Received 9 February 1996/Accepted 20 April 1996

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1996, p. 2338 2344 Vol. 62, No. 7 0099-2240/96/$04.00 0 Copyright 1996, American Society for Microbiology Characterization of Spirochetes Isolated from Ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and Comparison of the Sequences with Those of Borrelia burgdorferi Sensu Lato Strains MASAHITO FUKUNAGA, 1 * AKIKO HAMASE, 1 KEIJI OKADA, 1 HIROFUMI INOUE, 1 YASUTO TSURUTA, 1 KENJI MIYAMOTO, 2 AND MINORU NAKAO 2 Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-02, 1 and Department of Parasitology, Asahikawa Medical College, Asahikawa, Hokkaido 078, 2 Japan Received 9 February 1996/Accepted 20 April 1996 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%. Since the first description of Borrelia burgdorferi, a large number of isolates have been obtained from tick vectors, vertebrate reservoirs, and Lyme disease patients in the Northern Hemisphere (1 4, 30, 31, 48, 51). B. burgdorferi sensu lato has now been separated into three distinct species, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (7, 11, 24). It has been generally assumed that the spirochete B. burgdorferi sensu lato is transmitted to humans via the bite of infected ticks belonging to the Ixodes ricinus species complex, North American Ixodes scapularis and Ixodes pacificus, European Ixodes ricinus, and Asiatic Ixodes persulcatus (3, 21). Recently, two new nonpathogenic Borrelia species, Borrelia japonica, found solely in Ixodes ovatus ticks, and Borrelia andersonii, commonly associated with Ixodes dentatus ticks and their wildlife hosts, have been reported (25, 28). In addition, group DN127 isolates from Ixodes neotomae ticks in North America appear to form a divergent group of spirochetes (41). They differ from B. burgdorferi sensu lato strains in their immunological and genetic characteristics. DNA-DNA hybridization results and 16S rrna gene sequence analysis have shown that the group DN127 isolates are closely related to B. burgdorferi sensu stricto (6, 41). These results appear to have shown the presence of a specific relationship between Borrelia species and arthropod vectors. It is therefore of interest to elucidate how Borrelia species adapted to their specific vector ticks. An understanding of the phylogenetic positions of these Borrelia species may provide information about the evolutionary adaptation of Borrelia species to ticks. * Corresponding author. Mailing address: Laboratory of Molecular Microbiology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Sanzo 985, Gakuencho 1, Fukuyama, Hiroshima 729-02, Japan. Phone: 81 849 36 2111. Fax: 81 849 36 2024. Electronic mail address: mfukunag@ddbj.nig.ac.jp. Ixodes persulcatus, which belongs to the I. ricinus complex, serves as a tick vector for Lyme disease pathogens in Japan (32, 33). We analyzed a large number of spirochete isolates from I. persulcatus by rrna gene restriction fragment length polymorphism (RFLP ribotyping) and classified them into ribotypes II (B. garinii), III (B. afzelii), IV, V, and VI (15, 16, 37, 38). Although we regarded ribotypes IV, V, and VI as unknown species, our recent studies suggested that they are strains of B. garinii (13, 14). In Japan, 18 species of Ixodes have been recorded (27, 39, 44, 53). I. persulcatus and I. ovatus are the dominant species in the northern part of Japan (32, 33, 35), where a high prevalence of spirochete infection was documented in unfed adult ticks of both species (33). Spirochetes were also isolated from Ixodes tanuki, Ixodes turdus, and Ixodes columnae collected in various locales in Japan (20, 34). These ticks are relatively rare species and do not belong to the I. ricinus species complex. The early study showed that the spirochetes isolated from these ticks have heterogeneous outer surface proteins and uniform 41- kda flagellin proteins (34). We also found that protein profiles of some spirochete isolates from Clethrionomys rufocanus voles are quite similar to those of isolates from I. tanuki (34). However, the taxonomic positions of these isolates remain to be determined. In this study, we performed additional experiments to characterize these unidentified spirochete isolates. The analytical methods used were sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot) analysis, RFLP ribotyping, sequencing of the outer surface protein C (OspC) gene, determination of the G C content, and whole DNA-DNA hybridization. Furthermore, 16S rrna gene sequences of spirochetes isolated from rare ticks and small rodents were determined and compared with those of the strains belonging to known Borrelia species. The results indicate that 2338

VOL. 62, 1996 CHARACTERIZATION OF SPIROCHETES FROM RARE TICK SPECIES 2339 Species TABLE 1. Species, origins, RFLP ribotypes, and accession numbers of strains used in this study Strain or isolate Origin RFLP ribotype OspC sequence Accession no. of: 16S rrna sequence B. burgdorferi sensu stricto B31 Ixodes scapularis, USA I D49497 M88329 297 Patient, USA I 20004 Ixodes ricinus, France I M64310 Sh-2-82 Ixodes scapularis, USA ND a M60969 1352 Amblyomma americanum, USA ND M64309 B. garinii 20047 Ixodes ricinus, France II D49498 D67018 PBi Patient, Germany II X69595 G1 Patient, Germany II M64311 G2 Patient, Germany II M60967 B. afzelii VS461 Ixodes ricinus, Switzerland III D49379 PKo Patient, Germany ND X62162 IP3 Ixodes persulcatus, Russia III M75149 J1 Ixodes persulcatus, Japan III L46697 HT61 Ixodes persulcatus, Hokkaido, Japan III D67019 B. japonica HO14 Ixodes ovatus, Hokkaido, Japan X D50801 L40597 NT112 Ixodes ovatus, Nagano, Japan X IKA2 Ixodes ovatus, Shizuoka, Japan ND L40598 B. andersonii 21038 Ixodes dentatus, USA ND L46701 19857 Rabbit (kidney), USA ND L46688 Group Hk501 Hk501 Ixodes tanuki, Hokkaido, Japan VII D50803 D67023 Hk512 Ixodes tanuki, Hokkaido, Japan VII Hk513 Ixodes tanuki, Hokkaido, Japan VII Hk514 Ixodes tanuki, Hokkaido, Japan VII Hk515 Ixodes tanuki, Hokkaido, Japan VII Hk516 Ixodes tanuki, Hokkaido, Japan VII OR1eR Clethrionomys rufocanus, Hokkaido, Japan VII D50802 OR2eL Clethrionomys rufocanus, Hokkaido, Japan VII D67020 OR3eL Clethrionomys rufocanus, Hokkaido, Japan VII Group Ya501 Ya501 Ixodes turdus, Yamagata, Japan VIII D50800 D67022 Ac502 Ixodes turdus, Aichi, Japan VIII D50799 D67024 Ac503 Ixodes turdus, Aichi, Japan VIII Ac504 Ixodes turdus, Aichi, Japan VIII Yh501 Ixodes turdus, Yokohama, Japan VIII Kt501 Ixodes turdus, Kyoto, Japan VIII Borrelia sp. Am501 Ixodes columnae, Aomori, Japan IX D50798 D67021 DN127 Ixodes pacificus, USA ND L40596 B. hermsii HS1 Ornithodoros hermsi, USA ND M60968 a ND, not determined. some strains are different from hitherto known Borrelia species. MATERIALS AND METHODS Borrelia isolates and culture. Designation and origins of isolates used in this study are given in Table 1. The adult ticks of I. tanuki fed on raccoon dogs (Nyctereutes procyonoides), the adult ticks of I. turdus fed on passerine birds, and unfed nymph ticks of I. columnae were used as culture sources (34). The isolates were established by culturing the midgut tissues of these ticks or the earlobe tissues of C. rufocanus voles. Representative strains of B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. japonica used as controls are also listed in Table 1. All spirochetes were grown in BSKII medium at 31 C and harvested by centrifugation as described previously (16). SDS-PAGE and Western blotting. Whole-cell lysates of spirochetes were analyzed by SDS-PAGE and Western blotting as previously described (15, 36, 37). Monoclonal antibody (MAb) H9724, reactive with the periplasmic flagella of the genus Borrelia (8), and MAb H5332, specific for the outer surface protein A (OspA) of B. burgdorferi, were used as probes for Western blot analysis (9). Determination of the G C content. The G C content was determined by the high-performance liquid chromatography method as described in our previous paper (17). RFLP ribotype analysis. Spirochetal DNAs digested with EcoRV or HincII were electrophoresed in a 0.7% agarose gel and blotted. The 23S rrna gene fragments NP (about 200 bp of the 5 end of the gene) and Sty (about 300 bp of the 3 end of the gene) from B. burgdorferi sensu stricto B31 were labeled and used as hybridization probes as described previously (37). The other experimental conditions of the RFLP analysis were described in detail in our previous reports (16, 37). Whole DNA-DNA hybridization. The extent of DNA reassociation was determined by the membrane filter hybridization method as described previously (17). Cloning of an OspC gene and sequencing. PCR primers (5 -TAA TGA AAA AGA ATA CAT TAA GTG-3, located at positions 2 to 22, and 5 -TTA AGG TTT TTT TGG ACT TTC TGC-3, located at positions 639 to 616) were designated from the published OspC gene sequence from the German strain PKo (12) and used to amplify the whole OspC gene as described previously (13). The PCR-amplified product was purified and ligated into the pgem5zf vector by using the pgem-t vector system (Promega Biotec, Madison, Wis.) as specified by the manufacturer. Resulting recombinant DNAs were then introduced into competent cells of Escherichia coli HB101 (Takara Shuzo, Kyoto, Japan) as described previously (13). The double-stranded plasmid DNA was

2340 FUKUNAGA ET AL. APPL. ENVIRON. MICROBIOL. (DNASTAR Inc., Madison, Wis.), and nucleotide sequence pair similarity values of the sequences were calculated by using the CLUSTAL V method (19). Confidence intervals were assessed by the CLUSTAL V bootstrap analysis. Nucleotide sequence accession numbers. The OspC gene nucleotide sequences of the Borrelia strains have been assigned DDBJ/EMBL/GenBank accession numbers D50798 to D50803. As shown in Table 1, our previously published sequences and the other submitted sequences were used in this study (12, 13, 50 52). The 16S rrna gene sequences have been assigned DDBJ/EMBL/ GenBank accession numbers D67018 to D67024. The accession numbers of sequences used for phylogenetic analysis are also given in Table 1. RESULTS AND DISCUSSION FIG. 1. Coomassie brilliant blue-stained proteins in whole-cell lysates of borrelial strains (B31, 20047, VS461, Hk501, Hk512, OR1eR, Ya501, Ac502, Yh501, and Am501) in a discontinuous SDS-PAGE gel (12.5% polyacrylamide). An SDS-PAGE analysis and a Western blot analysis were performed as described previously (15, 34). Solid and open arrowheads indicate the MAb H9724-reactive flagellin and the MAb H5332-reactive OspA proteins, respectively. The reactivities with MAb H5332 were very weak in VS461, Ac502, and Am501 strains. extracted and purified as described previously (18). DNA sequencing was performed by the dideoxy chain termination method with an autoread sequencing kit in an automated sequencer (13). Sequencing-reaction mixtures were primed with the vector-encoded Universal primer, the RV primer, or a custom-synthesized primer (OspC nucleotides 321 to 298 of strain PKo, 5 -GGG TTG ATA TTG CAT AGG CTC CTG C-3 ). Both strands of two independent clones of each PCR product were sequenced, and no sequence discrepancies were observed between the two clones. Amplification, cloning, and sequencing of the 16S rrna gene. The nucleotide primer set (5 -GCT GGC AGT GCG TCT TAA GCA TGC-3, located at positions 35 to 58 in the E. coli numbering system, and 5 -GTG ACG GGC GGT GTG TAC AAG GCC C-3, located at positions 1408 to 1384 in the E. coli numbering system) was synthesized and used for PCR amplification. PCR amplification was performed as described previously (16). The DNA fragment obtained by PCR amplification was purified, ligated into the pgem5zf vector plasmid, and introduced into competent E. coli JM109 cells as described previously (13). Nucleotide sequencing was carried out by the dideoxy chain termination method primed with a series of custom-synthesized primers (17). Both strands of at least two independent clones were sequenced. Phylogenetic analyses were performed by using the DNASTAR program Borrelia isolates were examined by SDS-PAGE and Western blotting, and the resulting profiles are shown in Fig. 1. The protein profiles of Borrelia strains from three species of Japanese ticks and voles were found to differ from those of the representatives of known Borrelia species. The molecular sizes and amounts of proteins from the borrelial strains varied. The epitope for MAb H9724 was present in the 41-kDa flagellin protein of all isolates examined in this study. The strains of group Hk501 possessed the 32-kDa OspA protein reactive with MAb H5332. MAb H5332 also reacted with the 33-kDa OspA protein of Ya501 and reacted very weakly with the proteins of strains Ac502, Yh501, and Am501. The G C contents of the genomic DNAs of members of the genus Borrelia are 27 to 32 mol% (26). The G C contents of strains Hk501, OR1eR, Hk516, Ya501, Kt501, Yh501, Ac504, and Am501, isolated from three species of Japanese ticks and a vole, were all between 28.0 and 31.8 mol%. Previous reports demonstrated that our ribotyping scheme is able to distinguish four species within B. burgdorferi sensu lato (16, 36, 37). Ribotypes I, II, III, and X correspond to B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. japonica, respectively. Ribotypes IV, V, and VI designated for Borrelia species isolated from I. persulcatus ticks and Lyme disease patients are thought to be strains of B. garinii (13, 14). The RFLP profiles of the type strains of four species and some of the unidentified isolates are illustrated in Fig. 2. The unidentified spirochete strains showed characteristic RFLP profiles that were distinct from those of known Borrelia species. The profiles of six strains isolated from I. tanuki were identical to each other. Similarly, six isolates from I. turdus showed the same RFLP profile. Therefore, we selected strain Hk501 as a FIG. 2. Southern blot hybridization patterns of Borrelia species. Genomic DNAs were digested with EcoRV or HincII, electrophoresed in a 0.7% agarose gel, blotted, and probed with labeled rrna gene fragments. Probes NP and Sty were used as hybridization probes as described previously (18). The other experimental conditions of the RFLP analysis were described in detail in our previous reports (16, 37). Sizes of the fragments (kilobase pairs) are shown in the figure. Fluorescein-labeled lambda DNA digested with HindIII was loaded as size markers.

VOL. 62, 1996 CHARACTERIZATION OF SPIROCHETES FROM RARE TICK SPECIES 2341 FIG. 3. Partial deduced amino acid sequence of the OspC protein. Partial sequences of the OspC gene in B. burgdorferi sensu lato strains were determined as described in Materials and Methods. The predicted amino acid region 11 to 51 is represented in comparison with those of B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. japonica strains. Dots represent identical amino acids, dashes represent deleted amino acids, and lowercase letters represent amino acid differences. representative of the spirochete of I. tanuki and strain Ya501 as a representative of the spirochete of I. turdus. One isolate from I. columnae (Am501) also showed a quite distinct RFLP profile. The profiles of three isolates from C. rufocanus were identical to those of the isolates of group Hk501. On the basis of these RFLP profiles, we designated ribotypes VII (group Hk501), VIII (group Ya501), and IX (Am501). The ribotype classifications are also summarized in Table 1. Partial nucleotide sequences of the OspC gene were determined, and deduced amino acid sequences were aligned to characterize the unidentified Borrelia strains. It has been reported that, overall, OspC sequences were highly heterogeneous among various strains (22, 23, 46, 47, 52). However, the amino acid motif at positions 23 to 35 seems to be available for classifying Borrelia species (50). The deduced amino acid sequences (positions 11 to 51) are shown in Fig. 3. For the strains of known species (B31, HO14, VS461, PKo, 20047, and PBi), the variation of their sequences reflected the difference of species. As regards the unidentified isolates, the sequences of Hk501 and OR1eR were identical to each other and similar to those of B. afzelii strains. The sequences of Ya501 and Ac502 were different in only one amino acid residue and were similar to those of B. garinii strains. The sequence of Am501 closely resembled those of B. garinii strains. The relative binding ratios obtained in whole DNA-DNA hybridization experiments performed by the membrane filter method are shown in Table 2. The levels of DNA relatedness in Hk501, Ya501, and Am501 were less than 70% when compared with the type strains of B. garinii and B. afzelii. The result also indicated that Hk501 and Ya501 were not related to each other. The genomic DNA of strain Hk501, which was isolated from I. tanuki, was quite similar to the DNA of strain OR1eR, which was isolated from C. rufocanus earlobe tissue. The data presented in this report indicate that group Hk501 from I. tanuki and C. rufocanus and group Ya501 from I. turdus are divergent at the species level. The results of OspC gene sequencing suggest that isolate Am501 from I. columnae is related to B. garinii, although its level of DNA relatedness was less than 70% when compared with B. garinii. We cloned the 16S rrna gene of representatives of three new ribotype groups and two species, and the nucleotide sequences (1,366 or 1,367 nucleotides, nucleotides 35 to 1408 of the E. coli numbering system) were determined to quantitatively assess the phylogenetic divergence of the strains belonging to Borrelia species. About 90% of the whole 16S rrna gene sequence was then aligned and compared with previously published sequences of Borrelia species. Nucleotide sequence pair similarity values of the sequences are given in Table 3, and a neighbor-joining phylogenetic tree (42) constructed on the basis of the sequence similarity matrix is shown in Fig. 4. The branching patterns of the tree were evaluated by bootstrap analysis. Phylogenetic analysis placed the strains into a coherent cluster of the Lyme disease borreliae and related genomic groups. According to this tree, all strains of each species of Borrelia were assigned to the same cluster. The representatives of genomic groups Hk501 and Ya501 and strain Am501 formed a lineage distinct from the members of Lyme disease borreliae. North American B. andersonii strains isolated from I. dentatus and B. japonica HO14 and IKA2 were unambiguously clustered into the distinct group. The tree clearly shows that the strains of B. japonica and B. andersonii are not closely related to all members of B. burgdorferi sensu lato strains. B. japonica and B. andersonii seemed to have diverged deeply from B. burgdorferi sensu lato strains and have adapted to I. ovatus or I. dentatus ticks. Strain DN127, isolated from the I. pacificus tick, also diverged at a level consistent with the creation of a new species. However, the level of relatedness determined by DNA-DNA hybridization experiments was borderline (41). Further studies are needed to determine the taxonomic position of strain DN127. It is clear that the strains of group Hk501 are branched from the cluster consisting of B. afzelii strains and are distinguishable from B. afzelii strains. Strain Am501 is closely related to the group Hk501 strains, as indicated by the branch length in the tree. Whole-DNA-DNA hybridization results revealed that strains Am501, Hk501, and Ya501 were clearly distinct from all other known Borrelia species. However, our phylogenetic analysis based on the 16S rrna gene sequence reported here demonstrated that the Am501 seems to be related to the Hk501 and OR1eR and distantly separated from B. burgdorferi sensu stricto and B. garinii strains. Our results also clearly indicate that the strains of group Ya501 are unambiguously placed among new species, closer to the group Hk501 strains. The reported results confirm the previous assignment of strains to the different Borrelia species, which were based on the RFLP ribotype method and DNA relatedness. This observation also suggests that it is possible to rely on RFLP ribotype and/or OspC sequence for species identification and for studying the genetic relationship between closely related species but that these methods do not seem to be trustworthy when compared with analysis of 16S rrna gene sequences. For taxonomic and phylogenetic analysis, conventionally used tools are DNA-DNA hybridization and sequencing of the rrna gene (43, 49). In conclusion, 16S rrna gene sequence analysis TABLE 2. Levels of DNA relatedness among Borrelia species Source of unlabeled DNA Relative binding ratio (%) with following source of labeled DNA: 20047 VS461 Hk501 Ya501 B. burgdorferi B31 54 53 50 58 B. japonica HO14 52 62 51 48 B. garinii 20047 100 ND a ND ND B. afzelii VS461 58 100 ND ND Borrelia sp. strain Hk501 56 63 100 56 Borrelia sp. strain OR1eR 57 64 98 55 Borrelia sp. strain Ya501 51 47 55 100 Borrelia sp. strain Am501 53 57 56 48 a ND, not determined.

2342 FUKUNAGA ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. 16S rrna gene sequence similarity matrix for Borrelia strains % Similarity with strain: Strain B. burgdorferi B31 B. burgdorferi Sh-2-82 B. burgdorferi 1352 B. burgdorferi 20004 B. garinii 20047 B. garinii G1 B. garinii G2 B. afzelii IP3 B. afzelii J1 B. afzelii HT61 B. japonica HO14 B. japonica IKA2 B. andersonii 21038 B. andersonii 19857 Hk501 OR2eL Ya501 Ac502 Am501 DN127 B. hermsii HS1 B. burgdorferi B31 100 B. burgdorferi Sh-2-82 97.6 100 B. burgdorferi 1352 98.4 98.5 100 B. burgdorferi 20004 98.3 98.5 99.2 100 B. garinii 20047 98.9 97.0 97.9 97.7 100 B. garinii G1 97.6 97.7 98.5 98.4 98.3 100 B. garinii G2 97.6 97.6 98.4 98.3 98.1 99.1 100 B. afzelii IP3 97.2 97.5 98.1 98.1 97.3 98.0 98.0 100 B. afzelii J1 97.6 96.4 97.2 97.1 98.0 97.3 97.2 98.0 100 B. afzelii HT61 97.9 96.0 96.9 96.7 98.4 96.8 96.7 97.6 98.4 100 B. japonica HO14 95.5 94.4 95.3 95.1 95.6 95.1 95.0 94.9 95.3 95.9 100 B. japonica IKA2 95.3 93.4 94.3 94.1 95.8 94.5 94.4 94.0 94.5 96.1 97.2 100 B. andersonii 21038 97.6 97.2 98.1 98.0 97.6 97.7 97.6 98.0 97.7 97.4 95.0 94.4 100 B. andersonii 19857 97.7 97.5 98.3 98.1 97.6 98.1 98.0 98.1 97.8 97.5 95.3 94.7 99.1 100 Hk501 98.5 96.7 97.6 97.4 99.4 97.9 97.7 97.4 98.2 98.6 95.5 95.6 97.6 97.6 100 OR2eL 98.6 96.8 97.6 97.5 99.4 97.8 97.6 97.5 98.1 98.7 95.4 95.7 97.7 97.8 99.8 100 Ya501 98.1 97.1 97.9 97.8 98.5 97.5 97.4 97.7 98.0 97.8 95.6 94.9 97.7 97.8 98.5 98.6 100 Ac502 98.3 97.2 98.0 97.9 98.6 97.6 97.6 97.8 98.1 98.0 95.8 95.1 97.9 98.0 98.7 98.8 99.8 100 Am501 98.5 96.7 97.6 97.4 99.2 97.6 97.5 97.6 98.3 98.9 95.4 95.8 97.7 97.8 99.4 99.5 98.6 98.8 100 DN127 96.3 94.5 95.3 95.3 96.6 95.2 95.0 94.6 94.9 96.2 97.5 96.1 95.1 95.2 96.2 96.3 95.5 95.7 96.1 100 B. hermsii HS1 92.3 93.5 93.3 93.3 92.1 92.8 92.7 93.3 91.9 91.5 90.0 89.1 93.1 93.2 92.1 92.2 92.3 92.4 92.5 90.0 100 permitted us to quantitatively describe the phylogenetic relationships between the Borrelia strains obtained from rare species of ixodid ticks. The intergeneric relationship and other new species of Borrelia were indicated. Our findings also indicate that the Am501 strain would be a third new Borrelia species. However, the Am501 strain is the only isolate we have, and its flagellin gene sequence is quite similar to that of the European tick isolate VS116 (our unpublished result). Further epidemiologic surveys and comparison of genetic characteristics are thought to be needed for species assignment of this strain. It has been generally assumed that the ixodid tick serves as the most efficient vector for perpetuating the enzootic transmission cycles of B. burgdorferi sensu lato. As reported here, each set of spirochetes isolated from rare species of ixodid ticks were divided individual phylogenetic clusters. Each I. turdus, I. tanuki, and I. columnae tick harbored a characteristic spirochete. Adult I. tanuki ticks parasitized specifically on raccoon dogs, and their larvae and nymphs have been found on rodents (53). All developmental stages of I. turdus have been found on birds (53). It has also been shown that the B. andersonii strains were found to infect mainly I. dentatus ticks and cotton-tailed rabbits (5). On the other hand, B. japonica commonly infects I. ovatus ticks, and the I. ovatus ticks are frequently found on shrews in Japan (29, 33, 35, 36, 45). In contrast, three species of Borrelia, B. burgdorferi, B. garinii, and B. afzelii, are found in I. ricinus ticks and both B. garinii and B. afzelii are carried by I. persulcatus ticks (3, 7, 16, 21). Furthermore, B. garinii isolates are found in both I. ricinus and Ixodes uriae ticks (10, 40), and group DN127 isolates are also associated with different tick species, I. pacificus, I. neotomae, and I. scapularis (41). Hostparasite relationships may be evolutionarily related with the diversion of Borrelia species, but adaptation of Borrelia species to tick vectors remains to be investigated. FIG. 4. Phylogenetic tree for Borrelia strains. The phylogenetic tree was constructed as described in the text by using 16S rrna gene sequences. Bar, 1% difference between sequences, as determined by measuring the lengths of the horizontal lines connecting the species. Numbers of the branch nodes indicate the results of bootstrap analysis. The 16S rrna gene sequence from Borrelia hermsii HS1 served as an outgroup.

VOL. 62, 1996 CHARACTERIZATION OF SPIROCHETES FROM RARE TICK SPECIES 2343 The pathogenicity of spirochetes found from I. tanuki, I. turdus, and I. columnae is unknown. The medical importance of these spirochetes seems to be slight, because these ticks rarely bite humans. ACKNOWLEDGMENTS We express our gratitude to Guy Baranton, Institut Pasteur, for valuable suggestions on the manuscript. We greatly appreciate Yasutake Yanagihara and Toshiyuki Masuzawa, University of Shizuoka, for their constant encouragement and support; and Richard T. Marconi, Virginia Commonwealth University, for sending us his manuscript prior to publication. We also thank Yoko Makita and Yukie Takahashi for their technical assistance. This work was supported in part by a Grant-in-Aid for International Cooperative Research, Joint Research no. 06044191; by Scientific Research grants 05670218, 06640912, and 07640939 from the Ministry of Education, Science and Culture of Japan; and the Takeda Scientific Research Grant from the Takeda Science Foundation, 1995. REFERENCES 1. Adam, T., G. S. Gassman, C. Rasiah, and U. B. Göbel. 1991. Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources. Infect. Immun. 59:2579 2585. 2. Aeschlimann, A., E. Chamot, F. Gigon, J. P. Jeanneret, D. Kesseler, and C. Walther. 1986. B. burgdorferi in Switzerland. Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 263:450 458. 3. Anderson, J. F. 1989. Epizootiology of Borrelia in Ixodes tick vectors and reservoir hosts. Rev. Infect. Dis. 11(Suppl. 6):S1451 S1459. 4. Anderson, J. F., P. H. Duray, and L. A. Magnarelli. 1987. Prevalence of Borrelia burgdorferi in white-footed mice and Ixodes dammini at Fort McCoy, Wis. J. Clin. Microbiol. 25:1495 1497. 5. Anderson, J. F., L. A. Magnarelli, R. B. LeFebvre, T. G. Andreadis, J. B. McAninch, G. C. Perng, and R. C. Johnson. 1989. Antigenically variable Borrelia burgdorferi isolates from cottontail rabbits and Ixodes dentatus in rural and urban areas. J. Clin. Microbiol. 27:13 20. 6. Assous, M. V., D. Postic, G. Paul, P. Nevot, and G. Baranton. 1994. Individualisation of two new genomic groups among American Borrelia burgdorferi sensu lato strains. FEMS Microbiol. Lett. 121:93 98. 7. Baranton, G., D. Postic, I. Saint Girons, P. Boerlin, J. C. Piffaretti, M. Assous, and P. A. D. Grimont. 1992. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int. J. Syst. Bacteriol. 42:378 383. 8. Barbour, A. G., S. F. Hayes, R. A. Heiland, M. E. Schrumpf, and S. L. Tessier. 1986. Borrelia-specific monoclonal antibody binds to a flagellar epitope. Infect. Immun. 52:549 554. 9. Barbour, A. G., R. A. Heiland, and T. R. Howe. 1985. Heterogeneity of major proteins in Lyme disease Borrelia: a molecular analysis of North American and European isolates. J. Infect. Dis. 152:478 484. 10. Bunikis, J., B. Olsen, V. Fingerle, J. Bonnedahl, B. Wilske, and S. Bergström. 1996. Molecular polymorphism of the Lyme disease agent Borrelia garinii in Northern Europe is influenced by a novel enzootic Borrelia focus in the North Atlantic. I. Clin. Microbiol. 34:364 368. 11. Canica, M. M., F. Nato, L. du Merle, J. C. Mazie, G. Baranton, and D. Postic. 1993. Monoclonal antibodies for identification of Borrelia afzerii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand. J. Infect. Dis. 25:441 448. 12. Fuchs, R., S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek. 1992. Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pc) in Escherichia coli. Mol. Microbiol. 6:503 509. 13. Fukunaga, M., and A. Hamase. 1995. Outer surface protein C gene sequence analysis of Borrelia burgdorferi sensu lato isolates of Japan. J. Clin. Microbiol. 33:2415 2420. 14. Fukunaga, M., and Y. Koreki. 1996. A phylogenetic analysis of Borrelia burgdorferi sensu lato isolates associated with Lyme disease in Japan by flagellin gene sequence determination. Int. J. Syst. Bacteriol. 46:416 421. 15. Fukunaga, M., M. Sohnaka, M. Nakao, and K. Miyamoto. 1993. Evaluation of genetic divergence of borrelial isolates from Lyme disease patients in Hokkaido, Japan, by rrna gene probes. J. Clin. Microbiol. 31:2044 2048. 16. Fukunaga, M., M. Sohnaka, and Y. Yanagihara. 1993. Analysis of Borrelia species associated with Lyme disease by rrna gene restriction fragment length polymorphism. J. Gen. Microbiol. 139:1141 1146. 17. Fukunaga, M., Y. Takahashi, Y. Tsuruta, O. Matsushita, D. Ralph, M. McClelland, and M. Nakao. 1995. Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan. Int. J. Syst. Bacteriol. 45:804 810. 18. Fukunaga, M., Y. Yanagihara, and M. Sohnaka. 1992. The 23S/5S ribosomal RNA genes (rrl/rrf) are separate from the 16S ribosomal RNA gene (rrs) in Borrelia burgdorferi, the aetiological agent of Lyme disease. J. Gen. Microbiol. 138:871 877. 19. Higgins, D. G., A. J. Bleasby, and R. Fuchs. 1992. CLUSTAL V: improved software for multiple sequence alignment. Comput. Appl. Biosci. 8:189 191. 20. Ishiguro, F., H. Iida, X. Ma, Y. Yano, H. Fujita, and N. Takada. 1994. Diversity of Borrelia isolates found in rodent-tick relationship in Fukui Prefecture and some additional areas. Jpn. J. Sanit. Zool. 45:141 145. 21. Jaenson, T. G. T. 1991. The epidemiology of Lyme borreliosis. Parasitol. Today 7:39 45. 22. Jauris-Heipke, S., R. Fuchs, M. Motz, V. Preac-Mursic, E. Schwab, E. Soutschek, G. Will, and B. Wilske. 1993. Genetic heterogeneity of the genes coding for the outer surface protein C (OspC) and the flagellin of Borrelia burgdorferi. Med. Microbiol. Immunol. 182:37 50. 23. Jauris-Heipke, S., G. Liegel, V. Preac-Mursic, D. Rossler, E. Schwab, E. Soutschek, G. Will, and B. Wilske. 1995. Molecular analysis of genes encoding outer surface protein C (OspC) of Borrelia burgdorferi sensu lato: relationship to ospc genotype and evidence of lateral gene exchange of ospc. J. Clin. Microbiol. 33:1860 1866. 24. Johnson, R. C., F. W. Hyde, G. P. Schmid, and D. J. Brenner. 1984. Borrelia burgdorferi sp. nov.: etiological agent of Lyme disease. Int. J. Syst. Bacteriol. 34:496 497. 25. Kawabata, H., T. Masuzawa, and Y. Yanagihara. 1993. Genomic analysis of Borrelia japonica sp. nov. isolated from Ixodes ovatus in Japan. Microbiol. Immunol. 37:843 848. 26. Kelly, R. T. 1984. Borrelia, p. 57 62. In N. R. Krieg and J. G. Holt (ed.), Bergey s manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 27. Kitaoka, S. 1973. Ixodes asanumai sp. n. (Ixodoidea, Ixodidae) from the Amami-Oshima islands and Miyake island, Japan. Natl. Inst. Anim. Health Q. 13:137 141. 28. Marconi, R. T., D. Liveris, and I. Schwartz. 1995. Identification of novel insertion elements, RFLP patterns, and discontinuous 23S rrna in Lyme disease spirochetes: phylogenetic analyses of rrna genes and their intergenic spacers in Borrelia japonica sp. nov. and genetic group 21038 isolates. J. Clin. Microbiol. 33:2427 2434. 29. Masuzawa, T., Y. Okada, Y. Beppu, T. Oku, F. Kawamori, and Y. Yanagihara. 1991. Immunological properties of Borrelia burgdorferi isolated from the Ixodes ovatus in Shizuoka, Japan. Microbiol. Immunol. 35:913 919. 30. Matuschka, F. R., O. Fischer, M. Heiler, D. Richter, and A. Spielman. 1992. Capacity of European animals as reservoir hosts for the Lyme disease spirochete. J. Infect. Dis. 165:479 483. 31. Matuschka, F. R., R. Lange, A. Spielman, D. Richter, and P. Fischer. 1990. Subadult Ixodes ricinus (Acari: Ixodidae) on rodents in Berlin, West Germany. J. Med. Entomol. 27:385 390. 32. Miyamoto, K., M. Nakao, N. Sato, and M. Mori. 1991. Isolation of Lyme disease spirochetes from an ixodid tick in Hokkaido, Japan. Acta Trop. 49:65 68. 33. Miyamoto, K., M. Nakao, K. Uchikawa, and H. Fujita. 1992. Prevalence of Lyme borreliosis spirochetes in ixodid ticks of Japan, with special reference to a new potential vector, Ixodes ovatus (Acari: Ixodida). J. Med. Entomol. 29:216 220. 34. Nakao, M., and K. Miyamoto. 1993. Isolation of spirochetes from Japanese ixodid ticks, Ixodes tanuki, Ixodes turdus, and Ixodes columnae. Jpn. J. Sanit. Zool. 44:49 52. 35. Nakao, M., and K. Miyamoto. 1993. Long-tailed shrew, Sorex unguiculatus, as a potential reservoir of the spirochetes transmitted by Ixodes ovatus in Hokkaido, Japan. Jpn. J. Sanit. Zool. 44:237 245. 36. Nakao, M., K. Miyamoto, and M. Fukunaga. 1994. Borrelia japonica in nature: genotypic identification of spirochetes isolated from Japanese small mammals. Microbiol. Immunol. 38:805 808. 37. Nakao, M., K. Miyamoto, and M. Fukunaga. 1994. Lyme disease spirochetes in Japan: enzootic transmission cycles in birds, rodents, and Ixodes persulcatus ticks. J. Infect. Dis. 170:878 882. 38. Nakao, M., K. Miyamoto, M. Fukunaga, Y. Hashimoto, and H. Takahashi. 1994. Comparative studies on Borrelia afzelii isolated from a patient of Lyme disease, Ixodes persulcatus ticks, and Apodemus speciosus rodents in Jpn. Microbiol. Immunol. 38:413 420. 39. Nakao, M., K. Miyamoto, and S. Kitaoka. 1992. A new record of Ixodes pavlovskyi Pomerantev from Hokkaido, Japan. Jpn. J. Sanit. Zool. 43:229 234. 40. Olsen, B., T. G. T. Jaenson, L. Noppa, J. Bunikis, and S. Bergström. 1993. A Lyme borreliosis cycle in seabirds and Ixodes uriae ticks. Nature (London) 362:340 342. 41. Postic, D., M. V. Assous, P. A. D. Grimont, and G. Baranton. 1994. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf(5s)-rrl(23s) intergenic spacer amplicons. Int. J. Syst. Bacteriol. 44:743 752. 42. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406 425. 43. Stackebrandt, E., and B. M. Goebel. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rrna sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44:846 849.

2344 FUKUNAGA ET AL. APPL. ENVIRON. MICROBIOL. 44. Takada, N., and H. Fujita. 1992. Description of Ixodes columnae sp. nov., associated with a nymphal sp. N2 and a larval sp. L1 (Acari: Ixodidae). J. Acarol. Soc. Jpn. 1:37 44. 45. Takahashi, Y., M. Sohnaka, M. Nakao, K. Miyamoto, and M. Fukunaga. 1993. Characterization of Borrelia species isolated from ixodid ticks, Ixodes ovatus. Microbiol. Immunol. 37:721 727. 46. Theisen, M., M. Borre, M. J. Mathiesen, B. Mikkelsen, A. M. Lebech, and K. Hansen. 1995. Evolution of the Borrelia burgdorferi outer surface protein OspC. J. Bacteriol. 177:3036 3044. 47. Theisen, M., B. Frederiksen, A. M. Lebech, J. Vuust, and K. Hansen. 1993. Polymorphism in ospc gene of Borrelia burgdorferi and immunoreactivity of OspC protein: implications for taxonomy and for use of OspC protein as a diagnostic antigen. J. Clin. Microbiol. 31:2570 2576. 48. Wallich, R., C. Helmes, U. E. Schaible, Y. Lobet, S. E. Moter, M. D. Kramer, and M. M. Simon. 1992. Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes. Infect. Immun. 60:4856 4866. 49. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moor, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Trüper. 1987. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37:463 464. 50. Wilske, B., S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Rössler, E. Soutschek, and R. C. Johnson. 1995. Phenotypic analysis of outer surface protein C (OspC) of Borrelia burgdorferi sensu lato by monoclonal antibodies: relationship to genospecies and OspA serotype. J. Clin. Microbiol. 33:103 109. 51. Wilske, B., V. Preac-Mursic, U. B. Göbel, B. Graf, S. Jauris, E. Soutschek, E. Schwab, and G. Zumstein. 1993. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J. Clin. Microbiol. 31:340 350. 52. Wilske, B., V. Preac-Mursic, S. Jauris, A. Hofmann, I. Pradel, E. Soutschek, E. Schwab, G. Will, and G. Wanner. 1993. Immunological and molecular polymorphism of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi. Infect. Immun. 61:2182 2191. 53. Yamaguti, N., V. J. Tipton, H. L. Keegan, and S. Toshioka. 1971. Ticks of Japan, Korea, and the Ryukyu islands. Brigham Young Univ. Sci. Bull. Biol. Ser. 15:1 226.