Ticks (Acari: Ixodida) transmit a greater variety of pathogens than any other blood-feeding group of arthropods. While numerous microbes have been identified inhabiting Australian Ixodidae, some of which are related to globally important tick-borne pathogens, little is known about the bacterial communities within ticks collected from Australian wildlife. In this study, 1,019 ticks were identified on 221 hosts spanning 27 wildlife species. Next-generation sequencing was used to amplify the V1-2 hypervariable region of the bacterial 16S rRNA gene from 238 ticks; Amblyomma triguttatum (n=6), Bothriocroton auruginans (n=11), Bothriocroton concolor (n=20), Haemaphysalis bancrofti (n=10), Haemaphysalis bremneri (n=4), Haemaphysalis humerosa (n=13), Haemaphysalis longicornis (n=4), Ixodes antechini (n=29), Ixodes australiensis (n=26), Ixodes fecialis (n=13), Ixodes holocyclus (n=37), Ixodes myrmecobii (n=1), Ixodes ornithorhynchi (n=10), Ixodes tasmani (n=51) and Ixodes trichosuri (n=3). After bioinformatic analyses, over 14 million assigned bacterial sequences revealed the presence of recently described bacteria 'Candidatus Borrelia tachyglossi', 'Candidatus Neoehrlichia australis', 'Candidatus Neoehrlichia arcana' and 'Candidatus Ehrlichia ornithorhynchi'. Furthermore, three novel Anaplasmataceae species were identified in the present study including; a Neoehrlichia sp. in I. australiensis and I. fecialis collected from quenda (Isoodon fusciventer) (Western Australia), an Anaplasma sp. from one B. concolor from echidna (Tachyglossus aculeatus) (New South Wales), and an Ehrlichia sp. from a single I. fecialis parasitising a quenda (WA). This study highlights the diversity of bacterial genera harboured within wildlife ticks, which may prove to be of medical and/or veterinary importance in the future.

Natural landscape alterations as a consequence of urbanisation are one of the main drivers in the movements of wildlife into metropolitan and peri-urban areas. Worldwide, these wildlife species are highly adaptable and may be responsible for the transmission of tick-borne pathogens including piroplasms (Babesia, Theileria and Cytauxzoon spp.) that cause piroplasmosis in animals and occasionally in humans. Little is known about piroplasms in the ticks of urban wildlife in Australia. Ticks from long-nosed bandicoots (Perameles nasuta; n = 71), eastern-barred bandicoots (Perameles gunnii; n = 41), northern-brown bandicoots (Isoodon macrourus; n = 19), southern-brown bandicoots (Isoodon obesulus; n = 4), bandicoot sp. (n = 2), flying foxes (Pteropus sp.; n = 3), black rats (Rattus rattus; n = 7), bush rats (Rattus fuscipes; n = 4), brushtail possums (Trichosurus vulpecula; n = 19), ringtail possums (Pseudocheirus peregrinus; n = 12), short-eared possums (Trichosurus caninus; n = 6), possum sp. (Trichosurus sp.; n = 8), and red foxes (Vulpes vulpes; n = 12) were analysed using piroplasm-specific 18S primers and Sanger sequencing. Seven Ixodes tasmani ticks from long-nosed bandicoots and bandicoots sp., three I. tasmani ticks and one Ixodes holocyclus tick from brushtail possums, and one Haemaphysalis longicornis tick from a red fox were positive for piroplasms. New genotypes, with sequences sharing 98% nucleotide similarities with Theileria sp. K1 detected in a burrowing bettong (Bettongia lesueur), were identified from bandicoot ticks. New genotypes were detected in ticks from brushtail possums, which shared 98% similarity with a Babesia sp. (JQ682877) previously identified in marsupials. Theileria orientalis was identified in the H. longicornis tick from the red fox. Babesia and Theileria spp. in the ticks parasitizing bandicoots and brushtail possums clustered closely with respective Babesia and Theileria clades derived from Australian marsupials. This represents the first detection of piroplasms in ticks parasitizing brushtail possums and a red fox in Australia.

Advances in sequencing technologies have revealed the complex and diverse microbial communities present in ticks (Ixodida). As obligate blood-feeding arthropods, ticks are responsible for a number of infectious diseases that can affect humans, livestock, domestic animals and wildlife. While cases of human tick-borne diseases continue to increase in the northern hemisphere, there has been relatively little recognition of zoonotic tick-borne pathogens in Australia. Over the past 5 years, studies using high-throughput sequencing technologies have shown that Australian ticks harbour unique and diverse bacterial communities. In the present study, free-ranging wildlife (n=203), representing ten mammal species, were sampled from urban and peri-urban areas in New South Wales (NSW), Queensland (QLD) and Western Australia (WA). Bacterial metabarcoding targeting the 16S rRNA locus was used to characterize the microbiomes of three sample types collected from wildlife: blood, ticks and tissue samples. Further sequence information was obtained for selected taxa of interest. Six tick species were identified from wildlife: Amblyomma triguttatum, Ixodes antechini, Ixodes australiensis, Ixodes holocyclus, Ixodes tasmani and Ixodes trichosuri. Bacterial 16S rRNA metabarcoding was performed on 536 samples and 65 controls, generating over 100 million sequences. Alpha diversity was significantly different between the three sample types, with tissue samples displaying the highest alpha diversity (P<0.001). Proteobacteria was the most abundant taxon identified across all sample types (37.3 %). Beta diversity analysis and ordination revealed little overlap between the three sample types (P<0.001). Taxa of interest included Anaplasmataceae , Bartonella , Borrelia , Coxiellaceae , Francisella , Midichloria , Mycoplasma and Rickettsia . Anaplasmataceae bacteria were detected in 17.7% (95/536) of samples and included Anaplasma , Ehrlichia and Neoehrlichia species. In samples from NSW, ‘Ca. Neoehrlichia australis’, ‘Ca. Neoehrlichia arcana’, Neoehrlichia sp. and Ehrlichia sp. were identified. A putative novel Ehrlichia sp. was identified from WA and Anaplasma platys was identified from QLD. Nine rodent tissue samples were positive for a novel Borrelia sp. that formed a phylogenetically distinct clade separate from the Lyme Borrelia and relapsing fever groups. This novel clade included recently identified rodent-associated Borrelia genotypes, which were described from Spain and North America. Bartonella was identified in 12.9% (69/536) of samples. Over half of these positive samples were obtained from black rats (Rattus rattus), and the dominant bacterial species identified were Bartonella coopersplainsensis and Bartonella queenslandensis . The results from the present study show the value of using unbiased high-throughput sequencing applied to samples collected from wildlife. In addition to understanding the sylvatic cycle of known vector-associated pathogens, surveillance work is important to ensure preparedness for potential zoonotic spillover events.

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Invasive rodent species are known hosts for a diverse range of infectious microorganisms and have long been associated with the spread of disease globally. The present study describes molecular evidence for the presence of a Trypanosoma sp. from black rats (Rattus rattus) in northern Sydney, Australia. Sequences of the 18S ribosomal RNA (rRNA) locus were obtained in two out of eleven (18%) blood samples with subsequent phylogenetic analysis confirming the identity within the Trypanosoma lewisi clade.

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 novels, 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.

Ticks (Acari: Ixodida) transmit a greater variety of pathogens than any other blood-feeding 1 2 arthropod. While numerous microbes have been identified inhabiting Australian Ixodidae, some of which are related to globally important tick-borne pathogens, little is known about the bacterial communities within ticks collected from Australian wildlife. In this study, 1,019 ticks were identified on 221 hosts spanning 27 wildlife species. Next-generation sequencing was used to amplify the V1-2 hypervariable region of the bacterial 16S rRNA gene from 238 ticks; Amblyomma triguttatum (n=6), Bothriocroton auruginans (n=11), Bothriocroton concolor (n=20), Haemaphysalis bancrofti (n=10), Haemaphysalis bremneri (n=4), Haemaphysalis humerosa (n=13), Haemaphysalis longicornis (n=4), Ixodes antechini (n=29), Ixodes australiensis (n=26), Ixodes fecialis (n=13), Ixodes holocyclus (n=37), Ixodes myrmecobii (n=1), Ixodes ornithorhynchi (n=10), Ixodes tasmani (n=51) and Ixodes trichosuri (n=3). After bioinformatic analyses, over 14 million assigned bacterial sequences revealed the presence of newly described bacteria 'Ca. Borrelia tachyglossi', 'Ca. Neoehrlichia australis', 'Ca. Neoehrlichia arcana' and 'Ca. Ehrlichia ornithorhynchi'. Furthermore, three novel Anaplasmataceae species were identified including; a Neoehrlichia sp. in I. australiensis and I. fecialis ex quenda (Isoodon fusciventer) (Western Australia), an Anaplasma sp. from one B. concolor ex echidna (Tachyglossus aculeatus) (New SouthWales), and an Ehrlichia sp. from a single I. fecialis parasitising a quenda (WA). This study highlights the diversity of bacterial genera harboured within wildlife ticks, which may prove to be of medical and/or veterinary importance in the future.

Invasive rodent species are known hosts for a diverse range of infectious microorganisms and have long been associated with the spread of disease globally. The present study describes molecular evidence for the presence of a Trypanosoma sp. from black rats (Rattus rattus) in northern Sydney, Australia. Sequences of the 18S ribosomal RNA (rRNA) locus were obtained in two out of eleven (18%) blood samples with subsequent phylogenetic analysis confirming the identity within the Trypanosoma lewisi clade. Plowright RK (2019) Dynamic and integrative approaches to understanding pathogen spillover. Philos Trans R Soc Lond