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 approximate to4,000 unengorged chig gers obtained from the soil or ground surface in an endemic and a nonendemi c area of the Tsutsugamushi disease in Oita Prefecture, southwestern Japan, by nested polymerase chain reaction (PCR). Serotypes of O. tsutsugamushi w ere identified by restriction fragment-length polymorphism (RFLP) analysis. In the endemic area, 242 pools from five species [234 pools of Leptotrombi dium scutellare (Nagayo, Miyagawa, Mitamura, Tamiya and Tenjin),two L. pall idum (Nagayo, Miyagawa, Mitamura and Tamiya), four L, kitasatoi (Fukuzumi & Obata), one L. fuji (Kuwata, Berge and Philip), and one Neotombicula japon ica (Tanaka, Kaiwa Teramura and Kagaya)] were tested, and eight (seven pool s of L, scutellare and one hi japonica) were positive for O. tsutsugamushi. Among the seven positive pools of L, scutellare, the distribution of serot ypes was as follows: Kuroki (4), Gilliam (1), Karp (1), and Kawasaki (1). T he first two serotypes (Kuroki and Gilliam) were identified for the first t ime in this species. In the nonendemic area, 128 pools from eight species w ere tested, and 13 were positive for O, tsutsugamushi. The positive rate wa s as follows: L, pallidum (4/41), L, kitasatoi (1/18), Gahrliepia saduski W omersley (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 of the positive pools were Kuroki, except for two pools tone L. pallidum and one L. fuji), which were Gilliam (this s erotype was also detected for the first time in L, pallidum). Further analy sis 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) amo ng the positive samples. These data indicate that O. tsutsugamushi was wide ly distributed in various trombiculid species, even in the nonendemic area. The data are also suggestive of a possible horizontal transmission of O. t sutsugamushi among trombiculid species.