Isolation of , , and from Questing Ticks in Japan Using Arthropod Cells.

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from embryos (ISE6) and larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: , , and These included four previously validated rickettsial species namely ( = 2), ( = 3), ( = 2) and ( = 3) and one uncharacterized genotype sp. LON ( = 2). Four isolates of had the highest similarity with previously reported isolates: obtained from ticks in North America and sp. Bratislava 1 obtained from in Europe, while two isolates of showed 100% identity with sp. detected from at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.