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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.

Background Apicomplexan tick-borne pathogens that cause disease in companion animals include species of Babesia Starcovici, 1893, Cytauxzoon Neitz & Thomas, 1948, Hepatozoon Miller, 1908 and Theileria Bettencourt, Franca & Borges, 1907. The only apicomplexan tick-borne disease of companion animals that is known to occur in Australia is babesiosis, caused by Babesia canis vogeli Reichenow, 1937 and Babesia gibsoni Patton, 1910 . However, no molecular investigations have widely investigated members of Apicomplexa Levine, 1980 in Australian ticks that parasitise dogs, cats or horses, until this present investigation. Results Ticks ( n = 711) removed from dogs ( n = 498), cats ( n = 139) and horses ( n = 74) throughout Australia were screened for piroplasms and Hepatozoon spp. using conventional PCR and Sanger sequencing. The tick-borne pathogen B. vogeli was identified in two Rhipicephalus sanguineus Latreille ticks from dogs residing in the Northern Territory and Queensland (QLD). Theileria orientalis Yakimov & Sudachenkov, 1931 genotype Ikeda was detected in three Haemaphysalis longicornis Neumann ticks from dogs in New South Wales. Unexpectedly, the exotic tick-borne pathogen Hepatozoon canis James, 1905 was identified in an Ixodes holocyclus Neumann tick from a dog in QLD. Eight novel piroplasm and Hepatozoon species were identified and described in native ticks and named as follows: Babesia lohae n. sp., Babesia mackerrasorum n. sp., Hepatozoon banethi n. sp., Hepatozoon ewingi n. sp., Theileria apogeana n. sp., Theileria palmeri n. sp., Theileria paparinii n. sp. and Theileria worthingtonorum n. sp. Additionally, a novel cf. Sarcocystidae sp. sequence was obtained from Ixodes tasmani Neumann but could not be confidently identified at the genus level. Conclusions Novel species of parasites in ticks represent an unknown threat to the health of companion animals that are bitten by these native tick species. The vector potential of Australian ticks for the newly discovered apicomplexans needs to be assessed, and further clinical and molecular investigations of these parasites, particularly in blood samples from dogs, cats and horses, is required to determine their potential for pathogenicity.

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.

In a letter to the Editor, Harris considers the eight new species of Apicomplexa that were recently identified and named to be invalid on the basis that only molecular characters were provided in the species descriptions. In this response, we counter that the species names are valid as the descriptions have met the requirements of the International Code of Zoological Nomenclature ; molecular characters can be used to satisfy article 13.1.1 of the code.

[Cryptosporidium] is an important enteric parasite that can contribute large numbers of infectious oocysts to drinking water catchments. As a result of its resistance to disinfectants including chlorine, it has been responsible for numerous waterborne outbreaks of gastroenteritis. Wildlife and livestock play an important role in the transmission of [Cryptosporidium] in the environment. Studies conducted outside Australia have indicated that camels may also play a role in the transmission of zoonotic species of [Cryptosporidium]. Despite Australia being home to the world’s largest camel herd, nothing is known about the prevalence and species of [Cryptosporidium] infecting camels in this country. In the present study, [C. parvum] was identified by PCR amplification and sequencing of a formalin-fixed intestinal tissue specimen from a one-week old dromedary camel ([Camelus dromedarius]). Subtyping analysis at the glycoprotein 60 ([gp60]) locus identified [C. parvum] subtype IIaA17G2R1, which is a common zoonotic subtype reported in humans and animals worldwide. Histopathological findings also confirmed the presence of large numbers of variably-sized (1-3 µm in diameter) circular basophilic protozoa - consistent with [Cryptosporidium] spp.- adherent to the mucosal surface and occasionally free within the lumen. Further analysis of the prevalence and species of [Cryptosporidium] in camel populations across Australia are essential to better understand their potential for contamination of drinking water catchments.

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.