1.B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.

Borrelia isolated from various sources in Japan, including rare species of ixodid ticks, Ixodes tanuki, I. turdus, and I. columnae, were characterized by restriction fragment length polymorphism analysis and sequencing analysis of the 5S-23S rRNA intergenic spacer amplicon. Borrelia sp. isolated from I. tanuki, I. turdus and I. columnae generated restriction fragment length polymorphism patterns different from those of known B. burgdorferi sensu lato isolates previously reported. Furthermore, some B. afzelii and B. garinii isolated in Japan showed unique RFLP patterns which were not observed among European B. afzelii and B. garinii.

Restriction fragment length polymorphism (RFLP) analysis of the outer surface protein C (ospC) gene amplicon was used for rapid screening for genetic variability within Borrelia burgdorferi sensu stricto species and for detection of multiple borreliae in culture. Primers for the ospC gene amplified a fragment of about 600 bp from Borrelia cultures. After cleavage of the amplified products by MboI and DraI, eight different RFLP types were found among 13 B. burgdorferi sensu stricto strains from various sources and geographical areas, and three RFLP types were found among 10 representative isolates from skin biopsy specimens taken from patients residing on the eastern end of Long Island, New York (B. W. Berger, R. C. Johnson, C. Kodner, and L. Coleman, J. Clin. Microbiol. 30:359-361, 1992). These results suggested that the DNA organization of B. burgdorferi sensu stricto is heterogeneous not only globally but also within a localized geographical area and that the ospC-based typing approach could differentiate the B. burgdorferi sensu stricto. From the results obtained using mixed cultures of two different RFLP types of B. burgdorferi sensu stricto, contamination of at least 0.5% of different types of Borrelia cells in culture could be detected. This method could detect a multiple-B. burgdorferi sensu stricto infection in the bladders of mice experimentally infected with two different RFLP type strains. The present study showed that RFLP analysis of ospC-PCR products is a reliable method for epidemiological typing of B. burgdorferi sensu stricto and could be used for rapid detection of mixed Borrelia culture and multiple B. burgdorferi sensu stricto infections in animals, ticks, and patients.

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 by sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence. The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 703% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.

Borrelia afzelii, B. japonica, and 'B. tanukii' isolated from various sources and geographical origins in Japan were characterized by restriction fragment length polymorphism (RFLP) analysis and sequencing analysis of the outer surface protein C (OspC) amplicon. B. afzelii and 'B. tanukii' generated variable RFLP patterns and differences in ospC gene sequence were confirmed. In contrast, 26 isolates of B. japonica generated one OspC RFLP type, and sequence similarity between B. japonica ranged from 96.4 to 99.7%. These finding suggests that B. japonica is unique in comparison with other members of B. burgdorferi sensu lato species with respect to homogeneity of the ospC gene.