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Genotypic and phenotypic diversity among nine Swiss isolates of Borrelia burgdorferi
Zbl. Bakt. 279,173-179 (1993) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Genotypic and Phenotypic Diversity Among Nine Swiss Isolates of Borrelia burgdorferi JENNIFER MEISTER- TURNER 1, ELISABETH FILIPUZZI-JENNy 1 ,2, OLIVIER PETER 3 , ANNE GABRIELLE BRETZ3, MARGARETHA ST ALHAMMAR-CARLEMALM1+, an~ JURG MEYER l , h Biozentrum der Universitiit, Abt. Mikrobiologie, 4056 Basel, Switzerland Zahniirztliches Institut der Universitiit, Abt. Priiventivzahnmedizin und Orale Mikrobiologie, 4051 Basel, Switzerland 3 Institut Central des Hopitaux Valaisans, Microbiologie Clinique, 1950 Sion, Switzerland
1 2
With 4 Figures· Received July 15, 1992 . Revision received November 15, 1992 . Accepted January 28, 1993
Summary Nine strains of Borrelia burgdorferi isolated from ticks in the canton of Valais (Switzerland) were characterized genotypically by determining restriction fragment length polymorphisms (RFLP) and plasmid profiles. The strains were also compared with respect to presence and electrophoretic mobility of the outer surface proteins OspA and OspB, and immunoreactivity of OspA and a 12 kD antigen. By both approaches, three different patterns were observed resulting in identical grouping of the strains. However, RFLP's allowed determination of relationships among strains within a group and have shown that geographic distribution does not correlate with genotype. Zusammenfassung Neun Zecken-Isolate von Borrelia burgdorferi aus dem Kanton Wallis (Schweiz) wurden genotypisch durch Bestimmung der Restriktionsfragment-Liingenpolymorphismen und der Plasmidprofile charakterisiert. Diese Ergebnisse wurden mit der elektrophoretischen Untersuchung der iiugeren Oberfliichenproteine OspA und OspB sowie der Immunreaktivitiit von OspA und einem 12 kD Antigen verglichen. Aufgrund von drei verschiedenen Mustern der genotypischen sowie der phiinotypischen Merkmale wurden die Stiimme in die gleichen Gruppen eingeteilt. Dariiber hinaus zeigt die genotypische Analyse unterschiedliche Verwandtschaftsgrade zwischen Vertretern der gleichen Gruppe auf. Die genetische Verwandtschaft korreliert nicht mit der geographischen Verteilung der Isolate. + Present address: Institute of Medical Microbiology, University of Lund, 223 62 Lund, Sweden * Corresponding author
174
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Introduction
Borrelia burgdorferi is the etiological agent of Lyme borreliosis, an infectious disease of public concern, which is prevalent in Europe, North America and other regions with a moderate climate. This spirochete is transmitted by ticks of the genus Ixodes. The disease is characterized most frequently by distinctive skin lesions, the erythema chronicum migrans, but may involve a spectrum of neurological, arthritic orland further dermatological disorders (for review see 1). Differences in the frequency and severity of clinical symptoms between different geographic regions have been reported (1-3). They were suspected to be a consequence of genetic differences between European and American isolates of B. burgdorferi (4, 5). The main vector of B. burgdorferi in Europe is Ixodes ricinus. In Switzerland, between 3 and 55% of ticks of the populations studied were found to be infected (6, 7). Several authors (4, 8, 9) reported a high degree of antigenic variability among European isolates of B. burgdorferi, whereas most North American isolates appeared to be closely related to the type strain B31. Sero-grouping of B. burgdorferi strains was first attempted by Barbour (10) who described three groups based on the reactivity of the outer surface protein OspA with the monoclonal antibodies H5332 and H3TS, and also the presence of a 20kD protein which does not react with these monoclonal antibodies. An extensive analysis and sero-grouping of B. burgdorferi strains by Wilske et al. (11) using monoclonal antibodies as well as animal and human antisera led to a distinction of seven serogroups. Genetic studies have revealed the existence of at least three subgroups among B. burgdorferi isolates recovered from various sources (12-16). We intended to extend our knowledge on the variations among the Swiss tick isolates of B. burgdorferi and to correlate genetic and phenotypic markers. We selected nine isolates originating from I. ricin ius from the state (canton) of Valais (Switzerland). We determined restriction fragment length polymorphisms (RFLP's) as well as plasmid profiles and evaluated protein and antigenic profiles by SDS-PAGE and immunoblotting. Materials and Methods
Selection and designation of isolates. Of 627 I. ricinus examined, 210 (= 33.5%) were infected as judged by the presence of borrelias established by dark-field microscopy and indirect immunofluorescence assay. The midgut of 165 infected ticks was incubated in BSKII medium, resulting in 36 cultures of spirochetes, 21 of which could be maintained and identified as B. burgdorferi by immunofluorescence. The isolates VS3 (Sion, VS, CH\ VSI02, and VS286 (Rarogne, VS, CH), VS215 and VS219 (Martigny, VS, CH), VS185 (Sierre, VS, CH), VS461, VS464 and VS468 (Vouvry, VS, CH) were representative of different regions of the Valais (7). Cellular fractions obtained from low passage cultures were compared to the type strain B31. Determination of RFLP's. Purification of a chromosomal DNA fraction by CsCl gradient centrifugation, digestion with the restriction enzymes, BglII, BcoRI, HindIII, Asp700; and HaeIII (Boehringer, Mannheim), separation by agarose gel electrophoresis, transfer of the DNA to nylon (0.2 !-lm Pall Biodyne A) membranes, hybridization with seven 32P-labelled, cloned EcoRI fragments derived from the type strain B31, and preparations of autoradio1
Locality, State of Valais (VS), Switzerland (CH)
Borrelia burgdorferi Genotypic Diversity
175
grams were done as described before (13). If there were ambiguities as to whether bands of different strains had indeed distinguishable sizes, the digests were electrophoresed side by side and reevaluated. The sizes of hybridizing bands were recorded and used to determine the average fraction of fragments conserved with respect to size in each pairwise comparison of the patterns (18). Analysis of linear and circular plasm ids. Plasmid fractions enriched for linear and circular plasmids, respectively, were obtained separately by CsCI gradient centrifugation. Separation
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was achieved by agarose gel electrophoresis and field inn'i",ion gel electrophoresis, respectively, as described in (13). Protein and antigen analysis. The preparation of protein c,tracts from low passage cultures, electrophoresis on 12.5% polyacrylamide gels and tralhlt'i" to Immobilon membranes (Millipore) were done as described (17). The membranes wen: '!.lined with Coomassie blue or were used in immunoblot assays with monoclonal antibody H3 J.., , 19) reacting . with OspA, or D6 reacting with a 12 kD antigen (17). Results DNA characterization
Southern blots of chromosomal DNA fractions cleaved with one of five restriction endonucleases were hybridized with the seven different DNA probes derived from strain B31. An example is shown in Fig. 1. The results were used to determine the average fractions of fragments which had been conserved with respect to size in each pairwise comparison of the patterns (Fig. 2). Eight of the nine strains were found to belong to two clusters while isolate VS461 had a distinct RFLP pattern. The two isolates VS215 and VS219 appeared to be closely related to strain B31. The remaining six strains formed two related pairs (VS102NS185, VS3NS286) and two individual patterns (VS464, VS468), of which VS464 showed a considerable divergence from the others. Plasmid profiles represent less reliable markers for strain typing than chromosomal RFLP's, e.g. due to loss or dimerization of plasmids during cultivation (13). Plasmid analysis of low-passage cells has nevertheless revealed interesting correlations. The circular plasmid profiles showed size classes of 26-29 kb, 18-20 kb and about 8 kb (not shown) and allowed little distinction. The linear plasmids reflected the results obtained from the RFLP patterns: Strains VS215 and VS219 had indistinguishable profiles; as members of the B31-type cluster, they carried a 16 kb and a 50 kb species and four in the size range of 22-35 kb. The six members of the other group as well as
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Fig. 2. Phenogram depicting the relatedness among the ten strains analysed.
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Fig. 3. Separation of the linear plasmids carried by the strains studied by orthogonal field agarose gel electrophoresis. The sizes of representative plasmids were taken from (13).
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the distinct isolate VS461 lacked both the 16 kb and the 50 kb linear plasmids, but typically contained a larger one of about 58 kb (Fig. 3). Again the strains VS 102 and VS185 which were closely related on the basis of chromosomal RFLP's had the same plasmid profile. The other related pair, VS3 and VS286, however, had different sets of linear plasmids, as had strains VS464 and VS468. Noteworthy was the presence of a large linear plasmid species of about 70 kb in strain VS464.
Protein profiles and antigenic analysis The nine strains analysed showed three different patterns with respect to the electrophoretic mobility of the outer surface proteins OspA and OspB and to the immunoblot analysis (Fig. 4). They represented three of the four groups defined by Peter and Bretz (17). Strains VS215 and VS219, like the type strain B31, had a 34 kD OspB, a 31 kD OspA which reacted with the monoclonal antibody H3TS, and no signal was obtained with monoclonal antibody D6. Strain VS461 was unique, having a 32 kD OspA, a 35 kD OspB and did not react with both monoclonal antibodies. The six remaining strains, VS3, VS102, VS185, VS286, VS464, and VS468 appeared to lack OspB and had an OspA of 33 kD. Further, the monoclonal antibody D6 revealed one band of 12 kD, whereas H3TS did not bind to OspA.
Discussion The present study demonstrates the existence of different genotypic groups among
B. burgdorferi isolates from ticks collected in a relatively small part of Switzerland. It
also shows that the genotypic analysis leads to the same grouping of isolates as protein and antigen analysis (17). However, determination of RFLP's and plasmid profiles .revealed further differences between members of a group. Genetic relationship does not correlate with geographic distribution: the three strains VS461, VS464, VS468 from a minute forest of about 5000 m2 differed quite drastically in their RFLP patterns as well as their plasmid profiles. Similarly, strains VS102 and VS286 from the same forest were different, while their genetically closest relatives were isolates from ticks collected 20 km (VS3) and 35 km (VS185) away. Only one pair of strains derived from the same forest (VS215NS219) was hardly distinguishable. The two major RFLP patterns of eight of these Valais strains are those described for B. burgdorferi isolates from other parts of Switzerland (13) and correspond to the proposed species B. burgdorferi sensu stricto and Borrelia garinii (20). Strain VS461 was quite distinct from the other strains. Based on protein and antigen analysis and multilocus enzyme electrophoresis (19), it appeared to be related to several human skin isolates. On the other hand, several European cerebrospinal fluid isolates showed close relationship to strain VS102. Similar relationships were observed by DNA-DNA hybridization and by determination of rDNA restriction patterns (20). A correlation between the genotype of the infecting B. burgdorferi and the manifestations of the human infection could be firmly established, once a larger number of strains had been compared by the various approaches.
Acknowledgments. We thank Dina Bee for technical assistance. - The work was supported by grants of the Swiss National Science Foundation (No 31-25680.88, 3.587-0.87 and 31-9172.87). .
Borrelia burgdorferi Genotypic Diversity
179
References 1. Steere, A. c.: Lyme disease. N. Engl. J. Med. 321 (1989) 586-596 2. Asbrink, E. and I. Olsson: Clinical manifestations of erythema chronicum migrans
Afzelius in 161 patients: a comparison with Lyme disease. Acta Derm. Venereol. 65 (1985) 43-52 3. Stanek, G., G. Wewalka, V. Groh, R. Neumann, and W. Kristoferitsch: Differences between Lyme disease and European arthropod-borne Borrelia infections. Lancet i (1985) 401 4. Barbour, A. G., R. A. Heiland, and T. R. Howe: Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates. J. Infect. Dis. 152 (1985) 478-484 5. Karlson, M., K. Hovind-Hougen, B. Svenungsson, and G. Stiernstedt: Cultivation and characterization of spirochetes from cerebrospinal fluid of patients with Lyme borreliosis. J. Clin. Microbiol. 28 (1990) 473-479 6. Aeschlimann, A., E. Chamot, F. Gigon,]. P. ]eanneret, D. Kesseler, and Ch. Walther: B. burgdorferi in Switzerland. Zbl. Bakt. A 263 (1986) 450-458 7. Peter, 0.: Lyme borreliosis in the State of Valais, Switzerland. J. Int. Fed. Clin. Chern. 2 (1990) 121-124 8. Wilske, B., V. Preac-Mursic, and G. Schierz: Antigenic heterogeneity of European Borrelia burgdorferi strains isolated from patients and ticks. Lancet i (1985) 1099 9. Lane, R. S. and]. A. Pasocello: Antigenic characteristics of Borrelia burgdorferi isolates from ixodic ticks in Canada. J. Clin. Microbiol. 27 (1989) 2344-2349 10. Barbour, A. G.: A proposal for a serotyping system based upon major outer surface proteins of Borrelia burgdorferi. Lyme Borreliosis Newslett. 2 (1986) 7-9 11. Wilske, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer: Antigenic variability of Borrelia burgdorferi. Ann. N. Y. Acad. Sci. 539 (1988) 126-143 12. Postic, D., C. Edlinger, C. Richard, F. Grimont, J. Dufresne, P. Perolat, G. Baranton, and P. A. D. Grimont: Two genomic species in Borrelia burgdorferi. Res. Microbiol. 141 (1990) 465-475 13. Stalhammar-Carlemalm, M., E. Jenny, L. Gern, A. Aeschlimann, and]. Meyer: Plasmid analysis and restriction fragment length polymorphisms of chromosomal DNA allow a distinction between Borrelia burgdorferi strains. Zbl. Bakt. A 274 (1990) 28-39 14. Adam, T., G. S. Gassmann, C. Rasiah, and U. B. Gobel: Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources. Infect. Immun. 8 (1991) 2579-2585 15. Rosa, P. A., D. Hogan, and T. G. Schwan: Polymerase chain reaction analyses identify two distinct classes of Borrelia burgdorferi. J. Clin. Microbiol. 3 (1991) 524-532 16. Marconi, R. T. and C. F. Garon: Phylogenetic analysis of the genus Borrelia: a comparison of North American and European isolates of Borrelia burgdorferi. J. Bact. 174 (1992) 241-244 17. Peter, O. and A. G. Bretz: Polymorphism of outer surface proteins of Borrelia burgdorferi as a tool for classification. Zbl. Bakt. 277 (1992) 28-33 18. Harshman, L. and M. Riley: Conservation and variation of nucleotide sequences in Escherichia coli strains isolated from nature. J. Bact. 144 (1980) 560-568 19. Barbour, A. G., S. L. Tessier, and S. F. Hayes: Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45 (1984) 94-100 20. Baranton,G., D. Postic, I. Saint Girons, P. Boerlin, J. C. Piffaretti, M. Assous, and P. A. D. Grimont: Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int. J. System. Bact. 42 (1992) 378-383 21. Boerlin, P., O. Peter, A. G. Bretz, D. Postic, G. Baranton, and]. C. Piffaretti: Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis. Infect. Immun. 60 (1992) 1677-1683 Prof. Dr. J. Meyer, Zahniirztliches Institut der Universitiit Basel, Abt. Praventivzahnmedizin und Orale Mikrobiologie, Petersplatz 14, CH-4051 Basel, Switzerland 13 Zbl. Bakt. 279/2
Genotypic and Phenotypic Diversity Among Nine Swiss Isolates of Borrelia burgdorferi JENNIFER MEISTER- TURNER 1, ELISABETH FILIPUZZI-JENNy 1 ,2, OLIVIER PETER 3 , ANNE GABRIELLE BRETZ3, MARGARETHA ST ALHAMMAR-CARLEMALM1+, an~ JURG MEYER l , h Biozentrum der Universitiit, Abt. Mikrobiologie, 4056 Basel, Switzerland Zahniirztliches Institut der Universitiit, Abt. Priiventivzahnmedizin und Orale Mikrobiologie, 4051 Basel, Switzerland 3 Institut Central des Hopitaux Valaisans, Microbiologie Clinique, 1950 Sion, Switzerland
1 2
With 4 Figures· Received July 15, 1992 . Revision received November 15, 1992 . Accepted January 28, 1993
Summary Nine strains of Borrelia burgdorferi isolated from ticks in the canton of Valais (Switzerland) were characterized genotypically by determining restriction fragment length polymorphisms (RFLP) and plasmid profiles. The strains were also compared with respect to presence and electrophoretic mobility of the outer surface proteins OspA and OspB, and immunoreactivity of OspA and a 12 kD antigen. By both approaches, three different patterns were observed resulting in identical grouping of the strains. However, RFLP's allowed determination of relationships among strains within a group and have shown that geographic distribution does not correlate with genotype. Zusammenfassung Neun Zecken-Isolate von Borrelia burgdorferi aus dem Kanton Wallis (Schweiz) wurden genotypisch durch Bestimmung der Restriktionsfragment-Liingenpolymorphismen und der Plasmidprofile charakterisiert. Diese Ergebnisse wurden mit der elektrophoretischen Untersuchung der iiugeren Oberfliichenproteine OspA und OspB sowie der Immunreaktivitiit von OspA und einem 12 kD Antigen verglichen. Aufgrund von drei verschiedenen Mustern der genotypischen sowie der phiinotypischen Merkmale wurden die Stiimme in die gleichen Gruppen eingeteilt. Dariiber hinaus zeigt die genotypische Analyse unterschiedliche Verwandtschaftsgrade zwischen Vertretern der gleichen Gruppe auf. Die genetische Verwandtschaft korreliert nicht mit der geographischen Verteilung der Isolate. + Present address: Institute of Medical Microbiology, University of Lund, 223 62 Lund, Sweden * Corresponding author
174
J. Meister-Turner et al.
Introduction
Borrelia burgdorferi is the etiological agent of Lyme borreliosis, an infectious disease of public concern, which is prevalent in Europe, North America and other regions with a moderate climate. This spirochete is transmitted by ticks of the genus Ixodes. The disease is characterized most frequently by distinctive skin lesions, the erythema chronicum migrans, but may involve a spectrum of neurological, arthritic orland further dermatological disorders (for review see 1). Differences in the frequency and severity of clinical symptoms between different geographic regions have been reported (1-3). They were suspected to be a consequence of genetic differences between European and American isolates of B. burgdorferi (4, 5). The main vector of B. burgdorferi in Europe is Ixodes ricinus. In Switzerland, between 3 and 55% of ticks of the populations studied were found to be infected (6, 7). Several authors (4, 8, 9) reported a high degree of antigenic variability among European isolates of B. burgdorferi, whereas most North American isolates appeared to be closely related to the type strain B31. Sero-grouping of B. burgdorferi strains was first attempted by Barbour (10) who described three groups based on the reactivity of the outer surface protein OspA with the monoclonal antibodies H5332 and H3TS, and also the presence of a 20kD protein which does not react with these monoclonal antibodies. An extensive analysis and sero-grouping of B. burgdorferi strains by Wilske et al. (11) using monoclonal antibodies as well as animal and human antisera led to a distinction of seven serogroups. Genetic studies have revealed the existence of at least three subgroups among B. burgdorferi isolates recovered from various sources (12-16). We intended to extend our knowledge on the variations among the Swiss tick isolates of B. burgdorferi and to correlate genetic and phenotypic markers. We selected nine isolates originating from I. ricin ius from the state (canton) of Valais (Switzerland). We determined restriction fragment length polymorphisms (RFLP's) as well as plasmid profiles and evaluated protein and antigenic profiles by SDS-PAGE and immunoblotting. Materials and Methods
Selection and designation of isolates. Of 627 I. ricinus examined, 210 (= 33.5%) were infected as judged by the presence of borrelias established by dark-field microscopy and indirect immunofluorescence assay. The midgut of 165 infected ticks was incubated in BSKII medium, resulting in 36 cultures of spirochetes, 21 of which could be maintained and identified as B. burgdorferi by immunofluorescence. The isolates VS3 (Sion, VS, CH\ VSI02, and VS286 (Rarogne, VS, CH), VS215 and VS219 (Martigny, VS, CH), VS185 (Sierre, VS, CH), VS461, VS464 and VS468 (Vouvry, VS, CH) were representative of different regions of the Valais (7). Cellular fractions obtained from low passage cultures were compared to the type strain B31. Determination of RFLP's. Purification of a chromosomal DNA fraction by CsCl gradient centrifugation, digestion with the restriction enzymes, BglII, BcoRI, HindIII, Asp700; and HaeIII (Boehringer, Mannheim), separation by agarose gel electrophoresis, transfer of the DNA to nylon (0.2 !-lm Pall Biodyne A) membranes, hybridization with seven 32P-labelled, cloned EcoRI fragments derived from the type strain B31, and preparations of autoradio1
Locality, State of Valais (VS), Switzerland (CH)
Borrelia burgdorferi Genotypic Diversity
175
grams were done as described before (13). If there were ambiguities as to whether bands of different strains had indeed distinguishable sizes, the digests were electrophoresed side by side and reevaluated. The sizes of hybridizing bands were recorded and used to determine the average fraction of fragments conserved with respect to size in each pairwise comparison of the patterns (18). Analysis of linear and circular plasm ids. Plasmid fractions enriched for linear and circular plasmids, respectively, were obtained separately by CsCI gradient centrifugation. Separation
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176
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was achieved by agarose gel electrophoresis and field inn'i",ion gel electrophoresis, respectively, as described in (13). Protein and antigen analysis. The preparation of protein c,tracts from low passage cultures, electrophoresis on 12.5% polyacrylamide gels and tralhlt'i" to Immobilon membranes (Millipore) were done as described (17). The membranes wen: '!.lined with Coomassie blue or were used in immunoblot assays with monoclonal antibody H3 J.., , 19) reacting . with OspA, or D6 reacting with a 12 kD antigen (17). Results DNA characterization
Southern blots of chromosomal DNA fractions cleaved with one of five restriction endonucleases were hybridized with the seven different DNA probes derived from strain B31. An example is shown in Fig. 1. The results were used to determine the average fractions of fragments which had been conserved with respect to size in each pairwise comparison of the patterns (Fig. 2). Eight of the nine strains were found to belong to two clusters while isolate VS461 had a distinct RFLP pattern. The two isolates VS215 and VS219 appeared to be closely related to strain B31. The remaining six strains formed two related pairs (VS102NS185, VS3NS286) and two individual patterns (VS464, VS468), of which VS464 showed a considerable divergence from the others. Plasmid profiles represent less reliable markers for strain typing than chromosomal RFLP's, e.g. due to loss or dimerization of plasmids during cultivation (13). Plasmid analysis of low-passage cells has nevertheless revealed interesting correlations. The circular plasmid profiles showed size classes of 26-29 kb, 18-20 kb and about 8 kb (not shown) and allowed little distinction. The linear plasmids reflected the results obtained from the RFLP patterns: Strains VS215 and VS219 had indistinguishable profiles; as members of the B31-type cluster, they carried a 16 kb and a 50 kb species and four in the size range of 22-35 kb. The six members of the other group as well as
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Fig. 2. Phenogram depicting the relatedness among the ten strains analysed.
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178
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the distinct isolate VS461 lacked both the 16 kb and the 50 kb linear plasmids, but typically contained a larger one of about 58 kb (Fig. 3). Again the strains VS 102 and VS185 which were closely related on the basis of chromosomal RFLP's had the same plasmid profile. The other related pair, VS3 and VS286, however, had different sets of linear plasmids, as had strains VS464 and VS468. Noteworthy was the presence of a large linear plasmid species of about 70 kb in strain VS464.
Protein profiles and antigenic analysis The nine strains analysed showed three different patterns with respect to the electrophoretic mobility of the outer surface proteins OspA and OspB and to the immunoblot analysis (Fig. 4). They represented three of the four groups defined by Peter and Bretz (17). Strains VS215 and VS219, like the type strain B31, had a 34 kD OspB, a 31 kD OspA which reacted with the monoclonal antibody H3TS, and no signal was obtained with monoclonal antibody D6. Strain VS461 was unique, having a 32 kD OspA, a 35 kD OspB and did not react with both monoclonal antibodies. The six remaining strains, VS3, VS102, VS185, VS286, VS464, and VS468 appeared to lack OspB and had an OspA of 33 kD. Further, the monoclonal antibody D6 revealed one band of 12 kD, whereas H3TS did not bind to OspA.
Discussion The present study demonstrates the existence of different genotypic groups among
B. burgdorferi isolates from ticks collected in a relatively small part of Switzerland. It
also shows that the genotypic analysis leads to the same grouping of isolates as protein and antigen analysis (17). However, determination of RFLP's and plasmid profiles .revealed further differences between members of a group. Genetic relationship does not correlate with geographic distribution: the three strains VS461, VS464, VS468 from a minute forest of about 5000 m2 differed quite drastically in their RFLP patterns as well as their plasmid profiles. Similarly, strains VS102 and VS286 from the same forest were different, while their genetically closest relatives were isolates from ticks collected 20 km (VS3) and 35 km (VS185) away. Only one pair of strains derived from the same forest (VS215NS219) was hardly distinguishable. The two major RFLP patterns of eight of these Valais strains are those described for B. burgdorferi isolates from other parts of Switzerland (13) and correspond to the proposed species B. burgdorferi sensu stricto and Borrelia garinii (20). Strain VS461 was quite distinct from the other strains. Based on protein and antigen analysis and multilocus enzyme electrophoresis (19), it appeared to be related to several human skin isolates. On the other hand, several European cerebrospinal fluid isolates showed close relationship to strain VS102. Similar relationships were observed by DNA-DNA hybridization and by determination of rDNA restriction patterns (20). A correlation between the genotype of the infecting B. burgdorferi and the manifestations of the human infection could be firmly established, once a larger number of strains had been compared by the various approaches.
Acknowledgments. We thank Dina Bee for technical assistance. - The work was supported by grants of the Swiss National Science Foundation (No 31-25680.88, 3.587-0.87 and 31-9172.87). .
Borrelia burgdorferi Genotypic Diversity
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References 1. Steere, A. c.: Lyme disease. N. Engl. J. Med. 321 (1989) 586-596 2. Asbrink, E. and I. Olsson: Clinical manifestations of erythema chronicum migrans
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