We found Rickettsia raoultii infection in 6/261 brucellosis-negative patients with fever of unknown origin in brucellosis-endemic Inner Mongolia, China. We further identified Hyalomma asiaticum ticks associated with R. raoultii, H. marginatum ticks associated with R. aeschlimannii, and Dermacentor nuttalli ticks associated with both rickettsiae species in the autonomous region.
Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 10 copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.
Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1–V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.
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