Illuminating the Bacterial Microbiome of Australian Ticks with and -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 ( = 655) from companion animals (dogs, cats and horses). Bacterial NGS was used to identify most bacterial taxa and a -specific NGS assay was developed to identify species that were indistinguishable at the V1-2 regions of . Sanger sequencing of near full-length was used to confirm whether species detected by NGS were novel. The haemotropic bacterial pathogens , , " Mycoplasma haematoparvum" and were identified in (.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, from QLD, () from the NT, and 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 sequence databases revealed inconsistencies. The -specific gene NGS assay enabled the identification of co-infections with potentially novel species and genotypes most similar (97.9-99.1%) to and . " Rickettsia jingxinensis" was identified for the first time in Australia. Phylogenetic analysis of near full-length sequences confirmed a novel genus and species, two novel species, and two novel 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 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.