An epidemiological survey of zoonotic Babesia species in questing ticks in Hokkaido, Japan, using a newly developed PCR-sequencing approach.

Human babesiosis caused by tick-borne zoonotic Babesia species is a public health concern. However, the absence of diagnostic assays for several Babesia species limits surveillance efforts, leading to knowledge gap on endemic species and vectors. The present study aimed to develop a PCR-sequencing-based method for detecting zoonotic Babesia species and use it to survey questing ticks in Hokkaido, Japan. Through phylogenetic analysis of 18S rRNA sequences, we grouped zoonotic Babesia species into three: 1) Babesia microti and Babesia duncani, 2) Babesia divergens, Babesia venatorum, and Babesia odocoilei, and 3) Babesia crassa-like species and Babesia sp. KO1. Three 18S rRNA-based PCR assays were then developed for each group and used to screen DNA samples from 1456 questing ticks collected in Hokkaido, Japan. Of these ticks, 10 were positive in B. microti/B. duncani-PCR assay, including five Ixodes persulcatus, three Ixodes ovatus, and two Haemaphysalis japonica. Phylogenetic analysis of 18S rRNA sequences derived from the PCR amplicons confirmed that these three tick species harboured US- and Hobetsu-types of B. microti. Additionally, one I. persulcatus tested positive in B. divergens/B. venatorum/B. odocoilei-PCR assay, with phylogenetic analyses of 18S rRNA and beta-tubulin sequences suggesting the presence of a B. divergens-like species. All ticks were negative in B. crassa-like/Babesia sp. KO1 PCR assay. In conclusion, the present study, which developed a novel diagnostic approach to detect major zoonotic Babesia species, not only detected zoonotic Babesia in known tick vectors, but also identified H. japonica as a new potential vector of B. microti in Hokkaido.