Detection of Orientia tsutsugamushi (Rickettsiales: rickettsiaceae) in unengorged chiggers (Acari: Trombiculidae) from Oita Prefecture, Japan, by nested polymerase chain reaction.

The current study surveyed the 56-kDa type-specific antigen (TSA) gene DNAs of Orientia tsutsugamushi (Hayashi) in approximately 4.000 unengorged chiggers obtained from the soil or ground surface in an endemic and a nonendemic area of the Tsutsugamushi disease in Oita Prefecture, southwestern Japan, by nested polymerase chain reaction (PCR). Serotypes of O. tsutsugamushi were identified by restriction fragment-length polymorphism (RFLP) analysis. In the endemic area, 242 pools from five species [234 pools of Leptotrombidium scutellare (Nagayo, Miyagawa, Mitamura, Tamiya and Tenjin), two L. pallidum (Nagayo, Miyagawa, Mitamura and Tamiya), four L. kitasatoi (Fukuzumi & Obata), one L. fuji (Kuwata, Berge and Philip), and one Neotrombicula japonica (Tanaka, Kaiwa, Teramura and Kagaya)] were tested, and eight (seven pools of L. scutellare and one N. japonica) were positive for O. tsutsugamushi. Among the seven positive pools of L. scutellare, the distribution of serotypes was as follows: Kuroki (4), Gilliam (1), Karp (1), and Kawasaki (1). The first two serotypes (Kuroki and Gilliam) were identified for the first time in this species. In the nonendemic area, 128 pools from eight species were tested, and 13 were positive for O. tsutsugamushi. The positive rate was as follows: L. pallidum (4/41). L. kitasatoi (1/18), Gahrliepia saduski Womersley (2/10), L. fuji (4/50), L. himizu (Sasa, Kumada, Hayashi, Enomoto, Fukuzumi and Obata) (1/2), and Miyatrombicula kochiensis (Sasa, Kawashima and Egashira) (1/3). The latter three species were shown for the first time to harbor O. tsutsugamushi. All ofthe positive pools were Kuroki, except for two pools (one L. pallidum and one L. fuji), which were Gilliam (this serotype was also detected for the first time in L. pallidum). Further analysis revealed no differences in the nucleotide sequences (125 bp of variable domain 1 of TSA gene) of the same serotypes (i.e., Kuroki and Gilliam) among the positive samples. These data indicate that O. tsutsugamushi was widely distributed in various trombiculid species, even in the nonendemic area. The data are also suggestive of a possible horizontal transmission of O. tsutsugamushi among trombiculid species.