The ixodid tick Ixodes persulcatus is the most important vector of Lyme disease in Japan. Most spirochete isolates obtained from I. persulcatus ticks have been classified as Borrelia burgdorferi sensu lato because of their genetic, biological, and immunological characteristics. However, we found that a small number of isolates obtained from I. persulcatus contained a smaller 38-kDa endoflagellar protein and single 23s-5s rRNA gene unit. Representative isolate HT31T (T = type strain) had the same 23s rRNA gene physical map as Borrelia turicatae. The DNA base composition of strain HT31T was 28.6 mol% G+C. DNA-DNA hybridization experiments revealed that strain HT31T exhibited moderate levels of DNA relatedness (24 to 51%) with Borrelia hemsii, B. turicatae, Borrelia parkeri, and Borrelia coriaceae. However, the levels of DNA reassociation with the previously described Lyme disease borreliae (B. burgdorferi, Borrelia garinii, and Borrelia afielii) were only 8 to 13%. None of the previously described species examined exhibited a high level of DNA relatedness with strain HT31T. In addition, the 16s rRNA gene sequence (length, 1,368 nucleotides) of strain HT31T was determined and aligned with the 16s rRNA sequences of other Borrelia species. Distance matrix analyses were performed, and a phylogenetic tree was constructed. The results showed that isolate HT31T is only distantly related to both previously described Lyme disease borreliae and relapsing fever borreliae. Thus, the spirochetes isolated from I. persulcatus and closely related isolates should be classified as members of a new Borrelia species. We propose the name Borrelia rniyamotoi sp. nov. for this spirochete; strain HT31 is the type strain.We previously demonstrated the usefulness of a restriction fragment length polymorphism (RFLP) ribotyping system based on the 23s-5s rRNA gene repetition in Borrelia burgdorferi sensu lato associated with Lyme disease (14, 32). Many spirochete isolates were examined with our RFLP ribotyping system by using rRNA gene probes. The strains isolated in the United States and Europe were placed into three distinct RFLP groups. The North American isolates clustered in ribotype group I (B. burgdo$eri sensu stricto), and the European isolates were placed in ribotype groups I and I1 (Borrelia garinii) and ribotype group I11 (Borrelia afielii). These groups are completely consistent with the three previously described Lyme disease agent species (2,7,18). Our findings also showed that there are no B. burgdorferi sensu stricto representative strains in Japan and that some Japanese isolates belong to ribotype groups I1 and 111. In addition, most of the Japanese isolates produced RFLP patterns that were quite distinct from those of the North American and European isolates and were tentatively classified as ribotype group IV strains (14). Borrelia japonica is carried by Ixodes ovatus ticks, and it is thought that this microorganism is restricted to Japan. Moreover, some atypical spirochetes have been isolated in United States and Europe (...

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.

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