We determined nearly complete flagellin gene sequences for sensu lato isolates (11 isolates obtained from ticks and patients in Hokkaido, Japan, and 1 European isolate) representing six different restriction fragment length polymorphism (RFLP) ribotype groups following cloning of the PCR-amplified genes. These sequences were aligned with those of representatives of the three species associated with Lyme disease, and a phylogenetic tree was construced by the Clustal method. On the Lyme disease borrelia portion of the tree, the species were clearly delineated into three different phylogenetic groups, in complete agreement with the division of sensu lato into three species. A phylogenetic analysis revealed that the representatives of RFLP ribotype groups IV, V, and VI clustered tightly with each other and belonged on the same branch as . We used the criteria that are currently used to delineate bacterial species and determined the levels of DNA relatedness for these isolates. For the RFLP ribotype group IV, V, and VI isolates, the levels of DNA relatedness ranged from 79 to 88%, and the levels of relatedness to the reference strain of ranged from 70 to 80%. The levels of DNA relatedness of the RFLP ribotype group IV, V, and VI isolates to the representatives of other species associated with Lyme disease ranged from 53 to 66%. All of these findings indicate that the RFLP ribotype group IV, V, and VI isolates should be included in the species .


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