An invasive human commensal and a native marsupial maintain tick populations at the urban fringe.

Ticks (Acari: Ixodidae) are major disease vectors globally making it increasingly important to understand how altered vertebrate communities in urban areas shape tick population dynamics. In urban landscapes of Australia, little is known about which native and introduced small mammals maintain tick populations preventing host-targeted tick management and leading to human-wildlife conflict. Here, we determined (1) larval, nymphal, and adult tick burdens on host species and potential drivers, (2) the number of ticks supported by the different host populations, and (3) the proportion of medically significant tick species feeding on the different host species in Northern Sydney. We counted 3551 ticks on 241 mammals at 15 sites and found that long-nosed bandicoots (Perameles nasuta) hosted more ticks of all life stages than other small mammals but introduced black rats (Rattus rattus) were more abundant at most sites (33%-100%) and therefore important in supporting larval and nymphal ticks in our study areas. Black rats and bandicoots hosted a greater proportion of medically significant tick species including Ixodes holocyclus than other hosts. Our results show that an introduced human commensal contributes to maintaining urban tick populations and suggests ticks could be managed by controlling rat populations on urban fringes.

First Report of Candidatus Mycoplasma haemohominis Infection in Australia Causing Persistent Fever in an Animal Carer.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. METHODS: After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient's bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. RESULTS: WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. CONCLUSIONS: This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.

First report of Trypanosoma dionisii (Trypanosomatidae) identified in Australia.

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.

Molecular identification of the Trypanosoma (Herpetosoma) lewisi clade in black rats (Rattus rattus) from Australia.

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.

No evidence for widespread Babesia microti transmission in Australia.

BACKGROUND: A fatal case of autochthonous Babesia microti infection was reported in Australia in 2012. This has implications for Australian public health and, given that babesiosis is transfusion transmissible, has possible implications for Australian blood transfusion recipients. We investigated the seroprevalence of antibodies to B. microti in Australian blood donors and in patients with clinically suspected babesiosis. STUDY DESIGN AND METHODS: Plasma samples (n = 7,000) from donors donating in at-risk areas and clinical specimens from patients with clinically suspected babesiosis (n = 29) were tested for B. microti IgG by immunofluorescence assay (IFA). IFA initially reactive samples were tested for B. microti IgG and IgM by immunoblot and B. microti DNA by polymerase chain reaction. RESULTS: Although five donors were initially reactive for B. microti IgG by IFA, none was confirmed for B. microti IgG (zero estimate; 95% confidence interval, 0%-0.05%) and all were negative for B. microti DNA. None of the patient samples had B. microti IgG, IgM, or DNA. CONCLUSIONS: This study does not provide evidence for widespread exposure to B. microti in Australian blood donors at local theoretical risk, nor does it provide evidence of B. microti infection in Australian patients with clinically suspected babesiosis. Given that confirmed evidence of previous exposure to B. microti was not seen, these data suggest that transmission of this pathogen is currently uncommon in Australia and unlikely to pose a risk to transfusion safety at present.

Investigation of the morphological diversity of the potentially zoonotic Trypanosoma copemani in quokkas and Gilbert's potoroos.

Trypanosomes are blood-borne parasites that can cause severe disease in both humans and animals, yet little is known of the pathogenicity and life-cycles of trypanosomes in native Australian mammals. Trypanosoma copemani is known to be infective to a variety of Australian marsupials and has recently been shown to be potentially zoonotic as it is resistant to normal human serum. In the present study, in vivo and in vitro examination of blood and cultures from Australian marsupials was conducted using light microscopy, immunofluorescence, scanning electron microscopy and fluorescence in situ hybridization. Promastigote, sphaeromastigote and amastigote life-cycle stages were detected in vivo and in vitro. Novel trypanosome-like stages were also detected both in vivo and in vitro representing an oval stage, an extremely thin stage, an adherent stage and a tiny round stage. The tiny round and adherent stages appeared to adhere to erythrocytes causing potential haematological damage with clinical effects similar to haemolytic anaemia. The present study shows for the first time that trypomastigotes are not the only life-cycle stages circulating within the blood stream of trypanosome infected Australian native marsupials and provides insights into possible pathogenic mechanisms of this potentially zoonotic trypanosome species.

Molecular confirmation of the first autochthonous case of human babesiosis in Australia using a novel primer set for the beta-tubulin gene.

In 2012, the first autochthonous Australian case of human babesiosis was reported, after microscopic examinations of blood samples revealed intra-erythrocytic parasites in a hospitalized 56year-old man from NSW, who died in 2011 (Senanayake et al., 2012). Independent molecular analyses carried out in Australia and the USA, identified Babesia microti at the 18S ribosomal RNA (18S rRNA), and the beta-tubulin (ß-tubulin) gene loci. Here we present the details of a novel PCR-based assay for the ß-tubulin gene that was developed, during the original study, to corroborate the results obtained from the analysis of the 18S rDNA. The complete phylogenetic reconstruction, based on the two loci sequenced from the Australian clinical isolate, is also shown here for the first time.

Novel genotypes of Trypanosoma binneyi from wild platypuses (Ornithorhynchus anatinus) and identification of a leech as a potential vector.

Little is known about the prevalence and pathogenesis of trypanosomes in Australian monotremes, and few genetic characterisation studies have been conducted with these haemoparasites. During the present investigation, molecular and microscopic methods were used to screen peripheral blood (n=28) and ectoparasites (n=10 adult ticks; n=5 tick nymphs; n=1 leech; and n>500 tick eggs) collected from wild Tasmanian platypuses (Ornithorhynchus anatinus), for the presence of trypanosomatid-specific DNA and/or trypomastigotes. The genes for the small ribosomal subunit RNA (18S rDNA) and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) were amplified and sequenced, prior to conducting phylogenetic analyses. The detection rate of the parasite-specific 18S rDNA in platypus blood was 85.7% (n=24/28), and the leech was also positive at both loci. Microscopically, high parasitaemia and the presence of abundant trypomastigotes, morphologically consistent with Trypanosoma binneyi Mackerras (1959), were observed in the blood films. Phylogenetic analyses at the 18S locus revealed the existence of four trypanosomatid-like genotypes, with variable similarity to two previously-described genotypes of T. binneyi (range of genetic p-distance: 0.0-0.5%). For the gGAPDH locus, for which only one T. binneyi sequence is available in GenBank, three genotypes closely related T. binneyi were identified (range of genetic p-distance: 0.1-0.4%). The leech-derived trypanosome isolate was virtually identical (at the two loci studied) to the other parasites sequenced from infected platypuses; however, the molecular or morphological identification of the leech species was not possible. Although further studies are required, the molecular detection of trypanosomes in an aquatic leech removed from a platypus, suggests the possibility that these haematophagous hirudineans may be a vector for T. binneyi (and closely related genotypes).

Piroplasms of New Zealand seabirds.

Blood and ectoparasitic ticks were collected from migratory seabirds in New Zealand, including Australasian gannets (n = 13) from two sites and red-billed gulls (n = 9) and white-fronted terns (n = 2) from a third location. Blood smears were screened for parasite presence by microscopy, while DNA from blood samples was subjected to PCR for the presence of tick-transmitted protozoan haemoparasites belonging to the order Piroplasmida. Parasites were identified by comparing small subunit ribosomal RNA (18S rDNA) gene sequences to related sequences on GenBank. Analyses indicated that nine birds were infected with unknown variants of a Babesia poelea-like parasite (recorded as genotypes I and II), while four harboured a piroplasm that was genetically similar to Babesia kiwiensis. There was no parasite stratification by bird species; both the gannets and gulls were positive for all three parasites, while the terns were positive for the B. kiwiensis-like and the B. poelea-like (genotype I) parasites. The B. kiwiensis-like parasite found in the birds was also found in two species of ticks: Carios capensis and Ixodes eudyptidis. This represents the first report of Babesia-positive ticks parasitising seabirds in New Zealand. The lack of host specificity and evidence of wide ranging distributions of the three piroplasm genotypes suggests there is a high degree of haemoparasite transmission occurring naturally between New Zealand seabird populations and species.

Molecular analysis of human tick-bitten skin yields signatures associated with distinct spatial and temporal trajectories - A proof-of-concept study.

Tick-associated diseases present challenges due to tridirectional interactions among host-specific responses, tick toxins and salivary proteins as well as microbes. We aimed to uncover molecular mechanisms in tick-bitten skin samples (cases) and contralateral skin samples (controls) collected simultaneously from the same participants, using spatial transcriptomics. Cases and controls analysed using NanoString GeoMx Digital Spatial Profiler identified 274 upregulated and 840 downregulated differentially expressed genes (DEGs), revealing perturbations in keratinization and immune system regulation. Samples of skin biopsies taken within 72 h post tick-bite DEGs had changes in protein metabolism and viral infection pathways as compared to samples taken 3 months post tick-bite, which instead displayed significant perturbations in several epigenetic regulatory pathways, highlighting the temporal nature of the host response following tick bites. Within-individual signatures distinguished tick-bitten samples from controls and identified between-individual signatures, offering promise for future biomarker discovery to guide prognosis and therapy.

A systems biology approach to better understand human tick-borne diseases.

Tick-borne diseases (TBDs) are a growing global health concern. Despite extensive studies, ill-defined tick-associated pathologies remain with unknown aetiologies. Human immunological responses after tick bite, and inter-individual variations of immune-response phenotypes, are not well characterised. Current reductive experimental methodologies limit our understanding of more complex tick-associated illness, which results from the interactions between the host, tick, and microbes. An unbiased, systems-level integration of clinical metadata and biological host data - obtained via transcriptomics, proteomics, and metabolomics - offers to drive the data-informed generation of testable hypotheses in TBDs. Advanced computational tools have rendered meaningful analysis of such large data sets feasible. This review highlights the advantages of integrative system biology approaches as essential for understanding the complex pathobiology of TBDs.

Identification of novel Babesia and Theileria genotypes in the endangered marsupials, the woylie (Bettongia penicillata ogilbyi) and boodie (Bettongia lesueur).

Piroplasms, which include the genera Theileria and Babesia, are blood-borne parasites transmitted mainly by tick vectors. Relatively little is known about their prevalence and clinical impact in Australian marsupials. In the present study the occurrence and molecular phylogeny of these parasites were studied in both wild and captive marsupials from Western Australia (WA) and Queensland (QLD). Blood samples were screened by microscopy and molecular methods, using PCR and DNA sequencing of the 18S ribosomal RNA gene (18S rDNA). Overall, 7.1% of the blood samples (8/113) were positive for piroplasm 18S rDNA. Theileria and Babesia rDNA was detected in 0.9% (1/113) and 6.2% (7/113) of the animals, respectively. The single Theileria positive was identified in one of three boodies (Bettongia lesueur) screened from a wildlife rehabilitation centre in WA, while all seven Babesia positives were detected in WA in wild captured woylies (Bettongia penicillata ogilbyi). Small intraerythrocytic inclusions were observed in blood films made from six of these individuals. This is the first report of a Babesia sp. in woylies, and Theileria sp. in boodies. Phylogenetic analysis indicated that the woylie-derived Babesia was genetically distinct and most closely related to Babesia occultans, the causative agent of a benign form of cattle babesiosis (genetic similarity 98.4%). The Theileria identified was most closely related to the marsupial-derived species Theileria penicillata from the woylie, Theileria brachyuri from the quokka (Setonix brachyurus), and Theileria sp. from the long-nosed potoroo (Potorous tridactylus).

The Troublesome Ticks Research Protocol: Developing a Comprehensive, Multidiscipline Research Plan for Investigating Human Tick-Associated Disease in Australia.

In Australia, there is a paucity of data about the extent and impact of zoonotic tick-related illnesses. Even less is understood about a multifaceted illness referred to as Debilitating Symptom Complexes Attributed to Ticks (DSCATT). Here, we describe a research plan for investigating the aetiology, pathophysiology, and clinical outcomes of human tick-associated disease in Australia. Our approach focuses on the transmission of potential pathogens and the immunological responses of the patient after a tick bite. The protocol is strengthened by prospective data collection, the recruitment of two external matched control groups, and sophisticated integrative data analysis which, collectively, will allow the robust demonstration of associations between a tick bite and the development of clinical and pathological abnormalities. Various laboratory analyses are performed including metagenomics to investigate the potential transmission of bacteria, protozoa and/or viruses during tick bite. In addition, multi-omics technology is applied to investigate links between host immune responses and potential infectious and non-infectious disease causations. Psychometric profiling is also used to investigate whether psychological attributes influence symptom development. This research will fill important knowledge gaps about tick-borne diseases. Ultimately, we hope the results will promote improved diagnostic outcomes, and inform the safe management and treatment of patients bitten by ticks in Australia.

Illuminating the bacterial microbiome of Australian ticks with 16S and Rickettsia-specific next-generation sequencing.

Next-generation sequencing (NGS) studies show that mosquito and tick microbiomes influence the transmission of pathogens, opening new avenues for vector-borne pathogen control. Recent microbiological studies of Australian ticks highlight fundamental knowledge gaps of tick-borne agents. This investigation explored the composition, diversity and prevalence of bacteria in Australian ticks (n = 655) from companion animals (dogs, cats and horses). Bacterial 16S NGS was used to identify most bacterial taxa and a Rickettsia-specific NGS assay was developed to identify Rickettsia species that were indistinguishable at the V1-2 regions of 16S. Sanger sequencing of near full-length 16S was used to confirm whether species detected by 16S NGS were novel. The haemotropic bacterial pathogens Anaplasma platys, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum" and Coxiella burnetii were identified in Rhipicephalus sanguineus (s.l.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, Ixodes holocyclus from QLD, Rh. sanguineus (s.l.) from the NT, and I. holocyclus from QLD, respectively. Analysis of the control data showed that cross-talk compromises the detection of rare species as filtering thresholds for less abundant sequences had to be applied to mitigate false positives. A comparison of the taxonomic assignments made with 16S sequence databases revealed inconsistencies. The Rickettsia-specific citrate synthase gene NGS assay enabled the identification of Rickettsia co-infections with potentially novel species and genotypes most similar (97.9-99.1%) to Rickettsia raoultii and Rickettsia gravesii. "Candidatus Rickettsia jingxinensis" was identified for the first time in Australia. Phylogenetic analysis of near full-length 16S sequences confirmed a novel Coxiellaceae genus and species, two novel Francisella species, and two novel Francisella genotypes. Cross-talk raises concerns for the MiSeq platform as a diagnostic tool for clinical samples. This study provides recommendations for adjustments to Illumina's 16S metagenomic sequencing protocol that help track and reduce cross-talk from cross-contamination during library preparation. The inconsistencies in taxonomic assignment emphasise the need for curated and quality-checked sequence databases.

The bacterial biome of ticks and their wildlife hosts at the urban-wildland interface.

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.

Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia.

Vector-borne haemoprotozoans comprise a diverse group of eukaryote single-celled organisms transmitted by haematophagous (blood-feeding) invertebrates. They can cause debilitating diseases that impact wildlife, livestock, companion animals and humans. Recent research has shown that Australian wildlife host a diverse range of haemoprotozoan species; however, to date this work has primarily been confined to a few host species or isolated populations in rural habitats. There has been little investigation into the presence of these blood parasites in wildlife inhabiting urban and peri-urban areas. In this study, blood and tissue samples and ticks were collected from wildlife in New South Wales and Western Australia. Extracted DNA samples were screened with pan-specific molecular assays to determine the presence of haemoprotozoans using amplicon metabarcoding and Sanger sequencing approaches. In addition, light microscopy was performed on blood films. Eight haemoprotozoans were identified in the present study, which included species of Babesia, Hepatozoon, Theileria and Trypanosoma. Blood samples were collected from 134 animals; 70 black rats (Rattus), 18 common brush-tailed possums (Trichosurus vulpecula), two bush rats (Rattus fuscipes), 22 chuditch (Dasyurus geoffroii), 20 long-nosed bandicoots (Perameles nasuta), one quenda (Isoodon fusciventer) and one swamp rat (Rattus lutreolus). Molecular screening of DNA extracted from blood samples identified 52.2% (95% CI: 43.8-60.5%) of individuals were positive for at least one haemoprotozoan species, with 19.4% (95% CI: 13.4-26.7%) positive for more than one species. The present study provides the first sequences of Theileria cf. peramelis from black rats and long-nosed bandicoots. Babesia lohae was identified from brush-tailed possums. Two Hepatozoon genotypes were identified from black rats and bush rats. Black rats showed the highest haemoprotozoan diversity, with five species identified. No known human pathogens that have been described in the northern hemisphere were identified in the present study, and future work is required to understand the zoonotic potential of these microbes in Australia. This work represents the first large-scale body of research using molecular tools to investigate haemoprotozoans in animals at the urban-wildland interface. Further research is needed to investigate potential consequences of infection in wildlife, particularly effects of pathogen spillover from invasive black rats to native wildlife.

Blood Parasites in Endangered Wildlife-Trypanosomes Discovered During a Survey of Haemoprotozoa from the Tasmanian Devil.

The impact of emerging infectious diseases is increasingly recognised as a major threat to wildlife. Wild populations of the endangered Tasmanian devil, Sarcophilus harrisii, are experiencing devastating losses from a novel transmissible cancer, devil facial tumour disease (DFTD); however, despite the rapid decline of this species, there is currently no information on the presence of haemoprotozoan parasites. In the present study, 95 Tasmanian devil blood samples were collected from four populations in Tasmania, Australia, which underwent molecular screening to detect four major groups of haemoprotozoa: (i) trypanosomes, (ii) piroplasms, (iii) Hepatozoon, and (iv) haemosporidia. Sequence results revealed Trypanosoma infections in 32/95 individuals. Trypanosoma copemani was identified in 10 Tasmanian devils from three sites and a second Trypanosoma sp. was identified in 22 individuals that were grouped within the poorly described T. cyclops clade. A single blood sample was positive for Babesia sp., which most closely matched Babesia lohae. No other blood protozoan parasite DNA was detected. This study provides the first insight into haemoprotozoa from the Tasmanian devil and the first identification of Trypanosoma and Babesia in this carnivorous marsupial.

Bacterial community profiling highlights complex diversity and novel organisms in wildlife ticks.

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.

Theileria gilberti n. sp. (Apicomplexa: Theileriidae) in the Gilbert's potoroo (Potorous gilbertii).

The morphology and genetic characterisation of a new species of piroplasm identified in the blood of the Gilbert's potoroo (Potorous gilbertii) from the Two Peoples Bay Nature Reserve near Albany, Western Australia, is described from blood and tissue samples from 16 Gilbert's potoroos. Microscopy of blood showed these parasites are highly pleomorphic with a mean length of 1.8 mum and mean width of 0.85 mum. Phylogenetic analysis of 18S rRNA sequence data identified the piroplasm as a new species of Theileria that is closely related to other Australian marsupial piroplasm species. Based on biological and molecular data, it is proposed that the parasite from Gilbert's potoroo be given the name Theileria gilberti n. sp.

Automatic Barcode Gap Discovery reveals large COI intraspecific divergence in Australian Ixodidae.

Ticks (Ixodida) are haematophagous arthropods that transmit a number of pathogenic organisms, including bacteria, protozoa and viruses, to humans and animals. Globally, there are over 900 species of ticks and Australia has 73 described species, including five introduced and 68 native species. With the exception of only a few Australian tick species, there are still many unanswered questions regarding their taxonomy and systematics, and the phylogeny of Australian ticks is not properly resolved. In recent years, a putative link between tick bites and poorly defined tick-borne illness(es) has been identified (Graves Stenos 2017) and was the subject of a 2015 Australian Senate Inquiry into Lyme-like illnesses in Australia. There is an urgent need to further categorise Australian ticks, specifically hard ticks (Ixodidae), and accurate identification of Australian ticks is therefore of high importance.