Recent insights into the tick microbiome gained through next-generation sequencing

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

Detection and phylogenetic characterisation of novel Anaplasma and Ehrlichia species in Amblyomma triguttatum subsp. from four allopatric populations in Australia

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

A survey of ticks (Acari: Ixodidae) of companion animals in Australia

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

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 Illuminaʼs 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

Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification

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

Increased genetic diversity and prevalence of co-infection with Trypanosoma spp. in koalas (Phascolarctos cinereus) and their ticks identified using next-generation sequencing (NGS)

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

Identification of eukaryotic microorganisms with 18S rRNA next-generation sequencing in wastewater treatment plants, with a more targeted NGS approach required for Cryptosporidium detection

While some microbial eukaryotes can improve effluent quality in wastewater treatment plants (WWTPs), eukaryotic waterborne pathogens are a threat to public health. This study aimed to identify Eukarya, particularly faecal pathogens including Cryptosporidium, in different treatment stages (influent, intermediate and effluent) from four WWTPs in Western Australia (WA). Three WWTPs that utilise stabilisation ponds and one WWTP that uses activated sludge (oxidation ditch) treatment technologies were sampled. Eukaryotic 18S rRNA (18S) was targeted in the wastewater samples (n = 26) for next-generation sequencing (NGS), and a mammalian-blocking primer was used to reduce the amplification of mammalian DNA. Overall, bioinformatics analyses revealed 49 eukaryotic phyla in WWTP samples, and three of these phyla contained human intestinal parasites, which were primarily detected in the influent. These human intestinal parasites either had a low percent sequence composition or were not detected in the intermediate and effluent stages and included the amoebozoans Endolimax sp., Entamoeba sp. and Iodamoeba sp., the human pinworm Enterobius vermicularis (Nematoda), and Blastocystis sp. subtypes (Sarcomastigophora). Six Blastocystis subtypes and four Entamoeba species were identified by eukaryotic 18S NGS, however, Cryptosporidium sp. and Giardia sp. were not detected. Real-time polymerase chain reaction (PCR) also failed to detect Giardia, but Cryptosporidium-specific NGS detected Cryptosporidium in all WWTPs, and a total of nine species were identified, including five zoonotic pathogens. Although eukaryotic 18S NGS was able to identify some faecal pathogens, this study has demonstrated that more specific NGS approaches for pathogen detection are more sensitive and should be applied to future wastewater pathogen assessments

The Asian fish tapeworm (Schyzocotyle acheilognathi) discovered in Western Australia may pose a threat to the health of endemic native fishes

The Asian fish tapeworm (Schyzocotyle acheilognathi) is an important fish parasite with a wide host range that infects over 300 species of fish worldwide. Schyzocotyle acheilognathi has been reported from eastern coastal areas of Australia, but has not been previously reported in Western Australia (WA). During a control program for invasive freshwater fishes in south-western WA, a region with a unique and highly endangered freshwater fish fauna, tapeworms identified as S. acheilognathi from their distinctive scolex morphology were found at a prevalence of 3.3% in goldfish (Carassius auratus), 37.0% in koi carp (Cyprinus carpio haematopterus) and 65.0% in eastern gambusia (Gambusia holbrooki) in a small suburban lake to the north of Perth. For molecular confirmation, the 18S ribosomal RNA gene was targeted at hypervariable V4 region. Koi carp isolates were 100% identical to S. acheilognathi isolated from varying hosts, including the red shiner (Cyprinella lutrensis) and a human sample. Sequences obtained from two eastern gambusia were identified as S. acheilognathi, but formed a discrete cluster and may represent a novel genotype. Isolates from two other eastern gambusia and two goldfish formed a distinct clade with only 91.9% similarity to previously sequenced isolates of S. acheilognathi. This emphasizes the importance of molecular identification methods in addition to morphological identification. The presence and potential for transmission of these parasites in south-western WA may threaten the health of native fishes, which are immunologically naïve to this introduced parasite. Immediate control or containment measures should be implemented to halt the spread of these parasites

An Australian dog diagnosed with an exotic tick-borne infection: Should Australia still be considered free from Hepatozoon canis?

Recent molecular and sero-surveillance studies of the tick-borne pathogen Hepatozoon canis have identified new hosts, potential vector species, and have revealed that H. canis is more widespread than previously thought. We report the first diagnosed case of canine hepatozoonosis in Australia from a Maremma Sheepdog in Sarina, Queensland. Hepatozoon canis was detected with blood smear examination and 18S rRNA sequencing. It is unknown when or how the organism was introduced into Australia, which raises questions about border biosecurity policies and the H. canis infection status of its potential vectors and hosts in Australia. Surveillance for this pathogen is required to determine whether H. canis has established in Australia

Response to the Letter to the Editor by Harris

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