FEMS Microbiology Letters 142 (1996) 77-83

Transcription

1 ELSEVIER FEMS Microbiology Letters 142 (1996) MICROIOLOGY LETTERS Characterization of orrelia sp. isolated from Ixodes tanuki,, and I. columnae in Japan by restriction fragment length polymorphism of rrf (SS)-rrl (23s) intergenic spacer amplicons Toshiyuki Masuzawa *, Tetswro Komikado, Atswe Iwaki, Hiroyuki Suzuki, Kazuhide Kaneda, Yasutake Yanagihara epartment of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Shizuoka 422, Japan Received 7 May 1996; revised 13 June 1996; accepted 13 June 1996 Abstract orreliu isolated from various sources in Japan, including rare species of ixodid ticks, Zxodes tanuki,, and I. columnae, were characterized by restriction fragment length polymorphism analysis and sequencing analysis of the 5S-23s rrna intergenic spacer amplicon. orrelia sp. isolated from I. tan&, and I. columnae generated restriction fragment length polymorphism patterns different from those of known. burgdorferi sensu late isolates previously reported. Furthermore, some. afzelii and. garinii isolated in Japan showed unique RFLP patterns which were not observed among European. afzelii and. garinii. Keywords: Lyme disease; orrelia burgdorferi; Ixodes; Tick; rrna; Intergenic spacer; Polymerase chain reaction 1. Introduction Lyme disease is a multi-systemic disorder [l] caused by infection with orrelia burgdorferi sensu lato [2] which is transmitted by ticks of the Zxodes ricinus complex [3]. Many borrelia strains have been isolated from ixodid ticks, reservoir animals and Lyme disease patients.. burgdorferi sensu lato is classified into five genospecies,. burgdorferi sensu strict isolated in North America and Europe,. garinii [4] and. afzelii [5] isolated in Europe and * Corresponding author. Tel.: +81 (54) ; Fax: +81 (54) ; masuzawa@ys2.u-shizuoka-ken.ac.jp Japan,. japonica [6,7] isolated from I. ovatus in Japan, and. andersonii isolated from I. dentatus in North America [8]. Furthermore, Postic et al. [9] developed a simple and rapid method for identification of genospecies of. burgdorferi sensu lato based restriction fragment length polymorphism (ORFLP) analysis of the 5%23s rrna intergenic spacer amplicon and identified new genomic groups among isolates from North America and Europe. orrelia were isolated from I. persulcatus [ 1,l I], Z. ovatus [12,13], and small mammals [11,14,15], red fox [16] and patients with erythema migrans [17] in Japan. Also, spirochetes were isolated from rare ixodid tick species, I. tanuki,, and I. columnae collected in Japan [18]. Protein profiles of these spiro /96/$12. Copyright 1996 Federation of European Microbiological Societies. Published by Elsevier Science.V. PIISO (96)246-7

2 78 T Masuzowa et al. IFEMS Microbiology Letters 142 (1996) Table I Origins and RFLP patterns of. burgdorferi sensu late used in this study Genospecies or Strain Source genomic group Country/region RFLP pattern rlli MseI. burgdorferi 31 (ATCC3521) Ixodes dammini United States 297 Human spinal fluid United States A A A A. garinii 247 (CIP13362) I. ricinus France Pr Human spinal fluid Germany Pi Human spinal fluid Germany PHei Human spinal fluid Germany TN I. ririnus Germany T25 I. ricinus Germany. andersonii I. dentutus United States L Group N127 N127 I pactjicus United States I I Group VSI 16 VSll6 I. ricmus Switzerland F Group Poti2 Poti2 I. ricinus Portugal G G Japanese. garinii isolates HP13 I. prrsulcatus Hokkaido HTl8 I. persulcatus Hokkaido HT19 I. persulcatus Hokkaido HHI Human skin Hokkaido NT25 I. persulcatus Nagano NP8 I I. persulcutus Nagano NP76 I. persulcatus Nagano HP1 (JCM8952) I. persulcatus Hokkaido NP4 I. persulcatus Nagano HT59 I. persulcutus Hokkaido NT31 I. persulcatus Nagano ASF Apodemus speciosus Hokkaido C NT3 I I. prrsulcatu.s Nagano C NT24 I persulcatus Nagano C C C C. afzelii IPF (Jl) I. persulcatus Hokkaido Asal A. speciosus Hokkaido fox Vulpes vulpe5 Hokkaido Hfox V. vulpes Hokkaido JEMlO Human skin Hokkaido NT28 I. persulcatus Nagano N HT61 I. persulcatus Hokkaido N N N. japonica HOI4 (JCM8951) I. ovatus NO67 I. ovatus Hokkaido Nagano E E Isolates from I. tanuki Hk51 I. tanuki Hokkaido OR3eL Clethrionomys rufocanus Hokkaido Fi8lt I. tanuki Fukui TkAE8 I. tanuki Tokushima

3 T. Masuzawa et al. IFEMS Microbiology Letters I42 (1996) Table 1 (continued) Genospecies or genomic group Strain Source Country/region RFLP pattern rai MseI Isolates from Ya51 A~52 Yh51 Kt51 Yamagata Aicbi Yokohama Kyoto Isolates from I. columnae Am51 I. columnae Aomori Q, no rai restriction site. chetes were different from known. burgdorferi sensu lato isolates from I. persulcatus and I. ova&s. In this study, we applied RFLP analysis of the 5S-23s intergenic spacer amplicon for classification and further characterization of Japanese borreliae including these unique isolates. 2. Materials and methods 2.1. Strains and cultivation Table 1 shows the borrelia strains used in this study. These borrelia strains were cultivated at 32 C in arbour-stoenner-kelly II medium [19]. Genomic NA was extracted and purified by the method described previously [6] PCR and RFLP analysis Primers corresponding to the 3 end of the 5s rrna (rrj) [5 -CTGCGAGTTCGCGGGAGA-3 1 and the 5 end of the 23s rrna (rrl) [5 - TCCTAGGCATTCACCATA-3 1 as described previously [9] were synthesized using P-cyanoethyl phosphoramidite by a custom oligonucleotide synthesis service (Tanehashi Co., Tokyo, Japan). Two-ml aliquots of culture were washed and the cells were resuspended in 1 pl of water. The resultant cell suspensions were boiled at 1 C for 1 min. PCR was performed by the method previously described by Postic et al. [9]. The amplicon obtained after PCR was digested with MseI and ral according to the manufacturer s recommendations (New England iolabs, everly, MA, USA), and the digested NA was electrophoresed through a 16% polyacrylamide gel and subsequently stained with ethidium bromide. Marker 1 purchased from Nippon Gene Co. (Toyama, Japan) was used as a molecular mass marker Sequencing of ampll$ed products Each PCR product was cloned into the pcr@ii plasmid vector and the recombinant plasmids were transformed into E. coli INVa F using a TA cloning kit (Invitrogen Co., San iego, CA, USA) according to the manufacturer s instructions. The recombinant plasmids were extracted from E. coli cultures in L broth using the Wizard- 373 NA purification system (Promega Co., Madison, WI, USA), and sequenced by a dideoxy chain-termination method using the dye terminator-taq cycle sequencing kit by using model 373A NA-sequencer (Applied iosystems Inc., Foster city, CA, USA). At least two clones were sequenced for determination of each strain Nucleotide sequence accession numbers The intergenic spacer sequences have been assigned the following accession numbers: strains Am51 (8442), ASF (8443), Hk51 (8444), NT28 (8445), NP81 (8446), and Ya51 (8447) Results and discussion Previous reports have indicated that. garinii and. afzelii are present in Japan, but. burgdorferi

4 Table 2 Sequence similarity matrix of 5S-23s rrna gene intergenic spacer MseI Species NP81 NT29 ASF VS461 NT28 HO14 Nl27CA VSl16 UK Poti2 Poti CA2 Hk51 Ya51 Am51 RFLP Pattern 5 31 A. burgdorferi b 247. garinii NP81. garinii NT29 C. garinii R ASF C. garinii 2 VS461. afzelii NT28 N. afzelii HO14 E N127 I CA55 J 2515 K VS116 F UK F Poti2 G Poti3 H L L CA2 M Hk51 Ya51 P Am51 Q. japonica Group N 127 Group N127 Group N127 Group VSl16 Group VSl16 Group Poti2 Group Poti2 Group Group Group I. tanuki I. columnae a g g s g =: & 1

5 T. Masuzawa et al. IFEMS Microbiology Letters 142 (1996) Table 3 rai and MseI restriction fragments of 5S-23s intergenic spacer amplicons Genospecies or genomic group Amplicon size rai RFLP rai restriction MseI RFLP MseI restriction (bp) pattern fragment sixes (bp) pattern fragment sixes (bp). burgdorferi 254 A 144, 53, 29, 28 A 18, 51, 38, 29, 28. garinii , 52 18, 95, 5 NP , 52 18, 96, 51 NT C 144, 57, 52 C 18, 57, 5, 38 ASF 253 C 144, 57, 52 C 17, 57, 51, 38. a&iii VS , 52, 2 18, 68, 5, 2 NT N 144, 52, 3, 2 N 17, 51, 38, 3, 2. japonica 236 no fragment E 18, 78, 5 Group VS , 52 F 175, 5, 23, 7 Group Poti2 Potil and Poti2 257 G 145, 83, 29 G 18, 81, 39, 29 Poti3 255 H 158, 81, 16 H 18, 79, 52, 16 Group N127 N I 144, 53, 33, 27 I 18, 51, 38, 33, 27 California strains 226 J 144, 53, 29 J 18, 51, 38, K 173, 53, 27 K 18, 51, 34, 27, 17, 12, 4 Group andersonii 266 no fragment L 12, 67, 51, 28 CA2 255 M 146, 19 M 91, 5, 4, 28, 22, 17, 7 Isolates from Hk , 52, 2 174, 51, 2 I. tan& Isolates from Ya P 144, 81, 23 P 17, 51, 38, 21, 16, 8, 7 Isolates from Am no fragment Q 169, 51, 23, 6 I. columnae Exact fragment sizes were determined from the sequences. sensu stricto is not, by RFLP analyses of 16s rrna and flagellin genes [6], ribotype [2], PCR and RFLP analyses of OspA and Osp genes [21], and OspC sequence analysis [22] of Japanese isolates from various sources. When the PCR amplicon of the 5S-23s spacer was cleaved by MseI and rai,. burgdorferi sensu stricto,. garinii,. afzelii,. japonica,. andersonii, groups VS116, Poti2, and N127, generated independent fragments as described previously [9] and were distinguishable from each other. We confirmed the usefulness of this method for rapid screening and assessing the genetic diversity of orrelia strains. Japanese isolates from various sources including unique isolates from Z. tanuki,, and Z. columnue [ 181 were analyzed. Japanese isolates from I. persulcatus, wildlife and patients identified as. garinii were classified into two groups as previously reported [9]: group 1, rui pattern and MseI pattern, also observed among European, garinii, and group 2, rui pattern C and MseI pattern C, only observed among Japanese isolates (Table 1). Although Japanese strains identified as. afzelii for the most part generated RFLP pattern /, similarly to European. ufzelii, strains NT28 and HT61 isolated from Z. persulcatus, which was identified as. ufzelii by NA homology (unpublished result), PCR and RFLP analysis of OspA and Osp genes [21], RFLP ribotype analysis and flagellin gene sequencing [23], showed the unique RFLP pattern N /N. No European. ufzelii isolates generated this pattern [9]. This suggests that. ufzelii isolates in Japan are more heterogeneous than those in Europe. Similar findings were reported previously Spirochetes isolated from rare species of ixodid ticks, Z. tanuki, Z. columnae and Z. turdus, collected in Japan [ 181 were characterized. Since these spirochetes showed amplification by PCR of the intergenie spacer region, they had tandem repeats of the 23S-5S rrna gene. Consequently, these spirochetes should be classified as. burgdorferi sensu lato. Furthermore, these orrelia isolates generated RFLP patterns different from those previously reported. Table 2 shows the nucleotide sequence similarity

6 82 T. Masuzawa et al. IFEMS Microbiology Letters 142 (1996) 7743 of amplicons obtained from the representative isolates. These sequences were different from those of previously reported strains [9]. Relatively higher similarity values were observed among. garinii strains 247, NP81, NT29, and ASF. Similarity values of strains Hk51, Ya51 and Am51 ranged from 78.9 to 93.1%, 82.4 to 91.9%, and 82. to 95.3%, respectively, for all of the strains tested. The highest levels of similarity observed were between strain Am51 and strains VSl16 and UK of group VS116. These results suggest that strains Hk51 and Ya51 are genetically divergent from known species of. burgdorferi sensu lato and that Am51 is related to group VSl16. Table 3 shows the restriction fragment sizes determined from the sequence. Patterns N /N for strains NT28 and HT61, Or/O for isolates from I. tanuki, P /P for isolates from, and /Q for strain Am51 from I. columnae were different from 13 RFLP patterns, A /A to M /M, reported previously by Postic et al. [9]. Furthermore, phylogenetic analysis based on 16s rrna gene sequences distinguished orrelia strains isolated from I. tanuki,, and I. columnae from hitherto identified orrelia species [25]. In conclusion,. afzelii and. garinii isolated in Japan may be genetically more divergent than European species, and RFLP patterns of orrelia sp. isolated from I. tanuki, and L columnae are different from those of known orrelia species and genomic groups. Acknowledgments We thank G. aranton,. Postic, Pasteur Institute, M. Nakao, Asahikawa Medical College, N. Takada, Fukui Medical College, E. Isogai, Hokkaido Health Science University,. Wilske, University of Munich, and R.C. Johnson, University of Minnesota for providing orreliu strains. We are grateful to M. Fukunaga, Fukuyama University, for valuable discussion. This study was supported in part by Grant-in-Aid for Encouragement of Young Scientist (No ) Grant-in-Aid for Scientific Research (No ), and Grant-in-Aid for International Scientific Research Program; Joint Research (No ) from the Ministry of Education, Science and Culture, Japan. References 111 Steere, A.C. (1989) Lyme disease. New Engl. J. Med. 321, Johnson, R.C., Schmid, G.P., Hyde, F.W., Steingerwalt, A.G. and renner,.j. (1984) orrelio burgdorferi sp. no.: etiologic agent of Lyme disease. Int. J. Syst. acterial. 34, Anderson, J.F. (1989) Epizootiology of orrelia in Ixodes ticks vectors and reservoir hosts, Rev. Infect. is. 11, S1451ll459. [41 aranton, G., Postic,., Saint Girons, I., oerlin, P., Piffaretti, J.-C., Assous, M. and Grimont, P.A.. (1992) elineation of orrelia burgdorferi sensu stricto, orrelio garinii sp. [51 VI [71 PI no., and group VS461 associated with Lyme borreliosis. Int. J. Syst. acterial. 42, Canica, M.M., Nato, F., du Merle, L., Mazie, J.C., aranton, G. and Postic,. (1993) Monoclonal antibodies for identification of orrelia afzelii sp. nav. associated with late cuta- neous manifestations of Lyme borreliosis. Stand. J. Infect. is. 25, Kawabata, H., Masuzawa, T. and Yanagihara, Y. (1993) Genomic analysis of orrelia japonica sp. nav. isolated from Zxodes ova&s in Japan. Microbial. Immunol. 37, Postic,., elfaiza, J., Isogai, E., Saint Girons, I., Grimont, P. A.. and aranton, G. (1993) A new genomic species in orrelio burgdorferi sensu loto isolated from Japanese ticks. Res. Microbial. 144, Marconi, R.T., Liveris,. and Schwartz, I. (1995) Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23s rrna in Lyme disease spirochetes: Phylogenetic analysis of rrna genes and their intergenic spacers in orrelia juponica sp. nav. and genomic group 2138 (orreliu andersonii sp. nav.) isolates. J. Clin. Microbial. 33, [9] Postic,., Assous, M.V., Grimont, P.A.. and aranton, G. (1994) iversity of orrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5Strrl (23s) intergenic spacer amplicons. Int. J. Syst. acterial. 44, [lo] Masuzawa, T., Okada, Y., Yanagihara, Y. and Sato, N. (1991) Antigenic properties of orreliu burgdorferi isolated from Ixodes ovatus and Ixodes persulcatus in Hokkaido, Japan J. Clin. Microbial. 29, Miyamoto, K., Nakao, M., Sato, N. and Mori, M. (1991) Isolation of Lyme disease spirochetes from an ixodid tick in Hokkaido, Japan. Acta Trop. 49, Masuzawa, T., Okada, Y., eppu, Y., Oku, T., Kawamori, F. and Yanagihara, Y. (1991) Immunological properties of orrelia burgdorferi isolated from the Zxodes ovatus in Shizuoka, Japan. Microbial. Immunol. 35, [ 131 Miyamoto, K., Nakao, M., Uchikawa, K. and Fujita, H. (1992) Prevalence of Lyme borreliosis spirochetes in ixodid ticks of Japan, with special reference to a new potential vector, Zxodes ovam (Atari: Ixodidae). J. Med. Entomol. 29, [14] Nakao, M. and Miyamoto, K. (1993) Long-tailed shrew, Sorex unguiculatus, as a potential reservoir of the spirochetes

7 T. Masuzawa et al. I FEMS Microbiology Letters 142 (1996) transmitted by Ixodes ovatus in Hokkaido, Japan. Jpn. J. of orrelia species isolated from Ixodes persulcatus in Hokkai- Sanit. Zool. 44, do, Japan. J. Clin. Microbial. 31, [IS] Ishiguro, F., Takada, N., Nakata, K., Yano, Y., Suzuki, H., Masuzawa, T. and Yanagihara, Y. (1996) Reservoir competence of the vole, Clethrionomys rufocanus bedfordiae, for orrelia garinii or orrelia afzelii. Microbial. Immunol., 4, [16] Isogai, E., Isogai, H., Kawabata, H., Masuzawa, T., Yanagihara, Y., Kimura, K., Sakai, K., Azuma, Y., Fujii, N. and Ohno, S. (1994) Lyme disease spirochetes in a wild fox (Vu/pes vulpes schrencki) and in ticks. J. Wildl. is. 3, [17] Nakao, M., Miyamoto, K., Kawagishi, N., Hashimito, Y. and Iizuka, H. (1992) Comparison of orrelia burgdorferi isolated from humans and ixodid ticks in Hokkaido, Japan. Microbial. Immunol. 36, [IS] Nakao, M. and Miyamoto, K. (1993) Isolation of spirochetes from Japanese ixodid ticks, Ixodes tanuki, Ixodes turdus, and Ixodes columnne. Jpn. J. Sanit. Zool. 44, [19] arbour, A.G. (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J. iol. Med. 57, [2] Fukunaga, M., Sohnaka, M., Takahashi, Y., Nakao, M. and Miyamoto, K. (1993) Antigenic and genetic characterization [21] Kawabata, H., Tashibu, H., Yamada, K., Masuzawa, T. and Yanagihara, Y. (1994) Polymerase chain reaction analysis of orrelia species isolated in Japan. Microbial. Immunol. 38, [22] Fukunaga, M. and Hamase, H. (1995) Outer surface protein C gene sequence analysis of orrelia burgdorferi sensu lato isolates from Japan. J. Clin. Microbial. 33, [23] Fukunaga, M. and Koreki, Y. (1996) A phylogenetic analysis of orrelia burgdorferi sensu lato isolates associated with Lyme disease in Japan by flagellin gene sequence determina- tion. Int. J. Syst. acterial. 46, in press. [24] Fukunaga, M. and Takahashi, Y. (1994) Pulsed field gel electrophoresis analysis of orrelia burgdorferi sensu lato isolated in Japan and taxonomic implications with Lyme disease spi- rochetes. Microbial. Immunol. 38, [25] Fukunaga, M., Hamase, A., Okada, K., Inoue, H., Tsuruta, Y., Miyamoto, K. and Nakao, M. (1996) Characterization of spirochetes isolated form Ixodes tanuki, Ixodes turdus, and Ixodes columnae ticks in comparison with orrelia burgdorferi sensu lato strains. Appl. Environ. Microbial. 62, in press.