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Ticks are important medical and veterinary parasites that represent a substantial health threat to humans, companion animals, and livestock. Ixodiphagus wasps (Hymenoptera; Encyrtidae) are known endoparasitoids of ixodid (hard) and argasid (soft) ticks, with potential utility as natural biocontrol agents. Two species, Ixodiphagus brunneus and Ixodiphagus mysorensis, are previously recorded from Australia, however, the genus lacks formal revisionary work in Australia, and the validity and host ranges of these species remain uncertain. This work aimed to investigate the diversity of Ixodiphagus in Australasia and provide a molecular data resource for future work on these understudied endoparasitoids. We extracted DNA from archival Ixodiphagus specimens from Australian and New Zealand insect collections and performed high-throughput sequencing which resulted in complete or mostly complete mitochondrial genome sequences from 11 specimens, including I. brunneus, Ixodiphagus taiaroaensis, and a novel Ixodiphagus sp. reared from Rhipicephalus linnaei from Townsville, Australia. In addition, approximately 70% of the genome of the Wolbachia endosymbiont of I. brunneus was recovered. Finally, we screened 178 recently collected pooled tick samples from southern New South Wales, Australia, for Ixodiphagus spp. using 28S rRNA and cytochrome c oxidase subunit 1(COI) gene PCR, and recovered 14 positive samples. Phylogenetic analysis of Australasian Ixodiphagus spp. based on 28S rRNA and complete mitochondrial genome sequences determined that members of the Australasian fauna are distinct from Ixodiphagus hookeri (the only other Ixodiphagus species for which genetic data exists), and that at least two distinct species are present in Australia; I. brunneus identified from Ixodes holocyclus and Haemaphysalis bancrofti ticks, and an uncharacterised Ixodiphagus sp. found in Rhipicephalus linnaei ticks from northern Queensland. Furthermore, there was substantial genetic diversity at the 28S rRNA loci among I. brunneus samples, which may represent normal genetic variability or a secondary cryptic species. The molecular data generated here represents the first known for the genus Ixodiphagus in Australasia, doubling that of the world fauna, and provides the first known complete mitochondrial genomes for these important tick parasitoids., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tick-borne haemoparasites, including piroplasms and trypanosomes, are almost ubiquitous in Australian wildlife, with some associated with health impacts to individual animals and declining wildlife populations. An array of ecologically distinct piroplasm and trypanosome species occur throughout Australia although many of these species and their sylvatic ecologies are poorly characterised. Between May 2022 and October 2023, an anecdotally reported localised eastern grey kangaroo ( Macropus giganteus ) morbidity/mortality event occurred in coastal southern New South Wales, Australia, characterised by animals presenting with blindness, emaciation, lethargy, ataxia, and astasia. Here we used molecular techniques to identify tick-borne piroplasms ( Babesia and Theileria ) and trypanosomes in affected animals . Blood (n = 89) and liver (n = 19) samples were collected after the humane euthanasia of wild animals due to welfare concerns, and brief notes on the animal's health were recorded. In total, 20 (22.5%) animals were infected with tick-borne haemoparasites, including a novel Theileria sp. nov. (14, 15.7%), Babesia macropus (2, 2.2%), Trypanosoma gilletti (5, 5.6%), and Trypanosoma vegrandis (1, 1.1%). Liver samples were also screened for Wallal and Warego viruses due to animals' blindness, but were negative. This is the first report of T. gilletti and T. vegrandis in eastern grey kangaroos, although they have been previously reported in high numbers in ticks which commonly parasites this host. The novel Theileria sp. was previously reported in questing Ixodes holocyclus and in ticks from an opportunistically collected eastern grey kangaroo and red-necked wallaby ( Notamacropus rufogriseus ). However, we show for the first time this Theileria sp. can occur widely in eastern grey kangaroos. Ultimately, this small study did not intend, and is not able to draw inference regarding the pathogenicity of these haemoparasites to eastern grey kangaroos and it is likely that other factors, such as chronic Phalaris grass toxicity, had a role in this localised mortality/morbidity event., Competing Interests: The authors’ declare that no conflict of interest exists., (© 2024 The Authors.)

Feral deer are widespread throughout Australia with the capacity to impact livestock production via transmission of parasites. Samples of Dama dama (fallow deer), Rusa unicolor (sambar deer), Cervus elaphus (red deer) and an unidentified deer were sourced from various locations in south-eastern Australia for examination for parasites. Adult nematodes were collected from the lungs of all deer species across four separate geographical locations. The nematodes were identified as species of Dictyocaulus through both morphological and molecular means. Species identification based on morphological features was difficult, with many measurements from described species overlapping. Molecular analyses targeting three markers, namely 18S rRNA, ITS2, and cox1 revealed the presence of two distinct species: Dictyocaulus cervi and Dictyocaulus skrjabini . These are the first genetically confirmed reports of species of Dictyocaulus in feral deer in Australia, and although cross-transmission of species of Dictyocaulus with livestock has not yet been reported, it cannot be completely discounted without further research., Competing Interests: The authors have no affiliation with any organisation with a direct or indirect financial interest in the subject matter discussed in the manuscript., (© 2024 The Authors.)

We used entire mitochondrial (mt) genome sequences (14.5-15 kbp) to resolve the phylogeny of the four main lineages of the Haematobothrion ticks: Alloceraea, Archaeocroton, Bothriocroton and Haemaphysalis. In our phylogenetic trees, Alloceraea was the sister to Archaeocroton sphenodonti, a tick of an archetypal reptile, the tuatara, from New Zealand, to the exclusion of the rest of the species of Haemaphysalis. The mt genomes of all four of the Alloceraea species that have been sequenced so far had a substantial insert, 132-312 bp, between the tRNA-Glu (E) gene and the nad1 gene in their mt genomes. This insert was not found in any of the other eight subgenera of Haemaphysalis. The mt genomes of 13 species of Haemaphysalis from NCBI GenBank were added to the most recent data set on Haemaphysalis and its close relatives to help resolve the phylogeny of Haemaphysalis, including five new subgenera of Haemaphysalis not previously considered by other authors: Allophysalis (structurally primitive), Aboimisalis (structurally primitive), Herpetobia (structurally intermediate), Ornithophysalis (structurally advanced) and Segalia (structurally advanced). We elevated Alloceraea Schulze, 1919 to the status of genus because Alloceraea Schulze, 1919 is phylogenetically distinct from the other subgenera of Haemaphysalis. Moreover, we propose that the subgenus Allophysalis is the sister to the rest of the Haemaphysalis (14 subgenera) and that the 'structurally primitive' subgenera Hoogstraal and Kim comprise early diverging lineages. Our matrices of the pairwise genetic difference (percent) of mt genomes and partial 16S rRNA sequences indicated that the mt genome sequence of Al. kitaokai (gb# OM368280) may not be Al. kitaokai Hoogstraal, 1969 but rather another species of Alloceraea. In a similar way, the mt genome sequence of H. (Herpetobia) nepalensis Hoogstraal, 1962 (gb# NC_064124) was only 2% genetically different to that of H. (Allophysalis) tibetensis Hoogstraal, 1965 (gb# OM368293): this indicates to us that they are the same species. Alloceraea cretacea may be better placed in a genus other than Alloceraea Schulze, 1919. Reptiles may have been the host to the most recent common ancestor of Archaeocroton and Alloceraea., (© 2024 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Ticks have a profound impact on public health. Haemaphysalis is one of the most widespread genera in Asia, including Japan. The taxonomy and genetic differentiation of Haemaphysalis spp. is challenging. For instance, previous studies struggled to distinguish Haemaphysalis japonica and Haemaphysalis megaspinosa due to the dearth of nucleotide sequence polymorphisms in widely used barcoding genes. The classification of H. japonica japonica and its related sub-species Haemaphysalis japonica douglasi or Haemaphysalis jezoensis is also confused due to their high morphological similarity and a lack of molecular data that support the current classification. We used mitogenomes and microbiomes of H. japonica and H. megaspinosa to gain deeper insights into the phylogenetic relationships and genetic divergence between two species. Phylogenetic analyses of concatenated nucleotide sequences of protein-coding genes and ribosomal DNA genes distinguished H. japonica and H. megaspinosa as monophyletic clades, with further subdivision within the H. japonica clade. The 16S rRNA and NAD5 genes were valuable markers for distinguishing H. japonica and H. megaspinosa. Population genetic structure analyses indicated that genetic variation within populations accounted for a large proportion of the total variation compared to variation between populations. Microbiome analyses revealed differences in alpha and beta diversity between H. japonica and H. megaspinosa: H. japonica had the higher diversity. Coxiella sp., a likely endosymbiont, was found in both Haemaphysalis species. The abundance profiles of likely endosymbionts, pathogens, and commensals differed between H. japonica and H. megaspinosa: H. megaspinosa was more diverse., (© 2024. The Author(s).)

Background: Borrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia, once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards (Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia., Methods: We used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus-associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date., Results: The genomic architecture of Varanus-associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage., Conclusions: In this work we present four new genomes from three novel Borrelia, and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii, 'Candidatus Borrelia undatumii', and 'Candidatus Borrelia rubricentralis' for them., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2023 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

A new subgenus, Australixodes n. subgen., is described for the kiwi tick, Ixodes anatis Chilton, 1904. The subgenus Coxixodes Schulze, 1941 is validated for the platypus tick, Ixodes (Coxixodes) ornithorhynchi Lucas, 1846, sister group of the subgenus Endopalpiger Schulze, 1935. A phylogeny from mitochondrial genomes of 16 of the 22 subgenera of Ixodes (32 spp.) indicates, for the first time, the relationships of the subgenera of Ixodes Latreille, 1795, the largest genus of ticks.

Hoogstraal and Kim (1985) proposed from morphology, three groups of Haemaphysalis subgenera: (i) the "structurally advanced"; (ii) the "structurally intermediate"; and (iii) the "structurally primitive" subgenera. Nuclear gene phylogenies, however, did not indicate monophyly of these morphological groups but alas, only two mitochondrial (mt) genomes from the "structurally intermediate" subgenera had been sequenced. The phylogeny of Haemaphysalis has not yet been resolved. We aimed to resolve the phylogeny of the genus Haemaphysalis, with respect to the subgenus Alloceraea. We presented 15 newly sequenced and annotated mt genomes from 15 species of ticks, five species of which have not been sequenced before, and four new 18S rRNA and 28S rRNA nuclear gene sequences. Our datasets were constructed from 10 mt protein-coding genes, cox1, and the 18S and 28S nuclear rRNA genes. We found a 132-bp insertion between tRNA-Glu (E) gene and the nad1 gene in the mt genome of Haemaphysalis (Alloceraea) inermis that resembles insertions in H. (Alloceraea) kitaokai and Rhipicephalus (Boophilus) geigyi. Our mt phylogenies had the three species of Amblyomma (Aponomma) we sequenced embedded in the main clade of Amblyomma: Am. (Aponomma) fimbriatum, Am. (Aponomma) gervaisi and Am. (Aponomma) latum. This is further support for the hypothesis that the evolution of eyes appears to have occurred in the most-recent-common-ancestor of Amblyocephalus (i.e. Amblyomminae plus Rhipicephalinae) and that eyes were subsequently lost in the most-recent-common-ancestor of the subgenus Am. (Aponomma). Either Africaniella transversale or Robertsicus elaphensis, or perhaps Af. transversale plus Ro. elaphensis, appear to be the sister-group to the rest of the metastriate Ixodida. Our cox1 phylogenies did not indicate monophyly of the "structurally primitive", "structurally intermediate" nor the "structurally advanced" groups of Haemaphysalis subgenera. Indeed, the subgenus Alloceraea may be the only monophyletic subgenus of the genus Haemaphysalis sequenced thus far. All of our mt genome and cox1 phylogenies had the subgenus Alloceraea in a clade that was separate from the rest of the Haemaphysalis ticks. If Alloceraea is indeed the sister to the rest of the Haemaphysalis subgenera this would resonate with the argument of Hoogstraal and Kim (1985), that Alloceraea was a subgenus of "primitive" Haemaphysalis. Alectorobius capensis from Japan had a higher genetic-identity to A. sawaii, which was also from Japan, than to the A. capensis from South Africa. This indicates that A. capensis from Japan may be a cryptic species with respect to the A. capensis from South Africa., (Copyright © 2022. Published by Elsevier GmbH.)

Tick-borne zoonoses are emerging globally due to changes in climate and land use. While the zoonotic threats associated with ticks are well studied elsewhere, in Australia, the diversity of potentially zoonotic agents carried by ticks and their significance to human and animal health is not sufficiently understood. To this end, we used untargeted metatranscriptomics to audit the prokaryotic, eukaryotic and viral biomes of questing ticks and wildlife blood samples from two urban and rural sites in New South Wales, Australia. Ixodes holocyclus and Haemaphysalis bancrofti were the main tick species collected, and blood samples from Rattus rattus, Rattus fuscipes, Perameles nasuta and Trichosurus vulpecula were also collected and screened for tick-borne microorganisms using metatranscriptomics followed by conventional targeted PCR to identify important microbial taxa to the species level. Our analyses identified 32 unique tick-borne taxa, including 10 novel putative species. Overall, a wide range of tick-borne microorganisms were found in questing ticks including haemoprotozoa such as Babesia, Theileria, Hepatozoon and Trypanosoma spp., bacteria such as Borrelia, Rickettsia, Ehrlichia, Neoehrlichia and Anaplasma spp., and numerous viral taxa including Reoviridiae (including two coltiviruses) and a novel Flaviviridae-like jingmenvirus. Of note, a novel hard tick-borne relapsing fever Borrelia sp. was identified in questing H. bancrofti ticks which is closely related to, but distinct from, cervid-associated Borrelia spp. found throughout Asia. Notably, all tick-borne microorganisms were phylogenetically unique compared to their relatives found outside Australia, and no foreign tick-borne human pathogens such as Borrelia burgdorferi s.l. or Babesia microti were found. This work adds to the growing literature demonstrating that Australian ticks harbour a unique and endemic microbial fauna, including potentially zoonotic agents which should be further studied to determine their relative risk to human and animal health., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Recycled wastewater can carry human-infectious microbial pathogens and therefore wastewater treatment strategies must effectively eliminate pathogens before recycled wastewater is used to supplement drinking and agricultural water supplies. This study characterised the bacterial composition of four wastewater treatment plants (WWTPs) (three waste stabilisation ponds and one oxidation ditch WWTP using activated sludge treatment) in Western Australia. The hypervariable region 4 (V4) of the bacterial 16S rRNA (16S) gene was sequenced using next-generation sequencing (NGS) on the Illumina MiSeq platform. Sequences were pre-processed in USEARCH v10.0 and denoised into zero-radius taxonomic units (ZOTUs) with UNOISE3. Taxonomy was assigned to the ZOTUs using QIIME 2 and the Greengenes database and cross-checked with the NCBI nr/nt database. Bacterial composition of all WWTPs and treatment stages (influent, intermediate and effluent) were dominated by Proteobacteria (29.0-87.4%), particularly Betaproteobacteria (9.0-53.5%) and Gammaproteobacteria (8.6-34.6%). Nitrifying bacteria (Nitrospira spp.) were found only in the intermediate and effluent of the oxidation ditch WWTP, and denitrifying and floc-forming bacteria were detected in all WWTPs, particularly from the families Comamonadaceae and Rhodocyclales. Twelve pathogens were assigned taxonomy by the Greengenes database, but comparison of sequences from genera and families known to contain pathogens to the NCBI nr/nt database showed that only three pathogens (Arcobacter venerupis, Laribacter hongkongensis and Neisseria canis) could be identified in the dataset at the V4 region. Importantly, Enterobacteriaceae genera could not be differentiated. Family level taxa assigned by Greengenes database agreed with NCBI nr/nt in most cases, however, BLAST analyses revealed erroneous taxa in Greengenes database. This study highlights the importance of validating taxonomy of NGS sequences with databases such as NCBI nr/nt, and recommends including the V3 region of 16S in future short amplicon NGS studies that aim to identify bacterial enteric pathogens, as this will improve taxonomic resolution of most, but not all, Enterobacteriaceae species., (Copyright © 2019. Published by Elsevier B.V.)

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Borrelia are tick-borne bacteria that in humans are the aetiological agents of Lyme disease and relapsing fever. Here we present the first genomes of B. turcica and B. tachyglossi, members of a recently described and rapidly expanding Borrelia clade associated with reptile (B. turcica) or echidna (B. tachyglossi) hosts, transmitted by hard ticks, and of unknown pathogenicity. Borrelia tachyglossi and B. turcica genomes are similar to those of relapsing fever Borrelia species, containing a linear ~ 900 kb chromosome, a single long (> 70 kb) linear plasmid, and numerous short (< 40 kb) linear and circular plasmids, as well as a suite of housekeeping and macronutrient biosynthesis genes which are not found in Lyme disease Borrelia. Additionally, both B. tachyglossi and B. turcica contain paralogous vsp and vlp proteins homologous to those used in the multiphasic antigen-switching system used by relapsing fever Borrelia to evade vertebrate immune responses, although their number was greatly reduced compared to human-infectious species. However, B. tachyglossi and B. turcica chromosomes also contain numerous genes orthologous to Lyme disease Borrelia-specific genes, demonstrating a unique evolutionary, and potentially phenotypic link between these groups. Borrelia tachyglossi and B. turcica genomes also have unique genetic features, including degraded and deleted tRNA modification genes, and an expanded range of macronutrient salvage and biosynthesis genes compared to relapsing fever and Lyme disease Borrelia. These genomes and genomic comparisons provide an insight into the biology and evolutionary origin of these Borrelia, and provide a valuable resource for future work., (Copyright © 2018 Elsevier B.V. All rights reserved.)

As part of long-term monitoring of Cryptosporidium in water catchments serving Western Australia, New South Wales (Sydney) and Queensland, Australia, we characterised Cryptosporidium in a total of 5774 faecal samples from 17 known host species and 7 unknown bird samples, in 11 water catchment areas over a period of 30 months (July 2013 to December 2015). All samples were initially screened for Cryptosporidium spp. at the 18S rRNA locus using a quantitative PCR (qPCR). Positives samples were then typed by sequence analysis of an 825 bp fragment of the 18S gene and subtyped at the glycoprotein 60 (gp60) locus (832 bp). The overall prevalence of Cryptosporidium across the various hosts sampled was 18.3% (1054/5774; 95% CI, 17.3-19.3). Of these, 873 samples produced clean Sanger sequencing chromatograms, and the remaining 181 samples, which initially produced chromatograms suggesting the presence of multiple different sequences, were re-analysed by Next- Generation Sequencing (NGS) to resolve the presence of Cryptosporidium and the species composition of potential mixed infections. The overall prevalence of confirmed mixed infection was 1.7% (98/5774), and in the remaining 83 samples, NGS only detected one species of Cryptosporidium. Of the 17 Cryptosporidium species and four genotypes detected (Sanger sequencing combined with NGS), 13 are capable of infecting humans; C. parvum, C. hominis, C. ubiquitum, C. cuniculus, C. meleagridis, C. canis, C. felis, C. muris, C. suis, C. scrofarum, C. bovis, C. erinacei and C. fayeri. Oocyst numbers per gram of faeces (g -1 ) were also determined using qPCR, with medians varying from 6021-61,064 across the three states. The significant findings were the detection of C. hominis in cattle and kangaroo faeces and the high prevalence of C. parvum in cattle. In addition, two novel C. fayeri subtypes (IVaA11G3T1 and IVgA10G1T1R1) and one novel C. meleagridis subtype (IIIeA18G2R1) were identified. This is also the first report of C. erinacei in Australia. Future work to monitor the prevalence of Cryptosporidium species and subtypes in animals in these catchments is warranted., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Worldwide, Ehrlichia spp. are emerging infectious organisms of domestic animals and people, however, most Ehrlichia spp. naturally infect wildlife reservoirs causing mainly asymptomatic infections. Australian ecosystems have been under-explored for these potentially pathogenic organisms, and recent studies have identified a range of novel Ehrlichia, and their sister genera, Anaplasma and 'Candidatus Neoehrlichia' species, from native Australian ticks. We used bacterial 16S rRNA (16S) next-generation sequencing and genus-specific PCR to profile the bacterial communities in platypus (Ornithorhynchus anatinus) blood samples and platypus ticks (Ixodes ornithorhynchi), and identified a high prevalence of Ehrlichia sequences. We also observed Ehrlichia-like intra-neutrophilic inclusions (morulae) in PCR-positive stained platypus blood films that were consistent in morphology with other Ehrlichia spp. Bayesian phylogenetic analysis of 16S (1343 bp), gltA (1004 bp), and groEL (1074 bp) gene sequences group the platypus Ehrlichia with 'Candidatus Ehrlichia khabarensis' from far-eastern Russia, and demonstrate that the platypus Ehrlichia is clearly distinct from all other Ehrlichia spp. Enough genetic divergence exists to delineate this platypus Ehrlichia as a separate species that we propose to designate 'Candidatus Ehrlichia ornithorhynchi'. There is no evidence that 'Candidatus Ehrlichia ornithorhynchi' causes disease in wild platypuses, however, the organism does seem to be widespread in Australia, being found in both Queensland and Tasmania. 'Candidatus Ehrlichia ornithorhynchi' is the second native Australian Ehrlichia described and adds to the rapidly growing diversity of recently described native Australian tick-borne bacteria., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

In Western Australia a number of indigenous Trypanosoma spp. infect susceptible native marsupials, such as the woylie ( Bettongia penicillata ), brushtail possum ( Trichosurus vulpecula ), and chuditch (Dasyurus geoffroii ). Two genotypes of Trypanosoma copemani (identified as G1 and G2) have been found in the woylie, and G2 has been implicated in the decline of this host species, making its presence of particular interest. Here we used targeted amplicon next generation sequencing (NGS) of the Trypanosoma 18S rDNA loci on 70 Trypanosoma -positive marsupial blood samples, to identify T. copemani genotypes and multiple Trypanosoma infections (polyparasitism) in woylies and cohabiting species in Western Australia. Polyparasitism with Trypanosoma spp. was found in 50% of the wildlife sampled, and within species diversity was high, with 85 zero-radius operational taxonomic units (ZOTUs) identified in nine putative parasite species. Trypanosoma copemani was assigned 17 ZOTUs and was identified in 80% of samples. The most abundant ZOTU isolated (63%) differed slightly from the published genotype of G1, and G2 was the second most abundant ZOTU (14%). Trypanosome diversity was significantly greater in woylies than in brushtail possums, and parasite community composition also differed significantly between these host species. One novel Trypanosoma spp. genotype ( Trypanosoma sp. ANU2) was found in 20% of samples. A species of Crithidia was detected in a woylie, and two avian trypanosomes ( Trypanosoma avium and Trypanosoma sp. AAT) were identified in woylies for the first time.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

The morphological, biological, and molecular characterisation of a new Cryptosporidium species from the guinea pig (Cavia porcellus) are described, and the species name Cryptosporidium homai n. sp. is proposed. Histological analysis conducted on a post-mortem sample from a guinea pig euthanised due to respiratory distress, identified developmental stages of C. homai n. sp. (trophozoites and meronts) along the intestinal epithelium. Molecular analysis at 18S rRNA (18S), actin and hsp70 loci was then conducted on faeces from an additional 7 guinea pigs positive for C. homai n. sp. At the 18S, actin and hsp70 loci, C. homai n. sp. exhibited genetic distances ranging from 3.1% to 14.3%, 14.4% to 24.5%, and 6.6% to 20.9% from other Cryptosporidium spp., respectively. At the 18S locus, C. homai n. sp. shared 99.1% similarity with a previously described Cryptosporidium genotype in guinea pigs from Brazil and it is likely that they are the same species, however this cannot be confirmed as actin and hsp70 sequences from the Brazilian guinea pig genotype are not available. Phylogenetic analysis of concatenated 18S, actin and hsp70 sequences showed that C. homai n. sp. exhibited 9.1% to 17.3% genetic distance from all other Cryptosporidium spp. This clearly supports the validity of C. homai n. sp. as a separate species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The extent of within-host genetic diversity of parasites has implications for our understanding of the epidemiology, disease severity and evolution of parasite virulence. As with many other species, our understanding of the within-host diversity of the enteric parasite Cryptosporidium is changing. The present study compared Sanger and Next Generation Sequencing of glycoprotein 60 (gp60) amplicons from Cryptosporidium hominis (n=11), Cryptosporidium parvum (n=22) and Cryptosporidium cuniculus (n=8) DNA samples from Australia and China. Sanger sequencing identified only one gp60 subtype in each DNA sample: one C. hominis subtype (IbA10G2) (n=11), four C. parvum subtypes belonging to IIa (n=3) and IId (n=19) and one C. cuniculus subtype (VbA23) (n=8). Next Generation Sequencing identified the same subtypes initially identified by Sanger sequencing, but also identified additional gp60 subtypes in C. parvum and C. cuniculus but not in C. hominis, DNA samples. The number of C. parvum and C. cuniculus subtypes identified by Next Generation Sequencing within individual DNA samples ranged from two to four, and both C. parvum IIa and IId subtype families were identified within the one host in two samples. The finding of the present study has important implications for Cryptosporidium transmission tracking as well as vaccine and drug studies., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Anaplasma and Ehrlichia spp. are tick-borne pathogens that can cause severe disease in domestic animals, and several species are responsible for emerging zoonoses in the northern hemisphere. Until recently, the only members of these genera reported in Australia (A. marginale, A. centrale, and A. platys) were introduced from other continents, through the importation of domestic animals and their associated ticks. However, unique Anaplasma and Ehrlichia 16S rRNA gene sequences were recently detected for the first time in native Australian ticks, particularly in Amblyomma triguttatum subsp. ticks from southwest Western Australia (WA). We used molecular techniques to survey Am. triguttatum subsp. ticks from four allopatric populations in southern and western Australia for Anaplasma and Ehrlichia species, and described here the phylogeny of these novel organisms. An A. bovis variant (genotype Y11) was detected in ticks from two study sites; Yanchep National Park (12/280, 4.3%) and Barrow Island (1/69, 1.4%). Phylogenetic analysis of 16S rRNA and groEL gene sequences concluded that A. bovis genotype Y11 is a unique genetic variant, distinct from other A. bovis isolates worldwide. Additionally, a novel Ehrlichia species was detected in Am. triguttatum subsp. from three of the four study sites; Yanchep National Park (18/280, 6.4%), Bungendore Park (8/46, 17.4%), and Innes National Park (9/214, 4.2%), but not from Barrow Island. Phylogenetic analysis of 16S, groEL, gltA, and map1 gene sequences revealed that this Ehrlichia sp. is most closely related to, but clearly distinct from, E. ruminantium and Ehrlichia sp. Panola Mountain. We propose to designate this new species 'Candidatus Ehrlichia occidentalis'. Anaplasma bovis genotype Y11 and 'Candidatus E. occidentalis' are the first Anaplasma and Ehrlichia species to be recorded in native Australian ticks., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

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.

Q fever is an infectious disease with a global distribution caused by the intracellular bacterium, Coxiella burnetii, which has been detected in a large number of tick species worldwide, including the brown dog tick, Rhipicephalus sanguineus. Recent reports of a high seroprevalance of C. burnetii in Australian dogs, along with the identification of additional Coxiella species within R. sanguineus ticks, has prompted an investigation into the presence and identification of Coxiella species in R. sanguineus ticks in Australia. Using a combination of C. burnetii species-specific IS1111a transposase gene and Coxiella genus-specific 16S rRNA PCR assays, a Coxiella sp. was identified in 100% (n=199) of R. sanguineus ticks analysed, and C. burnetii was not detected in any R. sanguineus ticks studied. Phylogenetic analysis of the 16S rRNA gene revealed the Coxiella sequences were closely related to Coxiella sp. identified previously in R. sanguineus and R. turanicus ticks overseas. This study illustrates the value of using genus specific PCR assays to detect previously unreported bacterial species. Furthermore, the presence of an additional Coxiella sp. in Australia requires further investigation into its potential for contributing to serological cross-reactions during Q fever testing., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, two novel species of Anaplasmataceae were detected in the Australian paralysis tick, Ixodes holocyclus, by 16S rRNA gene metabarcoding. Analysis of these sequences suggested that these novel organisms are closely related to the genus 'Candidatus Neoehrlichia'. In this study, phylogenetic analysis of 16S rRNA (1264 bp), groESL (1047 bp) and gltA (561 bp) gene sequences, and concatenated (2872 bp) sequences, all concur that these novel species belong in the genus 'Candidatus Neoehrlichia' and are most closely related to, but distinct from the only other recognised members of this genus, 'Candidatus Neoehrlichia mikurensis' and 'Candidatus Neoehrlichia lotoris'. Based on their unique molecular signature, we propose to designate these species 'Candidatus Neoehrlichia australis' (reference strain HT41R) and 'Candidatus Neoehrlichia arcana' (reference strain HT94R). Identical 'Candidatus Neoehrlichia australis' 16S rRNA, groESL and gltA sequences were detected in 34/391 (8.7 %) individual Ixodes holocyclus ticks, and sequences were most similar to 'Candidatus Neoehrlichia lotoris' (96.2 %, 83.1 % and 67.2 %, respectively) and 'Candidatus Neoehrlichia mikurensis' (96.2 %, 84 % and 68.4 % respectively). Likewise, identical 'Candidatus Neoehrlichia arcana' 16S rRNA, groESL and gltA sequences were detected in 12/391 (3.1 %) Ixodes holocyclus ticks, and sequences were most similar to 'Candidatus Neoehrlichia lotoris' (98.5 %, 88.7 % and 79.3 %, respectively) and 'Candidatus Neoehrlichia mikurensis' (96.3 %, 84 % and 67.4 % respectively). These new species are the first Anaplasmataceae (except Wolbachia spp.) to be found to be endemic to Australia. The pathogenic consequences of these organisms are yet to be determined.

Background: To date, little has been documented about microorganisms harboured within Australian native ticks or their pathogenic potential. Recently, a Borrelia sp. related to the Relapsing Fever (RF) group was identified in a single tick removed from a wild echidna (Tachyglossus aculeatus). The present study investigated the presence of Borrelia in 97 Bothriocroton concolor ticks parasitizing echidnas in Queensland, New South Wales, and Victoria, Australia, using nested PCR with Borrelia-specific primers targeting the 16S rRNA (16S) and flaB genes., Results: Borrelia-specific PCR assays confirmed the presence of a novel Borrelia sp. related to the RF and reptile-associated (REP) spirochaetes in 38 (39 %) B. concolor ticks. This novel Borrelia sp. was identified in 41 % of the B. concolor ticks in Queensland and New South Wales, but not in any ticks from Victoria. The resulting flaB sequences (407 bp) were 88 and 86 % similar to the flaB sequences from Borrelia turcica and Borrelia hermsii, respectively. Of the ticks confirmed as Borrelia-positive following the flaB assay, 28 were positive with the 16S assay. Phylogenetic analysis of the 16S sequences (1097 bp) suggests that these sequences belong to a novel Borrelia sp., which forms a unique monophyletic clade that is similar to, but distinct from, RF Borrelia spp. and REP-associated Borrelia spp., Conclusions: We conclude that the novel Borrelia sp. identified in this study does not belong to the Borrelia burgdorferi (sensu lato) complex, and that the phylogenetic analysis of the partial 16S gene sequences suggests it forms a unique monophyletic cluster in the genus Borrelia, potentially forming a fourth major group in this genus associated with monotremes in Australia. However, a thorough molecular characterisation will be required to confirm the phylogenetic position of this unique Borrelia sp. The zoonotic potential and pathogenic consequences of this novel Borrelia sp. are unknown at the current time.

Background: Ticks are among the most important vectors of pathogens affecting companion animals, and also cause health problems such as tick paralysis, anaemia, dermatitis, and secondary infections. Twenty ixodid species have previously been recorded on dogs, cats, and horses in Australia, including Rhipicephalus sanguineus, Ixodes holocyclus and Haemaphysalis longicornis, which transmit tick-borne diseases. A survey of hard ticks (Acari: Ixodidae) was conducted during 2012-2015 to investigate tick species that infest dogs, cats, and horses in Australia., Methods: Individual tick specimens were collected from dogs, cats and horses across Australia and sample collection locations were mapped using QGIS software. Ticks were morphologically examined to determine species, instar and sex. The companion animal owners responded to questionnaires and data collected were summarised with SPSS software., Results: A total of 4765 individual ticks were identified in this study from 7/8 states and territories in Australia. Overall, 220 larvae, 805 nymphs, 1404 males, and 2336 females of 11 tick species were identified from 837 companion animal hosts. One novel host record was obtained during this study for Ixodes myrmecobii, which was found on Felis catus (domestic cat) in the town of Esperance, Western Australia. The most common tick species identified included R. sanguineus on dogs (73 %), I. holocyclus on cats (81 %) and H. longicornis on horses (60 %)., Conclusions: This study is the first of its kind to be conducted in Australia and our results contribute to the understanding of the species and distribution of ticks that parasitise dogs, cats, and horses in Australia. Records of R. sanguineus outside of the recorded distribution range emphasise the need for a systematic study of the habitat range of this species. Several incomplete descriptions of ixodid species encountered in this study hindered morphological identification.

In Australia, a conclusive aetiology of Lyme disease-like illness in human patients remains elusive, despite growing numbers of people presenting with symptoms attributed to tick bites. In the present study, we surveyed the microbial communities harboured by human-biting ticks from across Australia to identify bacteria that may contribute to this syndrome. Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes in DNA samples from individual Ixodes holocyclus (n = 279), Amblyomma triguttatum (n = 167), Haemaphysalis bancrofti (n = 7), and H. longicornis (n = 7) ticks. The 16S amplicons were sequenced on the Illumina MiSeq platform and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomies. Nested PCR and Sanger sequencing were used to confirm the NGS data and further analyse novel findings. All 460 ticks were negative for Borrelia spp. by both NGS and nested PCR analysis. Two novel "Candidatus Neoehrlichia" spp. were identified in 12.9% of I. holocyclus ticks. A novel Anaplasma sp. was identified in 1.8% of A. triguttatum ticks, and a novel Ehrlichia sp. was identified in both A. triguttatum (1.2%) ticks and a single I. holocyclus (0.6%) tick. Further phylogenetic analysis of novel "Ca. Neoehrlichia", Anaplasma and Ehrlichia based on 1,265 bp 16S rRNA gene sequences suggests that these are new species. Determining whether these newly discovered organisms cause disease in humans and animals, like closely related bacteria do abroad, is of public health importance and requires further investigation.

Background: The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks., Methods: Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont "Candidatus Midichloria mitochondrii" (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM., Results: Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel "Candidatus Neoehrlichia" spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai., Conclusions: Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel "Candidatus Neoehrlichia" spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This "pathogen blocking" could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV), a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2-5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking.