Polymorphism of Aeromonas spp. tRNA intergenic spacers

We investigated the length polymorphism of the intergenic spacers lying between tRNA genes of Aeromonas spp. A total of 69 strains representing all known genomic species of Aeromonas were used in the study. tDNA-PCR patterns were examined by Dice coefficient (S(D)) and unweighted pair group method of clustering (UPGMA). The strains were allocated into 15 groups at a similarity level of 70%. The strains belonging to seven genomic species: A. hydrophila (HG 1), A. caviae (HG 4), A. sobria (HG 7), A. veronii (HG 8/10), A. encheleia (HG 16), A. popoffii (HG 17), and A. culicicola (HG 18) formed distinct clusters. Our study revealed a genetic heterogeneity of the following species: A. bestiarum, A. salmonicida, A. media, A. eucrenophila, A. jandaei, A. schubertii, and A. allosaccharophila.     

Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish

Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.     

Identification of Aeromonas strains of different origin to the genomic species level

A total of 71 Aeromonas strains was identified with established genomic species by DNA–DNA hybridization. The strains were isolated from diarrhoeal stools, dead and live fish, drinking, lake, river and sea water, municipal sewage and aluminium rolling emulsion. The strains were allocated to seven hybridization groups (HGs) but the majority belonged to HG 4 (42%), HG 8/10 (30%) and HG 3 (18%). All strains were examined by 136 phenotypic tests. Useful phenotypic characters for separation of Aeromonas HG 1–3 genospecies were: utilization of DL -lactate, urocanic acid and growth at 40·5 °C. Few phenotypic differences were detected between strains of HG 4, HG 5 and HG 6. Most isolates of the Aer. veronii biotype sobria (HG 8) showed a characteristic biochemical profile: positive V.P. (Voges–Proskauer) reaction, oxidation of gluconate, production of gas from glucose, susceptibility to cephalotin, no hydrolysis of elastin, arbutin and aesculin, and no acid production from L -arabinose, arbutin and salicin.     

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.     

Characterization of Lactobacillus sake isolates from dry-cured sausages by restriction fragment length polymorphism analysis of the 16S rRNA gene

Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies using Eco RI and Hin dIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, Eco RI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When Hin dIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when Eco RI and Hin dIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake .     

Differentiation of closely related Carnobacterium food isolates based on 16S-23S ribosomal DNA intergenic spacer region polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763(T) and C. mobile DSM 4849(T) generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766(T) and C. piscicola NCDO 2762(T) were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA(Ile) and tRNA(Ala) genes. The M-ISR included one tRNA(Ala) gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.     

Biochemical and Molecular Characterization of Tetracycline-Resistant Aeromonas veronii Isolates from Catfish

Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.     

Marked genetic polymorphism of the swine steroid 21-hydroxylase gene, and its location between the SLA class I and class II regions

Restriction fragment length polymorphism (RFLP) analysis of the swine 21-hydroxylase (CYP21) region was conducted on 31 unrelated SLA class I typed pigs, mainly Large Whites, including 15 haplotypes. Ten haplotypes were from SLA genotypic homozygotes and five were from SLA class I phenotypic homozygotes. DNA digestion with Hin dIII, TaqI and PstI, and hybridization to a 4.5-kb swine CYP21 genomic probe yielded respectively two, four and three RFLP patterns. Six patterns were identified with combined RFLP. In addition, analysis of the CYP21 region in families comprising several SLA recombinants demonstrated that the CYP21 gene lies in the DNA segment between the SLA class I and class II regions. These overall results reinforce our previous conclusion about the existence in the pig of a single 21-hydroxylase gene. The characterization of at least six CYP21 allelic patterns provides a new tool for studying the associations between the SLA region and zootechnical traits.     

Pulsed-field gel electrophoresis in the study of mesophilic and psychrophilic Aeromonas spp.

Pulsed-field gel electrophoresis (PFGE) was used to study the genetic diversity of mesophilic Aeromonas hybridization group (HG) 1, HG 2, HG 3, HG 4, HG 5, HG 6, HG 7, HG 8/10and HG 11, psychrophilic Aeromonas salmonicida subsp. salmonicida and atypical Aerom. salmonicida strains. Xba I was chosen for restriction because it producedfragments whose numbers and size were appropriate for PFGE analysis of all studied HGs. Allmesophilic Aeromonas strains within an HG had different banding patterns. No sharedbands which could be used for identification of an HG were found. Pulsed-field gelelectrophoresis analysis further confirmed the known genetic homogeneity of Aerom.salmonicida subsp. salmonicida . Pulsed-field gel electrophoresis pattern analysissuggested that the genomic size of Aerom. salmonicida subsp. salmonicida issmaller than that of mesophilic Aeromonas spp. or atypical Aerom. salmonicida . Aeromonas salmonicida subsp. salmonicida had only one large restriction fragment (310kb) and lacked other large fragments (>160 kb). Although the PFGE patterns of atypical Aerom. salmonicida resembled the banding patterns of mesophilic Aeromonas spp.they had several small fragments (15–50 kb) shared with Aerom. salmonicida subsp. salmonicida suggesting genetic relatedness.     

Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tet A

Oxytetracycline-resistant (OT(r)) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OT(r) plasmids from these isolates was investigated. Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT(r) plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A. veronii b.v. sobria HG8 (six isolates), and A. caviae HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb EcoRI restriction fragment. Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT(r) plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A. salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E. coli strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn1721 in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

16S rDNA restriction fragment length polymorphism analysis of psychrotrophic vibrios from Japanese coastal water

Restriction fragment length polymorphism (RFLP) analysis was carried out for 136 natural isolates belonging to the family Vibrionaceae. These were collected from inshore areas of Japan, mainly in winter. Twenty-eight 16S rDNA genotypes were obtained by digestion with four restriction endonucleases (HhaI, DdeI, RsaI, and Sau3AI). To estimate the genetic relationships, 53 informative fragments were scored by their presence or absence. A dendrogram was constructed using the unweighted pair group method with the arithmetic averages algorithm. Five RFLP groups (groups I to V) were obtained. Group I corresponded to Vibrio splendidus-like strains. It was confirmed that this group was not only found in Otsuchi Bay, but also in broad coastal areas of Japan. Group II strains were not identified as previously known Vibrio species. Group III strains were regarded as members of the Vibrio main group, which is a major phylogenetic group deduced from 16S rRNA gene analysis in the family Vibrionaceae. The RFLP profile indicated that Group IV strains were closely related to V. hollisae. Group V strains showed RFLP patterns which have not been observed previously. From the clustering analysis, it was concluded that group V strains were not Vibrio species. Most of the isolates studied were not identified as previously described species. It suggests that many psychrotrophic vibrios in cold marine environments remain as unknown species.     

Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

Genotypic characteristics of the rrn operon and genome of indigenous soybean Bradyrhizobia in cropping zones of China

Four genetic assays, 16S rRNA restriction fragment length polymorphism (RFLP), 16S rRNA sequencing, 16S-23S rRNA intergenetic spacer (IGS) RFLP, and amplified fragment length polymorphism (AFLP), were conducted to determine the genotypic characteristics of 44 indigenous strains of Bradyrhizobium from soybean (Glycine max L.) cropping zones of China. The results generated from different assays showed that soybean bradyrhizobial isolates comprised four genomic groups. Group I was composed of strains mainly isolated from the North and Northeast plains of China. All four assays confirmed this group as phylogenetically divergent from all the reference strains. Strains of the group may represent a new species. Strains in Group II isolated from a variety of geographic regions were ascribed to B. liaoningense. Strains in Group III, mainly isolated from Central and East China, were closely related to the reference strains of B. japonicum. Strains in Group IV belonged to B. elkanii.     

Molecular diversity and phylogeny of rhizobia associated with wild legumes native to Xinjiang, China

A total of 111 rhizobial strains were isolated from wild legumes in Xinjiang, an isolated region of northwest China. Nine genomic species belonging to four genera of Rhizobium, Mesorhizobium, Ensifer, and Bradyrhizobium were defined among these strains based on the characterization of amplified 16S ribosomal DNA restriction analysis (ARDRA), restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA intergenic spacers (IGS), 16S rRNA gene sequencing and multilocus sequence analysis (MLSA). Twenty-five nodC types corresponding to eight phylogenetic clades were divided by RFLP and sequence analysis of the PCR-amplified nodC gene. The acid-producing Rhizobium and Mesorhizobium species were predominant, which may be related to both the local environments and the hosts sampled. The present study also showed the limitation of using nod genes to estimate the host specificity of rhizobia.     

Comparison of three molecular methods for typing Aeromonas popoffii isolates

Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763^T and C. mobile DSM 4849^T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766^T and C. piscicola NCDO 2762^T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA^Ile and tRNA^Ala genes. The M-ISR included one tRNA^Ala gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.     

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.

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%.