Infections with Trypanosoma spp. have been associated with poor health and decreased survival of koalas (Phascolarctos cinereus), particularly in the presence of concurrent pathogens such as Chlamydia and koala retrovirus. The present study describes the application of a next-generation sequencing (NGS)-based assay to characterise the prevalence and genetic diversity of trypanosome communities in koalas and two native species of ticks (Ixodes holocyclus and I. tasmani) removed from koala hosts. Among 168 koalas tested, 32.2% (95% CI: 25.2–39.8%) were positive for at least one Trypanosoma sp. Previously described Trypanosoma spp. from koalas were identified, including T. irwini (32.1%, 95% CI: 25.2–39.8%), T. gilletti (25%, 95% CI: 18.7–32.3%), T. copemani (27.4%, 95% CI: 20.8–34.8%) and T. vegrandis (10.1%, 95% CI: 6.0–15.7%). Trypanosoma noyesi was detected for the first time in koalas, although at a low prevalence (0.6% 95% CI: 0–3.3%), and a novel species (Trypanosoma sp. AB-2017) was identified at a prevalence of 4.8% (95% CI: 2.1–9.2%). Mixed infections with up to five species were present in 27.4% (95% CI: 21–35%) of the koalas, which was significantly higher than the prevalence of single infections 4.8% (95% CI: 2–9%). Overall, a considerably higher proportion (79.7%) of the Trypanosoma sequences isolated from koala blood samples were identified as T. irwini, suggesting this is the dominant species. Co-infections involving T. gilletti, T. irwini, T. copemani, T. vegrandis and Trypanosoma sp. AB-2017 were also detected in ticks, with T. gilletti and T. copemani being the dominant species within the invertebrate hosts. Direct Sanger sequencing of Trypanosoma 18S rRNA gene amplicons was also performed and results revealed that this method was only able to identify the genotypes with greater amount of reads (according to NGS) within koala samples, which highlights the advantages of NGS in detecting mixed infections. The present study provides new insights on the natural genetic diversity of Trypanosoma communities infecting koalas and constitutes a benchmark for future clinical and epidemiological studies required to quantify the contribution of trypanosome infections on koala survival rates.

Realizou-se um estudo epidemiológico observacional retrospectivo para caracterizar os acidentes escorpiônicos em Belo Horizonte, Minas Gerais, Brasil, entre 2005 e 2009. Foram coletados dados do Sistema Nacional de Informação de Agravos de Notificação (SINAN) e Sistema de Vigilância Epidemiológica (SISVE) e das fichas de atendimento do Hospital João XXIII. Observou-se um total de 2.769 casos no período (114,7/100 mil habitantes), tendência decrescente e maior ocorrência entre agosto e janeiro. A espécie Tityus serrulatus foi responsável pela maioria das picadas. Não houve diferença estatística entre os gêneros dos indivíduos acometidos e a faixa etária entre 55 e 64 anos foi a que apresentou maior risco para escorpionismo. Noventa e seis por cento dos quadros clínicos evoluíram para a cura, tendo sido observados dois óbitos. Alerta-se para necessidade de melhorias no processo de notificação de acidentes por escorpião e sugere-se considerar seus determinantes para o planejamento e direcionamento de intervenções por parte do serviço público de saúde.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Hoare, 1972; Baker, 1973;Marinkelle, 1976Marinkelle, , 1979 Gardner and Molyneux, 1988a, b; Hamanaka and Pinto Ada, 1993; Steindel et al., 1998; Barnabe et al., 2003; Grisard et al., 2003; Lisboa et al., 2008; Cottontail et al., 2009;Maia da Silva et al., 2009; Cavazzana et al., 2010; Garcia et al., 2012; Hamilton et al., 2012; Lima et al., 2012;Lima et al., 2013;Marcili et al., 2013; Silva-Iturriza et al., 2013; Cottontail et al., 2014; Ramirez et al., 2014).In Australia, three Trypanosoma spp. have been described in bats to date:Trypanosoma pteropi from the black flying fox (Pteropus gouldii) (Breinl, 1913; Mackerras, 1959), Trypanosoma hipposideri from the dusky horseshoe bat (Hipposideros bicolor albanensis) and Trypanosoma vegrandis, in pteropid bats (Yangochiroptera) and microbats In the present study, we describe the morphological and genetic characterisation of the novel trypanosome in the little red flying fox (Mackie et al., 2015), for which we proposed the name Trypanosoma teixeirae sp. n. Material and Methods Sample collectionA venous blood sample was collected from the cephalic vein of an adult female little red flying fox that presented to the Australia Zoo Wildlife Hospital (AZWH) in April, 2014.The flying fox had been rescued from the ground at Redcliffe in south-eastern Queensland, Australia and was moribund with anaemia and icterus. Clinical and pathological evidence of disease consistent with trypanosomosis in this flying fox was described by Mackie et al.. Morphological analysesThin blood smears were made from a drop of fresh blood and stained with Diff Quick (Siemens, Germany). After air-drying, the slides were then cover-slipped using DePeX 5 mounting medium Gurr (Merck Pty. Limited, Kilsyth, Victoria, Australia). Stained films were systematically examined using a BX50 microscope (Olympus, Japan) with screen views generated by a DP Controller (version 3.2.1.276, Olympus, Japan). Digital light micrograph images of any trypomastigotes observed were taken at x1000 magnification.Digital images of the organisms identified in the blood films were used to measure key morphological features such as total length (TL), width (W), posterior to kinetoplast (PK), kinetoplast to nucleus (KN), nucleus to anterior (NA) and free flagellum (FF), according to parameters described by Hoare (1972) and Mackerras (1959). Means and standard errors were calculated. The morphological measurements were taken using the software Image J (Abramoff et al., 2004).As two trypanosome species have previously been described in Australian bats based on morphol...

Case report An adult female Australian little red flying fox (Pteropus scapulatus) presented with icterus and anaemia. Examination of a blood smear revealed numerous trypanosomes 20.4–30.8 µm long with tapered ends. Necropsy and histological findings were consistent with trypanosome infection of lymphoid tissue and intravascular haemolysis. Sequence and phylogenetic analysis demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma minasense and Trypanosoma rangeli (with a genetic distance of 1% at the 18S rRNA locus for both). Conclusion To the authors’ knowledge this is the first report of a trypanosome infection associated with clinical disease in bats.

A molecular survey was conducted to provide baseline information on the prevalence, genetic diversity and potential clinical impacts of blood-borne and enteric protozoans in native wild mammals from the Northern Territory (NT). A total of 209 blood and 167 faecal samples were collected from four target species; the northern brown bandicoot (Isoodon macrourus), common brushtail possum (Trichosurus vulpecula), northern quoll (Dasyurus hallucatus) and brush-tailed rabbit-rat (Conilurus penicillatus). Blood samples were screened by PCR at the 18S rRNA gene for trypanosomes, piroplasms and haemogregarines, with faecal samples tested for Cryptosporidium spp. at the 18S rRNA locus, and for Giardia spp. at the glutamate dehydrogenase (gdh) and 18S rRNA loci. The potential clinical impact was investigated by associating clinical, haematological and biochemical parameters with presence or absence of infection. Overall, 22.5% (95% CI: 17.0-28.8%) of the animals tested were positive for haemoprotozoans. Trypanosomes were found in 26.6% (95% CI: 18.7-35.7%) of the bandicoots and were identified as Trypanosoma vegrandis G6, except for one unique genotype, most similar to T. vegrandis G3 (genetic distance=7%). The prevalence of trypanosomes in possums was 23.7% (95% CI: 11.4-40.2%), and the genotypes identified clustered within the T. noyesi clade. The presence of Babesia sp. and Hepatozoon sp. was confirmed in bandicoots only, both at a prevalence of 9.7% (95% CI: 2.7-9.2%). The total prevalence of intestinal protozoan parasites observed was relatively low (3%; 95% CI: 1.0-6.9%). No evidence of clinical disease associated with protozoan parasitic infection was observed, however bandicoots positive for Trypanosoma exhibited a significantly lower packed cell volume (PCV) compared to negative bandicoots (p=0.046). To the authors' knowledge, this is the first research conducted in the NT to characterise protozoan parasites in threatened native mammals using both molecular and morphological tools; and to assess the potential clinical impacts of these agents. The absence of clear signs of major morbidity in infected animals seems to exclude a direct association between infections with these agents and possible population decline events in northern Australian native mammals. However until the cause(s) of population decline are ascertained for each individual mammal species, further studies are required. The outcome of the present investigation may be used to inform wildlife conservation and zoonotic disease programs.

Bats (order Chiroptera) have been increasingly recognised as important reservoir hosts for human and animal pathogens worldwide. In this context, molecular and microscopy-based investigations to date have revealed remarkably high diversity of Trypanosoma spp. harboured by bats, including species of recognised medical and veterinary importance such as Trypanosoma cruzi and Trypanosoma evansi (aetiological agents of Chagas disease and Surra, respectively). This review synthesises current knowledge on the diversity, taxonomy, evolution and epidemiology of bat trypanosomes based on both molecular studies and morphological records. In addition, we use a One Health approach to discuss the significance of bats as reservoirs (and putative vectors) of T. cruzi, with a focus on the complex associations between intra-specific genetic diversity and eco-epidemiology of T. cruzi in sylvatic and domestic ecosystems. This article also highlights current knowledge gaps on the biological implications of trypanosome co-infections in a single host, as well as the prevalence, vectors, life-cycle, host-range and clinical impact of most bat trypanosomes recorded to date. Continuous research efforts involving molecular surveillance of bat trypanosomes are required for improved disease prevention and control, mitigation of biosecurity risks and potential spill-over events, ultimately ensuring the health of humans, domestic animals and wildlife globally.

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The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert’s potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.