Genetic Diversity of ônh ôborrelia burgdorferi ôns ô Sensu Lato Isolated in Far Eastern Russia

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1 Microbiol. Immunol., 41(8), , 1997 Genetic Diversity of ônh ôborrelia burgdorferi ôns ô Sensu Lato Isolated in Far Eastern Russia Toshiyuki Masuzawa*,1, Atsue Iwaki1, Yukita Sato2, Kenji Miyamoto2, Edward I. Korenberg3, and Yasutake Yanagihara1 1 Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Shizuoka 422, Japan, 2 Department of Parasitology, Asahikawa Medical College, Asahikawa, Hokkaido 078, Japan, and3gamaleya Institute of Epi demiologyand Microbiology, Russian Academy of Medical Sciences, Moscow , Russia Received March 12, 1997; in revised form, April 21, Accepted May 2, 1997 ônh ô Abstract ôns ô: One-hundred and fifty-seven ônh ôborrelia ôns ô isolated from adult ticks, ônh ôixodes persulcatus ôns ô, and wild rodents, ônh ô Clethrionomys rufocanus ôns ô and ônh ôapodemus peninsulae ôns ô, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S-23S rrna intergenic spacer. Some isolates showed unique RFLP patterns and were determined as ônh ôborrelia garinii ôns ô on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species-specific monoclonal antibodies (MAbs) and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as ônh ô Borrelia garinii ôns ô and ônh ôborrelia afzelii ôns ô, respectively, but no ônh ôborrelia burgdorferi ôns ô sensu stricto was detected. This finding is similar to the results obtained from ônh ôborrelia ôns ô surveys Hokkaido, Japan. of ônh ôi. persulcatus ôns ô and wild rodents in ônh ô Key words ôns ô: ônh ôborrelia ôns ô, Lyme disease, ônh ôixodes persulcatus ôns ô, rrna, Intergenic spacer, Far east, Russia Lyme borreliosis is the most prevalent tick-borne zoonotic disease (27) in Europe, North America and far eastern countries such as Japan, Russia, and China (1). Borrelia burgdorferi sensu lato are classified into seven species: B. burgdorferi (7, 13) sensu stricto isolated in North America and Europe; Borrelia garinii (3) and Borrelia afzelii (8) isolated in Europe and Japan; Borrelia japonica (14, 25), Borrelia tanukii (10, 17, 19) and Bor reliatrudae (10, 17) isolated from Ixodes ovatus, Ixodes tanuki and Ixodes turdus in Japan; and Borrelia ander soniiisolated from Ixodes dentatus in North America (15). Postic et al (24) developed a simple and rapid method for identification of species of B. burgdorferi sensu lato based on restriction fragment length poly morphism(rflp) analysis of the 5S-23S ribosomal RNA (rrna) intergenic spacer amplicon, and identified three genomic groups (Groups DN127, VS116 and Poti B2) among isolates from North America and Europe. We carried out a survey in the far eastern part of Russia in May Two-hundred and twenty-five of 846 Ixodes persulcatus collected in Khabarovsk, Vladi vostokand Yuzhno-Sakhalinsk were culture positive, and Borrelia was isolated from 20% of the rodents, *Address correspondence to Dr. Toshiyuki Masuzawa, Depart mentof Microbiology, School of Pharmaceutical Sciences, Uni versityof Shizuoka, 52-1 Yada, Shizuoka, Shizuoka 422, Japan. masuzawa@ys7.u-shizuoka-ken.ac.jp such as Apodemus peninsulae and Clethrionomys rufo canus(26). These isolates were characterized by ribo typinganalysis (12). However, some of the isolates showed unknown RFLP patterns which have not been observed previously among B. burgdorferi sensu lato iso lates,and some cultures showed mixtures of two differ entrflp patterns which might have resulted from mul tipleborrelia culturing (26). In this study, we characterized isolates in the far east ernpart of Russia by RFLP, sequencing analyses of the 5S-23S rrna intergenic spacer and on the basis of reactivity with monoclonal antibodies (MAbs). Materials and Methods Borrelia isolates. I. persulcatus were collected by flagging vegetation at three different woodland areas in the far eastern part of Russia, namely Khabarovsk, Vladivostok and Yuzhno-Sakhalinsk, from the end of May to the beginning of June At the same time, rodents were also captured by snap traps in Khabarovsk. Isolation and cultivation of Borrelia were performed Abbreviations: Hsp60, heat shock protein 60; MAb, mono clonalantibody; OspC, outer surface protein C; PCR, poly merasechain reaction; RFLP, restriction fragment length poly morphism;rrna, ribosomal RNA; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 595

2 596 T. MASUZAWA ET AL by a method described previously (21). In this study, 126 isolates from adult I. persulcatus and 31 isolates from the earlobes, heart muscles, and urinary bladders of 8 C. rufocanus and 2 A. peninsulae were examined. Borrelia were cultivated at 30C in Barbour-Stoenner-Kelly II medium (4). Monoclonal antibodies and Western blotting. MAb specific to the genus Borrelia, H9724 reactive to fla gellin(5), MAbs specific to B. burgdorferi sensu lato, O62 reactive to heat shock protein 60 (Hsp60) (16) and G7 reactive to outer surface protein C (OspC), MAb specific to B. garinii, D6 (3), MAb specific to B. afzelii, I (8), and MAb specific to B. japonica, O1441b reactive to flagellin (16), were used. SDS-PAGE and Western blotting were carried out according to a method described previously (18). PCR and RFLP analysis. Primers corresponding to the 3' end of the 5S rrna (rrf) [5'-CTGCGAGTTCGC- GGGAGA-3'] and the 5' end of the 23S rrna (rrl) [5'-TCCTAGGCATTCACCATA-3'], as described pre viously(17, 24), were synthesized using ƒà-cyanoethyl phosphoramidite by a custom oligonucleotide synthesis service (Bex Co., Tokyo). Two-milliliter aliquots of cultures were washed and the cells were resuspended in 100ƒÊl of water. The resultant cell suspensions were boiled at 100C for 10min. PCR was performed using a method previously described (24). The amplification products were digested with Mse I and Dra I according to the manufacturer's recommendations (New England Biolabs, Beverly, Ma., U.S.A.), and the digested DNA were electrophoresed through a 16% polyacrylamide gel. Marker 10 purchased from Nippon Gene Co. (Toya ma,japan) was used as the molecular weight marker. Sequencing of amplified products. Each PCR product was cloned into the pcrttm II plasmid vector and the recombinant plasmids were transformed into Escherichia coli INVƒ F' using a TA cloning kit (Invitrogen Co., San Diego, Calif., U.S.A.) according to the manufacturer's instructions. The recombinant plasmids were extracted from E. coli cultures using the WizardTM 373 DNA purification system (Promega Co., Madison, Wisc., U.S.A.), and sequenced by the dideoxy chain-termination method using a dye terminator-taq cycle sequencing kit and a model 373A DNA-sequencer (Applied Biosys temsinc., Foster City, Calif., U.S.A.). At least two clones were sequenced for determination of each strain. Nucleotide sequence accession numbers. The inter genicspacer sequences were assigned the following accession numbers: strains XB190 (D84402), XB141 (D84403) and XB37 (D84407). Results 5S-23S Intergenic Spacer RFLP Figure 1 shows representative RFLP patterns observed among the isolates from ticks. The arrow indicates the presence of DNA fragment. RFLP patterns found pre viouslyamong 7 species and 3 genomic groups of Lyme disease-related Borrelia are as follows: Pattern A, B. burgdorferi sensu stricto; Patterns B and C, B. garinii; Fig. 1. 5S-23S restriction patterns of representative tick isolates. DNA molecular size markers are indicated at the left. The arrow indi catesthe presence of DNA fragment. "?" indicates that RFLP pattern is not determined.

3 GENETIC DIVERSITY OF B. BURGDORFERI ISOLATED IN RUSSIA 597 Table 1. Dra I and Mse I restriction fragments of 5S-23S intergenic spacer amplicons Exact fragment sizes were determined from the sequences. Table 2. 5S-23S rrna intergenic spacer RFLP patterns of tick isolates and of rodent isolates NT, not tested;?, not determined. Patterns D and N, B. afzelii; Pattern E, B. japonica; Pat ternf, Group VS116; Patterns G and H, Group PoitB2; Patterns I, J and K, Group DN127; Patterns L and M, B. andersonii (Group 21123); Pattern O, B. tanukii; Pattern P, B. trudge; and Pattern Q, isolate from I. columnae (17, 24). We observed unique RFLP patterns Bv1, (B variation 1), Bv2 and Cv1, which have not been found previously (Fig. 1). Furthermore, mixtures of two different RFLP patterns (B+C, B+Bv1, B+Bv2, D+?, B+C, B+D and C+D) that might have resulted from multiple Bor reliaculturing were found (Fig. 1 and Table 2). To confirm the unique RFLP patterns (Bv1, BV2 and Cv1), the intergenic spacer sequences of representative strains XB190, XB141 and XB37 which showed these unique RFLP patterns, respectively, were determined. The sequence showed higher similarity values with B. garinii strains (GenBank/EMBL/DDBJ accession No. L30119), NP81 (No. D84406), NT29 (L30130), and ASF (No. D84403) in the range from 96.5 to 98.4%. In contrast, similarity values to other species and genomic groups ranged from 82.8 to 92.5%. Table 1 shows exact fragment sizes determined by the sequences. The results

4 598 T. MASUZAWA ET AL Table 3. 5S-23S rrna intergenic spacer RFLP patterns of tick isolates in the three survey areas?, not determined. indicated that RFLP patterns Bv1 and Bv2 were varia tionsof the RFLP pattern B group, the pattern Cv1 was a variation of the pattern C group and that these are classified as B. garinii. Reactivity of Borrelia Isolates with MAbs Table 2 summarizes RFLP patterns and Western blot tingresults. All isolates showed a 41 kda band reactive with MAb H9724 specific to genus Borrelia flagellin pro tein,a 20 to 23 kda band reactive with MAb G7 specif icto the OspC of B. burgdorferi sensu lato (data not pub lished)and 62 kda band reactive with MAb O62 specific to the Hsp60 of B. burgdorferi sensu lato (16). Previous results from ribotyping analysis suggested that some cultures from ticks and small rodents were multiple Borrelia cultures (26). Therefore, some of the cultures which showed a mixture of two different RFLP pat ternsresulted from multiple Borrelia culturing, and showed two bands reactive with MAb G7 (data not shown). On the basis of reactivity with species-specific MAbs, strains which showed patterns B, C, Bv1, Bv2 and Cv1, reacted with MAb D6 specific to B. garinii, but not with MAb I.17.3 specific to B. afzelii or MAb O1441b specific to B. japonica. On the other hand, all isolates which produced pattern D reacted with MAb I.17.3, but not MAbs D6 or O1441b, These results are in good agreement with the species determined by RFLP analy sis. The RFLP patterns mainly observed were pattern B, which is commonly found in European and Japanese B. garinii isolates, followed by pattern C, which is found among Japanese B. garinii isolates but not European B. garinii. 86.5% of the tick isolates were B. garinii and 12.7% were B. afzelii, but B. burgdorferi sensu stricto, which is commonly isolated from Ixodes ricinus in Europe, could not be detected. Similar results were obtained with the rodent isolates. In one culture from ticks and two from rodents, species could not be deter minedbecause of multiple RFLP patterns composed of unknown patterns. Table 3 shows the ratios of RFLP patterns observed in tick isolates from the three survey areas. RFLP patterns Bv1, Bv2, and Cv1 were only found in the Khabarovsk area. However, we found no significant differences in distribution of the Borrelia species among the three sur veyareas, Khabarovsk, Vladivostok and Sakhalin. Discussion We previously reported that 225 of 846 ticks (26.6%), 8 of 25C. rufocanus and 2 of 10 A. peninsulae were cul turepositive, and that we characterized 95 isolates from adult I. persulcatus and 32 isolates from various organs of the 2 A. peninsulae and 8 C. rufocanus by the ribo-

5 GENETIC DIVERSITY OF B. BURGDORFERI ISOLATED IN RUSSIA 599 typing method (26). From these results, 62 and 12% of the tick isolates, and 47 and 13% of the rodent isolates were identified as B. garinii and B. afzelii, respectively. However, some isolates showed unique and unclassified ribotyping patterns which might have resulted from multiple Borrelia culturing or unknown spirochetes such as Brevinema andersonii (2, 9). In this study, we attempted to characterize the unclas- sifiedisolates isolates from the far eastern part of Russia by analysis of the 5S-23S rrna intergenic spacer and on the basis of reactivity with MAbs. 86.5% of the isolates including a multiple Borrelia culture were identified as B. garinii. Remarkably, three unknown patterns (Bv1, (Bv,, Bv2 and Cv1) Cv,) were determined and identified as B. garinii. RFLP patterns Bv1, Bv, and Bv2 were variations of RFLP pattern B and pattern Cv1, Cv, was a variation of pattern C, and these were classified as B. garinii. This classification is supported by sequence homology values with B. garinii strains and reactivity with species-specific MAbs. The species determined by the PCR-RFLP method in this study and ribotyping analysis in a previous study (26) was consistent in 75 of 79 cultures used in both studies (26). Four cultures gave inconsistent results, however, the species determined based on reactivity with species- -specific MAbs and PCR-RFLP were in good agreement. The results suggest that the PCR-RFLP method is a useful and reliable method for the species determination of Lyme disease-related Borrelia. The genetic and immunological heterogeneity of B. garinii has been previously reported (6, 20, 28-30). This heterogeneity might explain the wide vector toler- anceof B. garinii for the two closely related tick species I. ricinus and I. persulcatus (6), and adaptation to birds and small rodents (22, 23). In one culture from ticks and two from rodents, Borrelia species could not be deter- minedbecause of multiple RFLP patterns composed of unknown patterns. These reacted with Borrelia-specif- icmab H9724 and B. burgdorferi sensu lato-specific MAbs 062 O62 and G7. We believe that these isolates rep- resentvariants of B. burgdorferi sensu lato. Other species and genomic groups related to Lyme disease borreliae previously observed in Europe, Japan and North America have not been found. Furthermore, no isolates were identified as Brevinema andersonii, which is a spirochete isolated from short-tailed shrew and white-footed mice in Connecticut and Minnesota, U.S.A. (2, 9), as positive reactions were observed with MAb H9724, specific to the genus Borrelia. Lyme disease Borrelia species in the far eastern part of Russia may resemble those of spirochetes isolated in Hokkaido (11, 22), in that B. garinii is the dominant species among the isolates there. In particular, B. garinii with RFLP pattern C are found only in Japan and the far eastern part of Russia, and not in Europe (17, 24). This result clearly indicates that the transmission of Borrelia occurred between the far eastern part of Russia and Japan. However, it is interesting that only pattern D of B. afzelii was found in Russia and not pattern N of B. afzelii, which is commonly observed in Japan but not in Europe (17). This finding may suggest that pattern N of B. afzelii developed phylogenetically after transmission of this Borrelia species to Japan. B, burgdorferi sensu stricto is commonly isolated from I. ricinus in Europe, but is not found in I. persulcatus in Japan or the far eastern part of Russia. These observations lead us to the speculation that I. persulcatus cannot be infected with B. burgdorferi sensu stricto, and I. ricinus cannot carry B. garinii of RFLP pattern C. This requires further testing in experimental transmission using laboratory tick colonies to confirm the transmission of these Borrelia species via ticks. Further studies are needed to determine the epidemiological and clinical importance of these Borrelia species in the far eastern part of Russia. We thank Drs. V.I. Volkov of the Khabarovsk Antiplague Station, L.I. Ivanov of the Khabarovsk Plague Control Station and R.N. Liberova of the Khabarovsk Regional Center of Sanitary Epidemiological Administration for arranging this survey in the Khabarovsk area. We are also grateful to Drs. A.G. Barbour (University of Texas, Health Science Center), E. Isogai (Health Science University of Hokkaido), D. Postic (Institute of Pas- teur)and and O. Peter (Institut Central des Hopitaux Valaisans) for providing MAbs. 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