Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.

The nucleotide sequences (534 to 546 bp) of the groEL gene, which encodes the 60-kDa heat shock protein GroEL, from 15 rickettsial strains were determined and compared. In the phylogenetic tree created by the unweighted pair group method with arithmetic averages and the neighbor-joining method, rickettsial strains could be distinguished from Ehrlichia strains. Five spotted fever group strains, four typhus group strains, and six scrub typhus group (STG) strains were differentiated as distinct entities. Unlike gltA and ompA gene analyses, differentiation between members of the genus Rickettsia and the STG rickettsiae by groEL gene analysis was possible. In comparison with 16S rRNA gene analysis, the groEL gene has a higher degree of divergence among the rickettsiae. We therefore successfully developed rapid differentiation methods, PCRrestriction fragment length polymorphism analysis and a species-specific PCR, based on the groEL gene sequences. Four Korean isolates were identified by these methods and groEL gene analysis. The results suggest that the groEL gene is useful for the identification and characterization of rickettsiae.

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The nucleotide sequences of the groEL genes, the flagellin genes, and the 16S rRNA genes from 22 reference strains of Borrelia were compared. groEL sequence analysis is useful not only in interspecies differentiation but also in intraspecies differentiation of Borrelia afzelii and Borrelia garinii isolates.Borrelia burgdorferi sensu lato, the causative spirochete of Lyme disease, is transmitted to humans and animals through Ixodes ticks (1, 3). Lyme disease is one of the most prevalent tick-borne infectious diseases in Europe and North America and now occurs all over the world (1, 12). Various DNA-based techniques have recently been developed for the species identification of B. burgdorferi because characterization and identification by conventional methods are time-consuming and expensive (2,5,6,11). A previous study proposed that groEL gene analysis is useful for the differentiation of B. burgdorferi sensu lato (9). In the present study, groEL gene analysis was compared with the sequence analyses of the 16S rRNA and the flagellin gene to determine the role of the groEL gene in defining evolutionary relationships among strains of B. burgdorferi sensu lato.Twelve Borrelia strains were recently isolated from Ixodes granulatus, Ixodes nipponensis (tick vectors for Lyme spirochetes in rare cases), and Ixodes persulcatus, and 11 strains were isolated from Apodemus agrarius (8,10). In previous studies, they were characterized as Borrelia afzelii, Borrelia garinii, and unclassified Haenam strains (8,10). In the present study, a comparative sequence analysis of the groEL gene from Korean isolates was performed to determine their relationships with the known species of the genus Borrelia.Twenty-two reference strains (Table 1) and 23 Korean isolates of the genus Borrelia were used in this study. The strains were cultivated at 32°C in Barbour-Stoenner-Kelly II (BSKII) medium. DNA was extracted by a modified version of a previously described method (4). The groEL genes of 22 reference strains and 23 Korean isolates and the flagellin genes and the 16S rRNA genes of 22 reference strains were amplified as presented in Table 2. The nucleotide sequences of the recombinant DNA were determined using the CEQ L DNA Analysis System and the CEQ 2000 Dye Terminator Cycle Sequencing kit (Beckman Coulter Inc., Fullerton, Calif.) with forward and reverse sequencing primers (M13) and sequencing primers ( Table 2). The multiple-alignment algorithm in the MegAlign software package (Windows version 3.12e; DNASTAR, Madison, Wis.) was used to align the sequences. All positions with alignment gaps were excluded from the pairwise sequence comparison. Phylogenetic trees were constructed by the unweighted pair group method with arithmetic averages using the MEGA program (7). A bootstrap analysis (100 replicates) was performed to evaluate the topology of the phylogenetic tree.In this study, interspecies differences in the groEL genes (positions 552 to 861 in B. burgdorferi B31 T numbering; 310 bp) of B. burgdorferi strains sensu lato were compared ...

In this study, two new duplex PCR methods based on the groEL gene were developed and investigated for the diagnosis of rickettsiae. The first duplex PCR assay amplified the 229‐bp and the 366‐bp DNAs of 6 strains including typhus group (TG) and spotted fever group (SFG) rickettsiae, and 5 scrub typhus group (STG) rickettsiae, respectively. The second duplex PCR assay amplified the 397‐bp and the 213‐bp DNAs of 6 Rickettsia strains and 5 STG strains. These duplex PCR methods could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of STG and other group rickettsiae in a single reaction.