Diversity of spotted fever group rickettsiae and their association with host ticks in Japan

Abstract

Spotted fever group (SFG) rickettsiae are obligate intracellular Gram-negative bacteria mainly associated with ticks. In Japan, several hundred cases of Japanese spotted fever, caused by Rickettsia japonica, are reported annually. Other Rickettsia species are also known to exist in ixodid ticks; however, their phylogenetic position and pathogenic potential are poorly understood. We conducted a nationwide cross-sectional survey on questing ticks to understand the overall diversity of SFG rickettsiae in Japan. Out of 2,189 individuals (19 tick species in 4 genera), 373 (17.0%) samples were positive for Rickettsia spp. as ascertained by real-time PCR amplification of the citrate synthase gene (gltA). Conventional PCR and sequencing analyses of gltA indicated the presence of 15 different genotypes of SFG rickettsiae. Based on the analysis of five additional genes, we characterised five Rickettsia species; R. asiatica, R. helvetica, R. monacensis (formerly reported as Rickettsia sp. In56 in Japan), R. tamurae, and Candidatus R. tarasevichiae and several unclassified SFG rickettsiae. We also found a strong association between rickettsial genotypes and their host tick species, while there was little association between rickettsial genotypes and their geographical origins. These observations suggested that most of the SFG rickettsiae have a limited host range and are maintained in certain tick species in the natural environment.

Figure 1

A phylogenetic tree of spotted fever group rickettsiae based on the gltA gene sequences (537 bp). The analysis was performed using a maximum likelihood method with the Kimura 2-parameter model. All bootstrap values from 1,000 replications are shown on the interior branch nodes. The sequences detected in this study are indicated in red. The number of samples positive for each genotype is indicated in the parentheses. The simplified tick phylogeny consisting of 12 tick species is indicated on the top right. The colour highlights in the column indicate the presence of infections in each tick species.

Figure 2

Phylogenetic trees based on the sequences of the ompA (493 bp) (A), ompB (780 bp) (B), htrA (465 bp) (C), sca4 (887 bp) (D), and 16S rRNA (1,245 bp) (E) genes. The analyses were performed using a maximum likelihood method with the Kimura 2-parameter model. All bootstrap values from 1,000 replications are shown on the interior branch nodes. The sequences obtained in this study are shown in red.

Figure 3

Sample collection sites.