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We compared the efficacy of three antibiotics (ceftriaxone, erythromycin and clarithromycin) against Borrelia garinii infection in mice. The nymphal ticks of Ixodes persulcatus infected with the strain JEM6 of Japanese B. garinii were allowed to feed on female C3H mice. The mice were treated with each of the antibiotics for 5 consecutive days 1, 3, or 7 weeks after tick detachment. The doses of antibiotics per day were as follows: 5 mg intraperitoneal injection of ceftriaxone, 2 mg intraperitoneal injection of erythromycin and 1 mg peroral administration of clarithromycin. The infection status in treated mice was monitored by culturing their earlobes, hearts and urinary bladders in BSK II medium. Ceftriaxone eliminated borreliae completely; however, a recurrence of infection was observed in mice treated with erythromycin and clarithromycin.

Based on the results of RFLP-ribotyping, whole DNA/DNA hybridization and phylogenetic analysis of the 16S rRNA gene, we previously defined two genomic groups of spirochetes closely related to Borrelia burgdorferi sensu lato: group Hk501 for strains isolated from Ixodes tanuki ticks and group Ya501 for strains isolated from Ixodes turdus ticks. In this report, we propose that group Hk501 should be classified as Borrelia tanukii sp. nov. and group Ya501 as Borrelia turdae sp. nov. The alignment of previously published Borrelia 16S rRNA gene sequences led us to design species-specific PCR primer sets. The primers allowed the rapid identification of B. tanukii and B. turdae.

We surveyed the natural distribution of Borrelia species associated with Lyme disease in the subalpine forests of Nagano prefecture, Japan, during 1993-94. Tick-derived isolates (n = 112) from Ixodes persulcatus and rodent-derived isolates (n = 55) from Apodemus argenteus, Apodemus speciosus, Eothenomys andersoni, Eothenomys smithii, and Microtus montebelli were classified by rRNA gene restriction fragment length polymorphism analysis (RFLP ribotyping). Ribotype group IV (an intraspecific variant of Borrelia garinii) was predominant among the tick isolates. It was also isolated repeatedly from the rodents. Ribotype group III (Borrelia afzelii) was detected in low frequencies among the tick and rodent isolates. The data suggest that humans are likely to be exposed to the group IV when they are bitten by I. persulcatus ticks.

Spirochetes were isolated from earlobe tissues of shrews (Sorex unguiculatus, Sorex caecutiens, and Crocidura dsienezumi), voles (Clethrionomys rufocanus), and mice (Apodemus argenteus and Apodemus speciosus) captured in various localities in Japan. The isolates were identified as Borrelia japonica by rRNA gene restriction fragment length polymorphism analysis. The data suggest that these small mammals are candidates of reservoir hosts for B. japonica.

The genospecies Borrelia afzelii was isolated from a patient of Lyme disease in Hokkaido, Japan, for the first time, by culturing the minced erythema lesion in BSK II medium. Two analytical methods, rRNA gene restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) using the specific primer set to amplify the 16S rRNA gene, revealed that this clinical isolate belongs to the group of B. afzelii. In our culture collection of spirochetes, part of the isolates from Ixodes persulcatus ticks, and from Apodemus speciosus rodents, were also classified as B. afzelii. These results strongly suggest that the agent pathogenic to humans is maintained in "rodent-tick" transmission cycle.

Results of a passive microcapsule agglutination test using 2 Japanese strains, Borrelia garinii and Borrelia afzelii, were comparable to that of the passive microcapsule agglutination test using a Swiss strain Borrelia burgdorferi IRS in detecting antibodies of an early stage of Lyme disease patients in Japan. MCAT with microcapsule sensitized strain IRS seems likely to become one of the important tools for the early diagnosis of Lyme disease in the world. The highest titer of MCAT was detected in the serum samples of patients which were taken around 2 weeks after erythema chronicum migrants. The test is very simple and can be employed easily in diagnostic laboratories.

Unfed adult Ixodes persulcatus ticks were collected from four locations of Nagano and Hokkaido in Japan. Infected Borrelia garinii were investigated by PCR-RFLP of the ospA and ospB gene sequences. The primer set amplified an approximately 1.6-kb DNA fragment (0.7-kb in some strains), and BsrI, BstYI, or NlaIII digestion of the product resulted in six distinctively different PCR-RFLP groups and two independent borrelial strains. The representatives in each PCR-RFLP group and individuals from the borrelial strains were sequenced, and their deduced amino acid sequences were aligned. A neighbor-joining phylogenetic analysis showed that the B. garinii OspA or OspB sequences were each divided into three major clusters including isolates from both the Nagano and Hokkaido locations. There was no local difference in OspA/B sequences between Nagano and Hokkaido. The osp gene of Borrelia burgdorferi sensu lato is highly heterogeneous, and this was also confirmed by our sequence analysis. Some strains of the different PCR-RFLP groups had closely related OspA sequences, while the OspB sequences of these strains were quite different. These findings suggested intraspecies gene exchange and recombination events between the two genes in B. garinii.

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.

Four spirochetal isolates (JEM1 to JEM4) were obtained from cutaneous lesions of patients manifesting erythema chronicum migrans in Hokkaido, Japan. In the protein profiles by SDS-PAGE and the reactivities with monoclonal antibodies (H5332 and H9724) by immunoblotting, all the human isolates were identical with the tick isolates from Ixodes persulcatus. These data indicate that I. persulcatus is an important vector of Lyme disease for humans in Japan.