Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Orientia tsutsugamushi is an obligate intracellular, Gramnegative bacterium that causes scrub typhus, an acute febrile illness characterized by a typical primary lesion (eschar), lymphadenopathy, and nonspecific symptoms such as chills, headache, and malaise (10, 24). The disease is endemic in Asian countries, including Japan, South Korea, China, Thailand, and Taiwan (6, 13). An estimated 1 million cases occur annually (22). However, the precise incidence of this disease is unknown, as the available diagnostic facilities in most of the region of endemicity are limited (32). In nature, the bacterial agent is maintained within a life cycle involving wild mammals and a mite vector, primarily of the Leptotrombidium genus (14). Humans are accidentally infected by the bites of a larval mite, known as a chigger, during feeding (30).The 56-kDa type-specific antigen (TSA) gene encodes a dominant membrane protein, which accounts for 10% to 15% of the total protein of O. tsutsugamushi (17). The deduced size of the 56-kDa protein varies from 516 to 541 amino acids, according to gene sequences deposited in the GenBank (11). It is highly immunogenic, and the sera of most patients with scrub typhus are reactive. Serological analyses show that the antigenicity of the 56-kDa protein is diverse and includes variants such as the representative Karp-, Gilliam-, and Kato-type strains and other isolates (2,8,18). The immune responses to the 56-kDa protein provide protection against Scrub typhus, suggesting that it is a potential candidate for vaccine development (3,16,33). No commercially licensed vaccine for scrub typhus is currently available.In the present study, we cloned and sequenced the 56-kDa TSA gene of O. tsutsugamushi isolated from the chigger mite obtained from a rodent in order to understand the genetic types as well as the relationships with other known strains. Our results demonstrated that the genotypes of O. tsutsugamushi isolates appeared highly diverse and widely and geographically distributed in Taiwan. MATERIALS AND METHODSStudy site and arthropod collection. All the arthropods used in this study were collected from rodents captured by randomly settin...