isolates, showed 100% sequence identity to T. copemani at the 18S rRNA locus, suggesting that the tick is a putative vector for T. copemani. ix Investigation of the life-cycle stages of native Australian trypanosomes using microscopy and in vitro culture of blood from quokkas and Gilbert’s potoroos revealed that native Australian trypanosomes are highly polymorphic, with three different trypomastigote blood stream forms detected within blood films, resembling slender, medium and broad stages. In addition, promastigote, sphaeromastigote and amastigote stages were observed directly within blood films and this is the first report of these stages in the circulatory system of Australian marsupials. Three novel trypanosome life- cycle forms representing an oval form, an extremely thin form and an adherent form were also identified both in vitro and in vivo,