During the spring to summer seasons (April to July) of 2012 and 2013, host-seeking adult ticks of I. persulcatus, I. pavlovskyi and I. ovatus were collected by flagging over vegetation from a total of 57 forested areas in Hokkaido (Figure 1 ). In these areas, no specific permissions were required for collection of ticks, and this study did not involve endangered or protected species. The collection of I. ovatus was discontinued in 2013 because of its extremely low prevalence of B. miyamotoi infection. The number of ticks examined, and the prevalence of Borrelia spp. among them were calculated for each district.
All of the ticks collected were subjected to a quantitative real-time PCR (qPCR) assay to detect spirochete DNA. The DNA for PCR templates was prepared from whole or half bodies of each tick by using sodium hydroxide (NaOH), as described previously [18] (link). Briefly, tick tissues including bacterial cells were lysed in 50 µL of 25 mM NaOH for 10 minutes at 95°C. After adding 4 µL of Tris-HCL (1 M, pH7.5) for neutralization, the lysate was centrifuged at 4°C, and the resulting supernatant was used as template DNA for qPCR. A preliminary study suggested that the sensitivity of qPCR using the lysate was equal to that using DNA extracted by DNeasy Tissue Kit (Qiagen, CA, USA) (Data not shown).
Parts of ticks collected were used to cultivate B. miyamotoi in modified Barbour-Stoenner-Kelly medium (BSK-M: using minimal essential medium alpha [BioWest, Germany] as a substitute for CMRL-1066) under microaerophilic conditions [19] (link), [20] (link) (Table S1 ). The surface of ticks was sterilized by washing with 0.1% sodium hypochlorite, then with 80% ethyl alcohol. The washed tick was longitudinally bisected using a disposable knife (ELP No. 10, Akiyama Medical MFG. CO., LTD., Tokyo, Japan), and one half was inoculated into BSK-M medium. The remaining half was used to prepare the PCR template, as described above. Tick samples which were shown by qPCR to be positive for B. miyamotoi and negative for LD borreliae, were cultivated at 30°C for 4 weeks, and then the growth of spirochetes was examined by dark-field microscopy every two weeks.
All of the ticks collected were subjected to a quantitative real-time PCR (qPCR) assay to detect spirochete DNA. The DNA for PCR templates was prepared from whole or half bodies of each tick by using sodium hydroxide (NaOH), as described previously [18] (link). Briefly, tick tissues including bacterial cells were lysed in 50 µL of 25 mM NaOH for 10 minutes at 95°C. After adding 4 µL of Tris-HCL (1 M, pH7.5) for neutralization, the lysate was centrifuged at 4°C, and the resulting supernatant was used as template DNA for qPCR. A preliminary study suggested that the sensitivity of qPCR using the lysate was equal to that using DNA extracted by DNeasy Tissue Kit (Qiagen, CA, USA) (Data not shown).
Parts of ticks collected were used to cultivate B. miyamotoi in modified Barbour-Stoenner-Kelly medium (BSK-M: using minimal essential medium alpha [BioWest, Germany] as a substitute for CMRL-1066) under microaerophilic conditions [19] (link), [20] (link) (
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