RESEARCH REPOSITORY This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination. The definitive version is available at: https://doi.org/10.1017/S0031182020001845 Austen, J.M., Van Kampen, E., Egan, S.L., O'Dea, M.A., Jackson, B., Ryan, U.M., Irwin, P.J. and Prada, D. (2020) First report of Trypanosoma dionisii (Trypanosomatidae) identified in Australia. Parasitology https://researchrepository.murdoch.edu.au/id/eprint/58241 Copyright: © 2020 The Authors It is posted here for your personal use. No further distribution is permitted. First report of Trypanosoma dionisii (Trypanosomatidae) identified in Australia Jill M. Austen1, Esther Van Kampen2, Siobhon L. Egan1, Mark A. O‟Dea2, Bethany Jackson2, Una M. Ryan1, Peter J. Irwin1, Diana Prada2 Author affiliations 1Vector and Waterborne Pathogens Research Group, College of Science, Health, Engineering and Education, Murdoch University, Perth 6150, Australia 2School of Veterinary Medicine, Murdoch University, Perth, WA 6150, Australia Correspondence: Dr. Jill M. Austen; ORCID ID: https://orcid.org/0000-0002-1826-1634 Vector and Waterborne Pathogens Research Group, College of Science, Health, Education and Engineering, Murdoch University, Perth, WA, 6150, Australia Email: [email protected] Tel: +61 8 9360 6314. Fax: +61 8 9310 4144. Running title: Trypanosoma dionisii identified and characterised in native Australian bats This is an Accepted Manuscript for Parasitology. This version may be subject to change during the production process. DOI: 10.1017/S0031182020001845 Downloaded from https://www.cambridge.org/core. Murdoch University, on 19 Oct 2020 at 05:31:09, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182020001845 Abstract Trypanosomes are blood-borne parasites that can infect a variety of different vertebrates, including animals and humans. This study aims to broaden scientific knowledge about the presence and biodiversity of trypanosomes in Australian bats. Molecular and morphological analysis was performed on 86 blood samples collected from seven different species of microbats in Western Australia. Phylogenetic analysis on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) sequences identified Trypanosoma dionisii in five different Australian native species of microbats; Chalinolobus gouldii, Chalinolobus morio, Nyctophilus geoffroyi, Nyctophilus major and Scotorepens balstoni. In addition, two novel, genetically distinct T. dionisii genotypes were detected and named T. dionisii genotype Aus 1 and T. dionisii genotype Aus 2. Genotype Aus 2 was the most prevalent and infected 20.9% (18/86) of bats in the present study, while genotype Aus 1 was less prevalent and was identified in 5.8% (5/86) of Australian bats. Morphological analysis was conducted on trypomastigotes identified in blood films, with morphological parameters consistent with trypanosome species in the subgenus Schizotrypanum. This is the first report of T. dionisii in Australia and in Australian native bats, which further contributes to the global distribution of this cosmopolitan bat trypanosome. Key words Australia, Trypanosoma dionisii, microbats, Chalinolobus gouldii, Chalinolobus morio, Nyctophilus geoffroyi, Nyctophilus major, Scotorepens balstoni 1 Downloaded from https://www.cambridge.org/core. Murdoch University, on 19 Oct 2020 at 05:31:09, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182020001845 Introduction Since the 1900s, numerous trypanosome species have been identified worldwide, infecting all classes of vertebrates, with the majority appearing to cause no disease to their hosts (Hoare, 1972; Tyler and Engman, 2001; Hamilton et al., 2007). Bats (order: Chiroptera) evolved ~50- 52 million years ago during the early Eocene period and comprise ~20% of extant mammals (Teeling et al., 2005). Bats are among the most common hosts of a large variety of trypanosome species worldwide, with the subgenus Schizotrypanum comprising the majority of the trypanosomes reported in bats throughout South America, Europe, Africa and Asia, (Hamilton et al., 2012b; Lima et al., 2013; Mafie et al., 2018; Wang et al., 2019). Trypanosomes in Chiroptera were first described in Miniopterus schreibersii from Italy by Dionisi in 1899 (Baker et al., 1972). Since then over 30 species of trypanosomes have been reported in more than 100 bat species, including well defined Trypanosoma cruzi, Trypanosoma vespertilionis, Trypanosoma rangeli and the globally distributed Trypanosoma dionisii (Beltz, 2017). Relatively little is known about Trypanosoma species in native Australian bat species. Trypanosoma pteropi was the first described trypanosome in Pteropus sp. from north Queensland (Breinl, 1913). Similar trypanosomes were characterised by Mackerras (1959) in Pteropus gouldii, however they were described as smaller in length, had a pointed anterior and a rounded kinetoplast, similar to T. vespertilionis from European bats, which shares biological characteristics with T. cruzi with both species having the ability to reproduce in organs (Mackerras, 1959). Mackerras (1959) also described a very tiny and slender trypanosome named Trypanosoma hipposideri from Hipposideros ater captured in northern Queensland. It was reported to be smaller than T. vespertilionis and T. pteropi, with a large distinctive kinetoplast and delicate short free flagellum. More recently, the first molecular 2 Downloaded from https://www.cambridge.org/core. Murdoch University, on 19 Oct 2020 at 05:31:09, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182020001845 study identified a high prevalence of Trypanosoma vegrandis in two species of pteropid bats (Pteropus alecto and Pteropus scapulatus) and two species of microbats (Chalinolobus gouldii and Nyctophilus geoffroyi) from Western Australian (Austen et al., 2015). Trypanosoma teixeirae from a native Australian bat, P. scapulatus was also recently described by Mackie et al. (2017) and further characterized by Barbosa et al. (2016). This trypanosome is longer in length than both T. pteropi and T. hipposideri and genetically similar to Trypanosoma rangeli and Trypanosoma minasense (Mackie et al., 2017). Interestingly T. teixeirae is the first bat trypanosome in Australia to cause clinical disease in its host, with descriptions of icterus and anaemia (Mackie et al., 2017). The aim of the present study was to investigate trypanosome species in seven species of microbats from the South-West Botanical Province of Western Australia, to gain insight into the prevalence and genetic diversity of trypanosomes in these species. Methods Field sample collection Animal work was performed with approval from the Murdoch University Animal Ethics Committee under permit #R2882/16. Field work was conducted between the 30th of November to the 9th of December 2016 at Charles Darwin Reserve (-29.581817, 117.001820) and Mount Gibson Wildlife Sanctuary (-29.630577, 117.277211), located within the south- west region of Western Australia, a globally recognised biodiversity hotspot (Myers et al., 2000). Bats were caught using harp traps (Faunatech, Australia) and mist nets (Ecotone, Australia) set over water points at sunset. The mist nets were monitored constantly with bats removed from the nets immediately after capture. Harp traps were left unmonitored for approximately three hours and then checked for individuals captured in the protective 3 Downloaded from https://www.cambridge.org/core. Murdoch University, on 19 Oct 2020 at 05:31:09, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182020001845 bagging. Sampling occurred at the capture site with no individuals being held for greater than two hours and handling limited to 5 minutes per individual to minimise stress. Palpably pregnant females were released without sampling. Bats were identified to species level using published keys (Churchill, 2008). Blood samples were collected from 86 microbats representing 7 known species; Chalinolobus gouldii (n=53), Chalinolobus morio (n=3), Nyctophilus geoffroyi (n=16), Nyctophilus major (n=1), Ozimops sp. (n=2), Scotorepens balstoni (n=7) and Vespadelus baverstocki (n=4). Ten microlitres of blood was collected from the uropatagial vein using a 25 gauge needle and a micropipette, and then diluted in sterile 1:10 phosphate buffered saline. Individual blood smears were made, air dried, and fixed in 100% methanol. Blood samples were stored at 4°C in the field and later stored at − 20 °C until use. DNA extraction and molecular analysis DNA was extracted from ethylene diamine tetraacetic acid (EDTA)-preserved whole blood using a NucleoMag VET kit (Macherey-Nagel, Germany) on an automated MagMax 96 processor (Life Technologies Corporation, U.S.A.) according to the manufacturer‟s instructions. Partial fragments of Trypanosoma spp. DNA were amplified from the 18S rDNA and gGAPDH loci using primers previously described by Maslov et al. (1996) and McInnes et al. (2009), (Table 1). Initially, samples were screened using a single round PCR to amplify a small fragment (~350bp) of Trypanosoma DNA using the 18S rDNA primer set S825F and S662R. Once sequences were obtained, a subsample of positives representing different genotypes in the initial