A survey was performed for Lyme disease borrelia in the southern part of China, in Zhejiang, Sichuan and Anhui provinces, along the Yangtze River valley, in May of 1997 and 1998. Twenty isolates from Ixodes granulatus, Ixodes ovatus, Apodemus agrarius and Niviventer confucianus were obtained. These isolates were characterized by RFLP of the 5S-23S rDNA intergenic spacer, sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene, DNA-DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. Six isolates from A. agrarius, five from I. granulatus collected in Zhejiang province and one from N. confucianus in Sichuan province were highly similar to strains 10MT and 5MT isolated in Korea and classified as Borrelia valaisiana. Four isolates from A. agrarius and I. granulatus collected in Zhejiang province generated unique RFLP patterns and phylogenetic analysis of the 16S rDNA and flagellin gene sequences suggested that the isolates should be classified as B. valaisiana. Furthermore, three isolates (CMN1a, CNM2, CMN3T) from N. confucianus captured in Sichuan province and one (CWO1) from I. ovatus in Anhui province showed lower 165 rDNA sequence similarity (less than 99.0%) to sequences of previously described Lyme disease-related Borrelia species. DNA-DNA hybridization results revealed that strains CMN3T and CMN1a were clearly distinct from all other known Lyme disease Borrelia species. Electron microscope observation showed the spirochaetes to be morphologically similar to those of Borrelia, but the cells contained only four periplasmic flagella inserted at each end of the spirochaetes. Based on these results, a new Borrelia species, Borrelia sinica sp. nov., is proposed. Strain CMN3T is the type strain of this new species.
In 2000 and 2001, a survey was conducted of Borrelia isolated from various mammals in the southernmost islands of Japan, including Okinawa (main island), Izena, Iriomote and Ishigaki. Isolates obtained from the ear tissues of Suncus murinus (10 isolates), Mus calori (four isolates), Rattus norvegicus (one isolate) and Crocidura watasei (one isolate), were characterized by RFLP of the 5S-23S rDNA intergenic spacer and sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene. While these isolates showed identical RFLPs to Borrelia valaisiana found in Korea, Taiwan and the southern and central parts of China, their RFLP patterns differed from those of B. valaisiana found in European countries, and strain Am501 isolated from Ixodes columnae in Japan. It was found that these isolates clustered with each other on a phylogenetic tree based on flagellin gene and 16S rDNA sequences, but were relatively divergent from the European B. valaisiana and strain Am501. These findings suggest that these isolates found in East Asia should be classified as a new genomospecies of Borrelia burgdorferi sensu lato.
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