- Email: [email protected]
Identification of Borrelia burgdorferi sensu lato Species in Europe
Zent.bl. Bakteriol. 287, 190-195 © Gustav Fischer Verlag 1998
Zentralblatt fUr
Bakt......sie
Research paper
Identification of Borrelia burgdorferi sensu lato Species in Europe I. Saint Girons, L. Gem, J. S. Gray, E. C. Guy, E. Korenberg, P. A. Nuttall, S. G. T. Rijpkema, A. Schonberg, G. Stanek, and D. Postic
Summary Characterisation at the species level of 142 Borrelia isolates obtained from ticks, hu mans and rodents in Western Europe was carried out and their geographical distribu tion was described. Borrelia garinii was the predominant species representing 44% of the isolates and B. afzelii and B. burgdorferi sensu stricto constituted 27% and 19% of isolates respectively. B. valaisiana, (formerly group VS116) constituted 10.5% of isolates. Some differences in the Borrelia species distribution were observed from one country to another, possibly linked to different sources of samples. In the human sam ples, which were mostly collected in Austria, B. afzelii was preferentially isolated from skin and B. garinii from CSF. B. afzelii was consistently isolated from rodents captured in Switzerland, but one isolate of B. garinii was obtained from a rodent in Austria. B. garinii was by far the most abundant species isolated from Ixodes ricinus ticks in all studied countries. B. valaisiana was isolated from I. ricinus ticks collected from vege tation and from I. ricinus engorged on birds.
Introduction The aim of this study was to identify at a species level Borrelia burgdorferi sen su lato strains isolated from ticks, rodents, and humans in different European countries in order to determine the distribution of the various species and to shed light on their relationship to clinical presentations and to host associations.
Methods Members of the European Union Concerted Action on Lyme Borreliosis (EU CALB) isolated strains of B. burgdorferi sensu lato (B. burgdorferi s.l.) and then dispatched them to the Pasteur Institute, Paris, France, in order to have
Identification of Borrelia burgdorferi sensu lato Species in Europe
191
them characterised by genomic tools, and to Neuchatel, Switzerland, for phe notypic characterisation. Three different characterisation methods were used. 1. Polymerase chain reaction followed by analysis of the restriction frag ment length polymorphism (PCR-RFLP) on rrf-rrl ribosomal spacer. This method is based on the unique organisation of ribosomal genes in B. burgdor feri s.1. There is only one copy of rrs (165) rRNA gene, whereas there are two copies tandemly duplicated of rrl (23S) rRNA gene and rrf (5S) rRNA gene. Thus, the use of primers chosen in conserved genes allows the amplification of the variable spacer between the two copies of these genes (1). The amplifi cation is specific for DNA from B. burgdorferi s.1. In a second step, each spe cies is characterised by a specific MseI restriction pattern. 2. Pulsed field gel electrophoresis (PFGE) B. burgdorferi s.1. isolates were analysed after MluI (a rare cutting enzyme) digestion of genomic DNA in agarose plugs, followed by PFGE. A previous study had shown that PFGE patterns permitted the identification of Borrelia species from the presence of fragments specific for each species and also per mitted the definition of subgroups within a Borrelia species (2). 3. Reactivity with monoclonal antibodies (Mabs) The reactivity of Borrelia lysates with Mabs H3TS, D6 and 117.3, specific for B. burgdorferi sensu stricto (B. burgdorferi s.s.), B. garinii, and B. afzelii respectively, was studied in Western-blot experiments (3).
Results A total of 142 Borrelia strains from 140 samples were studied from Switzer land, UK, Ireland, Germany, the Netherlands, Austria, France, and Slovakia (Table 1). Germany, Switzerland and Austria provided most of the isolates. All strains were identified by PCR-RFLP, 61 of them were additionally identified by PFGE and 65 strains were tested in western-blot experiments. The results obtained by the three methods were well correlated. The analysis of PFGE pat terns confirmed the genetic diversity within both B. burgdorferi s.s. and B. ga rinii species. The majority of isolates were obtained from I. ricinus, except for Austrian strains, which were mostly isolated from humans. A few strains were obtained from rodents mostly in Switzerland (Table 2). Among the 142 strains, 62 were B. garinii, 38 were B. afzelii, 27 were B. burgdorferi sensu stricto and 15 were B. valaisiana (formerly the genomospecies VS116) (4). Among the 28 human skin samples, 15 were identified as B. afzelii, 9 as B. garinii and 4 as B. burg dorferi s.s. The 7 cerebrospinal fluid (CSF) isolates were all identified as B. ga rinii. In two human isolates, a mixture of Borrelia species were found by PCR-RFLP; B. burgdorferi s.s. was associated with B. afzelii or with B. gari nii. Only one of these species was identified by PFGE. This fact can be ex plained by the need of subculture for PFGE, leading to disappearance of one
192
I. Saint Girons et al.
Table 1. B. burgdorferi s.l. isolates from western European countries
Borrelia burgdorferi s.s. Austria France Germany Ireland Netherlands Slovakia Switzerland
UK
Total isolates
7 3 9 2 1 5 27
Borrelia garinii
Borrelia Borrelia afzelii valaisiana
17 1 26 2 4
13
11 111
18
5
62
38
15
34 31
4
Totals 37 4 42 8 8 1 39 3 142
Table 2. Source of Borrelia burgdorferi s.l. isolates
Borrelia burgdorferi s.s. 4
Borrelia garinii
Borrelia Borrelia afzelii valaisiana
Totals
Human skin Human CSF Human other Rodents Birds Ticks
18
46
12
1 14
28 7 4 12 1 90
Total isolates
27
62
38
15
142
9
7
4
15
11
of the species to the benefit of the other. Four Borrelia species were identified from I. ricinus ticks in all countries (Table 2). B.garinii was by far the most abundant and the other species were equally distributed. B. valaisiana was iso lated from ticks in Germany, the Netherlands and Ireland and from one bird in Switzerland. All eleven isolates from rodents (Clethrionomys glareolus) captured in Switzerland were B. afzelii. B. burgdorferi s.s. was isolated from Apodemus sylvaticus in Austria.
Discussion This study has confirmed the greater frequency, as determined by cultivation, of B. garinii in the west of Europe, followed by B. afzelii, B. burgdorferi s.s. and B. valaisiana. It is likely that the distribution of species is influenced by
Identification of Borrelia burgdorferi sensu lato Species in Europe
193
geography and also by the nature of the habitat, in that the greatest variety of species was found in heterogeneous woodland. B. valaisiana was the spe cies least often isolated in this study. However, a previous PCR-based study using the reverse line blot method (5) conducted in Ireland (6), showed that B. valaisiana was the most prevalent species (34.6%) in unfed I. ricinus ticks, whereas B.garinii and B. burgdorferi s.s. were found in 24.3% and 18.4% respectively. B. afzelii was uncommon and found in only 3.7%. A previous study had resulted in the sole isolation of B. garinii from Irish ticks (7). The apparent discrepancy between the isolation rate of B. valaisiana in Ireland (only one strain) and the high prevalence of this species found by PCR, might suggest that this species is more fastidious in culture than the others. B. lusi taniae (formerly the genomospecies PotiB2) (8) was not isolated in the present study. B. garinii was isolated from all 7 CSF samples. B. afzelii was most often iso lated from 28 human skin isolates, although B. garinii and B. burgdorferi s.s. were also found. B. burgdorferi s.s. was also found in heart muscle (1 sample) and synovial fluid (2 samples) . These observations support the general view that neuroborreliosis is most often caused by B. garinii, skin manifestations by B. afzelii and musculoskeletal symptoms by B. burgdorferi s.s. (9, 10). Most of the skin lesions were erythema migrans, which explains the isolation of all three main species, even if B. afzelii was the most frequent. B. valaisiana was not found in human tissues in this study. However DNA from this species has been recovered by PCR both from ticks and human patients (11) suggesting that B. valaisiana is not only present in nature, but may also be pathogenic for humans. This present work can be compared to an independent study (12) carried out in collaboration with the Russian participant of EUCALB (Prof. E. Koren berg), in which 365 isolates from 12 areas in Russia and from New Indepen dent States were identified by the same laboratory. They were mostly obtained from I. persulcatus ticks, but also from I. ricinus, I. trianguliceps, and small mammals. It was found that B. garinii was most commonly isolated (56.1 %), followed by B. afzelii (32.9%) . The previously reported absence of B. burg dorferi s.s. from I. persulcatus was confirmed in this study. The frequency of B. burgdorferi s.s. seems to decrease from west to east and no B. burgdorferi s.s. has ever been isolated from Russia. B. valaisiana and B.lusitaniae were rarely isolated and always from I. ricinus. Mixed infections often occurred in both hosts and vectors. They can be easily detected by PCR-RFLP on inter genic spacer rrf-rrl from low passage strains, but after a few subcultures one species overcomes the others. A total of 62 Borrelia strains were isolated from rodents in Russia and Eastern Europe. B. garinii was identified in 39 cases and B. afzelii in 13 cases. In the present study, only a few animal samples were available, coming from the same area in Switzerland. B. afzelii was identified in 11 cases out 12. It is of interest to note that in Russia, where B. garinii was predominant, the samples were obtained from the urinary bladder of rodents. In contrast, in Switzerland, where B. afzelii was predominant, the samples
194
I. Saint Girons et al.
were obtained from rodent ears. The organ tropism of different Borrelia spe cies being different, the nature of the samples might result in different results. Furthermore, it should be noted that such differing organ tropisms may also affect the efficiency of transmission of genomospecies to ticks. An interesting finding of the present study was the association of B. valai siana and birds. Amongst 15 B. valaisiana strains, 4 were isolated from I. ric inus engorged on birds (Turdus merula) and one strain was isolated directly from a bird. A larger study has shown that birds could constitute the reser voir of B. valaisiana (13).
Acknowledgements We thank M. Labuda, C. Perez-Eid and A. van den Bogaard for supplying strains.
References 1. Postic, D., M. V. Assous, P. A. D. Grimont, and G. Baranton: 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 (1994) 743-752 2. Belfaiza,]., D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons: Genomic fin gerprinting of Borrelia burgdorferi sensu lato by pulsed field gel electrophoresis. J. Clin. Microbiol. 31 (1993) 2873-2877 3. Humair, P.-E, O. Piter, R. Wallich, and L. Gern: Strain variation of Lyme disease spirochetes isolated from I. ricinus ticks and rodents collected in two endemic areas in Switzerland. J. Med. Entomol. 32 (1995) 433-438 4. Wang, G., A. P van Dam, A. Le Fleche, D. Postic, O. Peter, G. Baranton, R. de Boer, L. Spanjaard, and J. Dankert: Genetic and phenotypic analysis of Borrelia valaisia na sp. nov. (Borrelia genomic groups VS116 and M19). Int. J. Syst. Bacteriol. 47 (1997) 926-932 5. Rijpkema, S. G. T., M.]. C. H. Molkenboer, L. M. Schouls, F.Jongejan, and J. E P. Schellekens: Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterisation of the amplified intergenic spacer region between 5S and 23S rRNA genes. J. Clin. Mi crobiol. 33 (1995) 3091-3095 6. Kirstein, E, S. Rijpkema, M. Molkenboer, and]. S. Gray: The distribution and prev alence of B. burgdorferi genomospecies in Ixodes ricinus ticks in Ireland. Europ. J. Epidemiol. 13 (1997) 67-72 7. Gray, J. S., A. Schonberg, D. Postic, J. Belfaiza, and I. Saint Girons: First isolation and characterisation of Borrelia garinii, the agent of Lyme borreliosis, from Irish ticks. Irish J. Med. Science. 165 (1996) 24-26 8. Le Fleche, A., D. Postic, K. Girardet, O. Piter, and G. Baranton: Characterisation of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis. Int. J. Syst. Bacteriol. 47 (1997) 921-925 9. Assous, M. v., D. Postic, G. Paul, P. Nlivot, and G. Baranton: Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Bor relia strains used as antigens. Eur. J. Clin. Microbiol. Infect. Dis. 12 (1993) 261-268
Identification of Borrelia burgdorferi sensu lato Species in Europe
195
10. van Dam, A. P., H. Kuiper, K. Vas, A. Widjojokusumo, B. M. de Jongh, L. Span jaard, A. C. P. Ramselaar, M. D. Kramer, and]. Dankert: Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin. Infect. Dis. 17 (1993) 708-717 11. Rijpkema, S. G. T., D.]. Tazelaar, M.]. C. H. Molkenboer, G. T. Noordhoek, G. Plantinga, L. M. Schouls, and J. F. P. Schellekens: Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clin. Microbiol. Infect- 3 (1997) 109-116 12. Postic, D., E. Korenberg, N. Gorelova, Y. V. Kovalevski, E. Bellenger, and G. Ba ranton: Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates. Res. Microbiol. 148 (1997) 691-702 13. Humair, P. F., D. Postic, R. Wallich, and L. Gern: An avian reservoir (Turdus me rula) of the Lyme disease spirochete. Zent.bl. Bakteriol. in press (1997)
Zentralblatt fUr
Bakt......sie
Research paper
Identification of Borrelia burgdorferi sensu lato Species in Europe I. Saint Girons, L. Gem, J. S. Gray, E. C. Guy, E. Korenberg, P. A. Nuttall, S. G. T. Rijpkema, A. Schonberg, G. Stanek, and D. Postic
Summary Characterisation at the species level of 142 Borrelia isolates obtained from ticks, hu mans and rodents in Western Europe was carried out and their geographical distribu tion was described. Borrelia garinii was the predominant species representing 44% of the isolates and B. afzelii and B. burgdorferi sensu stricto constituted 27% and 19% of isolates respectively. B. valaisiana, (formerly group VS116) constituted 10.5% of isolates. Some differences in the Borrelia species distribution were observed from one country to another, possibly linked to different sources of samples. In the human sam ples, which were mostly collected in Austria, B. afzelii was preferentially isolated from skin and B. garinii from CSF. B. afzelii was consistently isolated from rodents captured in Switzerland, but one isolate of B. garinii was obtained from a rodent in Austria. B. garinii was by far the most abundant species isolated from Ixodes ricinus ticks in all studied countries. B. valaisiana was isolated from I. ricinus ticks collected from vege tation and from I. ricinus engorged on birds.
Introduction The aim of this study was to identify at a species level Borrelia burgdorferi sen su lato strains isolated from ticks, rodents, and humans in different European countries in order to determine the distribution of the various species and to shed light on their relationship to clinical presentations and to host associations.
Methods Members of the European Union Concerted Action on Lyme Borreliosis (EU CALB) isolated strains of B. burgdorferi sensu lato (B. burgdorferi s.l.) and then dispatched them to the Pasteur Institute, Paris, France, in order to have
Identification of Borrelia burgdorferi sensu lato Species in Europe
191
them characterised by genomic tools, and to Neuchatel, Switzerland, for phe notypic characterisation. Three different characterisation methods were used. 1. Polymerase chain reaction followed by analysis of the restriction frag ment length polymorphism (PCR-RFLP) on rrf-rrl ribosomal spacer. This method is based on the unique organisation of ribosomal genes in B. burgdor feri s.1. There is only one copy of rrs (165) rRNA gene, whereas there are two copies tandemly duplicated of rrl (23S) rRNA gene and rrf (5S) rRNA gene. Thus, the use of primers chosen in conserved genes allows the amplification of the variable spacer between the two copies of these genes (1). The amplifi cation is specific for DNA from B. burgdorferi s.1. In a second step, each spe cies is characterised by a specific MseI restriction pattern. 2. Pulsed field gel electrophoresis (PFGE) B. burgdorferi s.1. isolates were analysed after MluI (a rare cutting enzyme) digestion of genomic DNA in agarose plugs, followed by PFGE. A previous study had shown that PFGE patterns permitted the identification of Borrelia species from the presence of fragments specific for each species and also per mitted the definition of subgroups within a Borrelia species (2). 3. Reactivity with monoclonal antibodies (Mabs) The reactivity of Borrelia lysates with Mabs H3TS, D6 and 117.3, specific for B. burgdorferi sensu stricto (B. burgdorferi s.s.), B. garinii, and B. afzelii respectively, was studied in Western-blot experiments (3).
Results A total of 142 Borrelia strains from 140 samples were studied from Switzer land, UK, Ireland, Germany, the Netherlands, Austria, France, and Slovakia (Table 1). Germany, Switzerland and Austria provided most of the isolates. All strains were identified by PCR-RFLP, 61 of them were additionally identified by PFGE and 65 strains were tested in western-blot experiments. The results obtained by the three methods were well correlated. The analysis of PFGE pat terns confirmed the genetic diversity within both B. burgdorferi s.s. and B. ga rinii species. The majority of isolates were obtained from I. ricinus, except for Austrian strains, which were mostly isolated from humans. A few strains were obtained from rodents mostly in Switzerland (Table 2). Among the 142 strains, 62 were B. garinii, 38 were B. afzelii, 27 were B. burgdorferi sensu stricto and 15 were B. valaisiana (formerly the genomospecies VS116) (4). Among the 28 human skin samples, 15 were identified as B. afzelii, 9 as B. garinii and 4 as B. burg dorferi s.s. The 7 cerebrospinal fluid (CSF) isolates were all identified as B. ga rinii. In two human isolates, a mixture of Borrelia species were found by PCR-RFLP; B. burgdorferi s.s. was associated with B. afzelii or with B. gari nii. Only one of these species was identified by PFGE. This fact can be ex plained by the need of subculture for PFGE, leading to disappearance of one
192
I. Saint Girons et al.
Table 1. B. burgdorferi s.l. isolates from western European countries
Borrelia burgdorferi s.s. Austria France Germany Ireland Netherlands Slovakia Switzerland
UK
Total isolates
7 3 9 2 1 5 27
Borrelia garinii
Borrelia Borrelia afzelii valaisiana
17 1 26 2 4
13
11 111
18
5
62
38
15
34 31
4
Totals 37 4 42 8 8 1 39 3 142
Table 2. Source of Borrelia burgdorferi s.l. isolates
Borrelia burgdorferi s.s. 4
Borrelia garinii
Borrelia Borrelia afzelii valaisiana
Totals
Human skin Human CSF Human other Rodents Birds Ticks
18
46
12
1 14
28 7 4 12 1 90
Total isolates
27
62
38
15
142
9
7
4
15
11
of the species to the benefit of the other. Four Borrelia species were identified from I. ricinus ticks in all countries (Table 2). B.garinii was by far the most abundant and the other species were equally distributed. B. valaisiana was iso lated from ticks in Germany, the Netherlands and Ireland and from one bird in Switzerland. All eleven isolates from rodents (Clethrionomys glareolus) captured in Switzerland were B. afzelii. B. burgdorferi s.s. was isolated from Apodemus sylvaticus in Austria.
Discussion This study has confirmed the greater frequency, as determined by cultivation, of B. garinii in the west of Europe, followed by B. afzelii, B. burgdorferi s.s. and B. valaisiana. It is likely that the distribution of species is influenced by
Identification of Borrelia burgdorferi sensu lato Species in Europe
193
geography and also by the nature of the habitat, in that the greatest variety of species was found in heterogeneous woodland. B. valaisiana was the spe cies least often isolated in this study. However, a previous PCR-based study using the reverse line blot method (5) conducted in Ireland (6), showed that B. valaisiana was the most prevalent species (34.6%) in unfed I. ricinus ticks, whereas B.garinii and B. burgdorferi s.s. were found in 24.3% and 18.4% respectively. B. afzelii was uncommon and found in only 3.7%. A previous study had resulted in the sole isolation of B. garinii from Irish ticks (7). The apparent discrepancy between the isolation rate of B. valaisiana in Ireland (only one strain) and the high prevalence of this species found by PCR, might suggest that this species is more fastidious in culture than the others. B. lusi taniae (formerly the genomospecies PotiB2) (8) was not isolated in the present study. B. garinii was isolated from all 7 CSF samples. B. afzelii was most often iso lated from 28 human skin isolates, although B. garinii and B. burgdorferi s.s. were also found. B. burgdorferi s.s. was also found in heart muscle (1 sample) and synovial fluid (2 samples) . These observations support the general view that neuroborreliosis is most often caused by B. garinii, skin manifestations by B. afzelii and musculoskeletal symptoms by B. burgdorferi s.s. (9, 10). Most of the skin lesions were erythema migrans, which explains the isolation of all three main species, even if B. afzelii was the most frequent. B. valaisiana was not found in human tissues in this study. However DNA from this species has been recovered by PCR both from ticks and human patients (11) suggesting that B. valaisiana is not only present in nature, but may also be pathogenic for humans. This present work can be compared to an independent study (12) carried out in collaboration with the Russian participant of EUCALB (Prof. E. Koren berg), in which 365 isolates from 12 areas in Russia and from New Indepen dent States were identified by the same laboratory. They were mostly obtained from I. persulcatus ticks, but also from I. ricinus, I. trianguliceps, and small mammals. It was found that B. garinii was most commonly isolated (56.1 %), followed by B. afzelii (32.9%) . The previously reported absence of B. burg dorferi s.s. from I. persulcatus was confirmed in this study. The frequency of B. burgdorferi s.s. seems to decrease from west to east and no B. burgdorferi s.s. has ever been isolated from Russia. B. valaisiana and B.lusitaniae were rarely isolated and always from I. ricinus. Mixed infections often occurred in both hosts and vectors. They can be easily detected by PCR-RFLP on inter genic spacer rrf-rrl from low passage strains, but after a few subcultures one species overcomes the others. A total of 62 Borrelia strains were isolated from rodents in Russia and Eastern Europe. B. garinii was identified in 39 cases and B. afzelii in 13 cases. In the present study, only a few animal samples were available, coming from the same area in Switzerland. B. afzelii was identified in 11 cases out 12. It is of interest to note that in Russia, where B. garinii was predominant, the samples were obtained from the urinary bladder of rodents. In contrast, in Switzerland, where B. afzelii was predominant, the samples
194
I. Saint Girons et al.
were obtained from rodent ears. The organ tropism of different Borrelia spe cies being different, the nature of the samples might result in different results. Furthermore, it should be noted that such differing organ tropisms may also affect the efficiency of transmission of genomospecies to ticks. An interesting finding of the present study was the association of B. valai siana and birds. Amongst 15 B. valaisiana strains, 4 were isolated from I. ric inus engorged on birds (Turdus merula) and one strain was isolated directly from a bird. A larger study has shown that birds could constitute the reser voir of B. valaisiana (13).
Acknowledgements We thank M. Labuda, C. Perez-Eid and A. van den Bogaard for supplying strains.
References 1. Postic, D., M. V. Assous, P. A. D. Grimont, and G. Baranton: 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 (1994) 743-752 2. Belfaiza,]., D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons: Genomic fin gerprinting of Borrelia burgdorferi sensu lato by pulsed field gel electrophoresis. J. Clin. Microbiol. 31 (1993) 2873-2877 3. Humair, P.-E, O. Piter, R. Wallich, and L. Gern: Strain variation of Lyme disease spirochetes isolated from I. ricinus ticks and rodents collected in two endemic areas in Switzerland. J. Med. Entomol. 32 (1995) 433-438 4. Wang, G., A. P van Dam, A. Le Fleche, D. Postic, O. Peter, G. Baranton, R. de Boer, L. Spanjaard, and J. Dankert: Genetic and phenotypic analysis of Borrelia valaisia na sp. nov. (Borrelia genomic groups VS116 and M19). Int. J. Syst. Bacteriol. 47 (1997) 926-932 5. Rijpkema, S. G. T., M.]. C. H. Molkenboer, L. M. Schouls, F.Jongejan, and J. E P. Schellekens: Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterisation of the amplified intergenic spacer region between 5S and 23S rRNA genes. J. Clin. Mi crobiol. 33 (1995) 3091-3095 6. Kirstein, E, S. Rijpkema, M. Molkenboer, and]. S. Gray: The distribution and prev alence of B. burgdorferi genomospecies in Ixodes ricinus ticks in Ireland. Europ. J. Epidemiol. 13 (1997) 67-72 7. Gray, J. S., A. Schonberg, D. Postic, J. Belfaiza, and I. Saint Girons: First isolation and characterisation of Borrelia garinii, the agent of Lyme borreliosis, from Irish ticks. Irish J. Med. Science. 165 (1996) 24-26 8. Le Fleche, A., D. Postic, K. Girardet, O. Piter, and G. Baranton: Characterisation of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis. Int. J. Syst. Bacteriol. 47 (1997) 921-925 9. Assous, M. v., D. Postic, G. Paul, P. Nlivot, and G. Baranton: Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Bor relia strains used as antigens. Eur. J. Clin. Microbiol. Infect. Dis. 12 (1993) 261-268
Identification of Borrelia burgdorferi sensu lato Species in Europe
195
10. van Dam, A. P., H. Kuiper, K. Vas, A. Widjojokusumo, B. M. de Jongh, L. Span jaard, A. C. P. Ramselaar, M. D. Kramer, and]. Dankert: Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin. Infect. Dis. 17 (1993) 708-717 11. Rijpkema, S. G. T., D.]. Tazelaar, M.]. C. H. Molkenboer, G. T. Noordhoek, G. Plantinga, L. M. Schouls, and J. F. P. Schellekens: Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clin. Microbiol. Infect- 3 (1997) 109-116 12. Postic, D., E. Korenberg, N. Gorelova, Y. V. Kovalevski, E. Bellenger, and G. Ba ranton: Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates. Res. Microbiol. 148 (1997) 691-702 13. Humair, P. F., D. Postic, R. Wallich, and L. Gern: An avian reservoir (Turdus me rula) of the Lyme disease spirochete. Zent.bl. Bakteriol. in press (1997)