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Cryptosporidium Spp. in Wild and Captive Australian Flying Foxes

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Cryptosporidium Spp. in Wild and Captive Australian Flying Foxes Cryptosporidium spp. in wild and captive Australian flying foxes (genus: Pteropus) Sabine Eva Schiller, Bachelor of Philosophy Macquarie University, North Ryde, NSW, 2109, Australia Department of Biological Sciences Tel.: 61 2 9850 9259; Fax: 61 2 9850 8245 Submission date: 09.10.2015 1 Table of Contents Title Page……………………………………………………………………………………………………………………………………….….1 Table of Contents……………………………………………………………………………………………………………………………2-3 Abstract………………………………………………………………………………………………………………..……………………………4 Declaration………………………………………………………………………………………………………………………………………..5 Acknowledgements……………………………………………………………………………………………………………………………6 Abbreviations…………………………………………………………………………………………………………………………………….7 1. Introduction .................................................................................................................................... 8 1.1. Biology of Cryptosporidium .................................................................................................... 9 1.1.1. Cryptosporidium taxonomy and species identification....................................................... 9 1.1.2. Cryptosporidium: host specificity and cryptosporidiosis .................................................. 10 1.1.3. Cryptosporidium lifecycle .................................................................................................. 11 1.1.4. Sources and transmission pathways ................................................................................. 14 1.2. Zooanthroponotic pathogen transmission in wildlife and livestock ..................................... 14 1.3. Bats as reservoirs for zoonotic pathogens ............................................................................ 15 1.4. Australian flying foxes ........................................................................................................... 18 1.5. Impacts of ecological alterations on pathogen transmission ............................................... 20 1.6. Study aims ............................................................................................................................. 21 2. Materials and methods ................................................................................................................. 23 2.1. Sites and sample collection ................................................................................................... 23 2.2. DNA extraction and PCR screening for Cryptosporidium characterisation ........................... 24 2.3. PCR screening at the actin locus ........................................................................................... 25 2.4. Additional screening at the actin locus ................................................................................. 25 2.5. PCR screening at the glycoprotein 60 (gp60) locus .............................................................. 26 2.6. PCR purification protocols .................................................................................................... 26 2.7. DNA sequencing and sequence analysis ............................................................................... 26 2.8. Cryptosporidium 18s clone library ........................................................................................ 26 2.9. Statistical analysis ................................................................................................................. 27 3. Results ........................................................................................................................................... 27 3.1. Cryptosporidium detection ................................................................................................... 27 3.2. Cryptosporidium sp. identification ........................................................................................ 28 3.3. Cryptosporidium sp. confirmation targeting actin and gp60 ................................................ 29 4. Discussion ...................................................................................................................................... 32 4.1. Conclusion ............................................................................................................................. 35 2 5. References....................................................................................................................................36 5.1. Highlights .............................................................................................................................. 44 5.2. Graphical abstract ................................................................................................................. 44 Supplementary 1. PCR primers and corresponding primer sequences...................................45 6. Appendix ....................................................................................................................................... 46 List of Tables Table 1. Cryptosporidium sp. identified in animals and humans ............................................. 12 Table 2. Reports of potential anthropogenic parasite transmission in wildlife and livestock 16 Table 3. Protozoan parasites identified in megabats .............................................................. 22 Table 4. Positive PCR results for loci tested across sampling sites. ......................................... 28 Table 5. Differences among partical 18s rDNA regions for Cryptosporidium bat genotypes..30 Table 6. Species identification for cloned isolates ................................................................... 31 List of Figures Figure 1. Cryptosporidium lifecycle .......................................................................................... 11 Figure 2. Australian flying fox distribution............................................................................... 19 Figure 3. Sampling locations for wild and captive flying fox populations in NSW and QLD .... 24 3 1 Abstract 2 Spillover of zoonotic pathogens from wildlife to humans is a primary threat to global health, but 3 the potential impacts of reverse pathogen transmission (zooanthroponosis) is still largely 4 unexplored. Increasing establishment of wildlife species in regional and urban Australia potentially 5 increases risk of human-borne pathogen spillover at the human/wildlife interface. To explore this 6 issue, the occurrence of the protozoan parasite Cryptosporidium was investigated in urbanised 7 flying fox populations. 8 Cryptosporidium infects a wide range of vertebrates, with species varying in host specificity. In 9 humans, C. hominis and C. parvum are responsible for the majority of infections. PCR screening of 10 faecal samples (n = 281) from seven wild and two captive flying fox populations identified the 11 presence of Cryptosporidium in 3.2% of samples, with a prevalence of 1.7% in wild versus 6.3% in 12 captive individuals (χ² = 3.708, DF = 1, p= 0.054). Using multilocus sequencing (18s rRNA, actin and 13 gp60) C. hominis was identified in captive animals (n = 2) and four novel Cryptosporidium 14 genotypes in wild and captive animals (n = 7). This is the first study to report the presence of 15 Cryptosporidium spp. in Australian flying foxes and findings indicate zooanthroponotic 16 transmission of Cryptosporidium from humans to flying foxes. 17 4 1 This thesis is written in the form of a journal article from International Journal of Parasitology: 2 Parasites and Wildlife, including an extended introduction/literature review (section1) Declaration 3 I wish to acknowledge the following assistance in the research detailed in this report: Dr. Michelle Power (Principal supervisor) Dr. Koa Webster Jennefer Maclean, Tolga Bat Hospital (Sample collection) Kerryn Parry-Jones, Wambina Flying Fox Sanctuary (Sample collection) Tim Pearson, Macquarie University (Sample collection) 4 I hereby declare that the work in this thesis entitled “Cryptosporidium spp. in wild and captive 5 Australian flying foxes (genus: Pteropus)” is an original piece of research conducted as part of a 6 nine month Masters of Research project. It has not previously been submitted for a degree to any 7 university other than Macquarie University. Any assistance that I have received during this project has been appropriately acknowledged along with all relevant information sources and literature in this thesis. Funding for this project was provided by the Ian Potter Foundation and Macquarie University. I hereby declare that I am not aware of any conflict of interest in relation to this project. All other research described in this report is my own original work. Sabine Eva Schiller (Student ID: 41967062) 09.10.2015 5 Acknowledgements Firstly I would like to thank my supervisor Dr. Michelle Power for her invaluable support and guidance during my Masters year. Thank you for sharing your vast knowledge and for teaching me so many different lab techniques. Your attention to detail is remarkable and I have benefitted greatly from your assistance throughout this project. I would also like to extend a big thank you to Dr. Koa Webster for providing me with regular feedback which has been much appreciated. I would like to particularly thank you for your help with calculating PCR concentrations. I would also like to thank Elke Vermeulen for all her help in the lab and for answering my many questions. I will never forget our time at the ASP Conference in Auckland. I am also extremely grateful to Matt Lott for his advice and support and to Amy Asher for being a lovely lab mate. Also a big thank
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