Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya

Abstract

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Keywords: Neotrombicula and Microtrombicula chiggers; Orientia chuto; Orientia tsutsugamushi; chiggers; rodents; scrub typhus.

FIG 1

Agarose gel electrophoresis of TSA47 gene (lanes 1 to 4) amplified by nested PCR with species-specific primers. M: 1 Kb Plus DNA ladder (Invitrogen, CA, USA). Lane 1, O. tsutsugamushi positive control amplified with O. tsutsugamushi primers. Lane 2, lack of amplification of chigger mite sample by O. tsutsugamushi primers. Lane 3, lack of amplification of O. tsutsugamushi positive control by O. chuto primers. Lane 4, amplification of chigger mite sample by O. chuto primers. Lane 5, amplification of O. tsutsugamushi positive control with genus-specific TSA56 primers. The Orientia genus TSA56 primers did not amplify the chigger mite sample (lane 6).

FIG 2

Phylogenetic tree of the study strain and prototype Orientia strains. The tree was constructed using the maximum likelihood method, based on 850 bp sequence of the TSA47 gene. The red triangle indicates the sequence from this study.

FIG 3

Evolutionary tree of Orientia species based on the 441-bp sequence of the 16S rRNA of the study OTU. The tree was generated using the maximum likelihood method in MEGA v 7.0 software. The red triangle corresponds to the sequence of the study OTU.