Daniele Postic

Delineation of Borrelia burgdorferi Sensu Stricto, Borrelia garinii sp. nov., and Group VS461 Associated with Lyme Borreliosis

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.

Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.

Borrelia isolates associated with Lyme borreliosis were previously divided into 3 genospecies, B. burgdorferi sensu stricto, B. garinii and group VS461, on the basis of DNA homology. B. burgdorferi sensu stricto and B. garinii were identified by monoclonal antibodies (MAbs), H3TS and D6 respectively, but no MAbs were available to identify group VS461. Two MAbs were produced, I 17.3 and J 8.3 which reacted with OspB and OspA proteins, respectively, of strains belonging to group VS461, which should be named B. afzelii sp. nov. 24 strains were assigned to B. afzelii sp. nov., 11 of them being isolated from skin lesions, 6 from acrodermatitis chronica atrophicans (ACA) and 5 from erythema chronicum migrans (ECM). Although quite unknown in the USA, ACA has frequently been reported in northern Europe where B. afzelii sp. nov. is commonly isolated. This study documents the involvement of B. afzelii sp. nov. as a specific aetiological agent of ACA.

GENOMIC FINGERPRINTING BY ARBITRARILY PRIMED POLYMERASE CHAIN REACTION RESOLVES BORRELIA BURGDORFERI INTO THREE DISTINCT PHYLETIC GROUPS

The causative agent of Lyme disease, Borrelia burgdorferi, was first identified by Burgdorfer et al. in 1982 (W. Burgdorfer, A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis, Science 216:1317-1319, 1982) and was isolated by Barbour et al. in 1983 (A. G. Barbour, W. Burgdorfer, S. E. Hayes, O. Peter, and A. Aeschlimann, Curr. Microbiol. 8:123-126, 1983). Since then, a large number of isolates have been collected, and there have been questions regarding the relationships among the various strains. Using genomic fingerprinting by an arbitrarily primed polymerase chain reaction, we resolved into three groups a collection of Eurasian and North American isolates of spirochetes that are generally categorized as B. burgdorferi. Group I strains have been identified in both North America and Eurasia, while strains belonging to Borrelia groups II and III have been found only in Eurasia. These same three groups have also been delineated by Baranton et al. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:370-375, 1992) by independent methods. Two isolates are distinct from all of the other strains in our collection but are clearly members of the genus Borrelia.

Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis

We determined the complete sequence of the rrs gene from five strains of genomic species PotiB2. Both distance and parsimony methods were used to infer the evolutionary relationships of the rrs gene sequence of this genomic species in comparison with the rrs gene sequence of Borrelia valaisiana and the rrs gene sequences of Borrelia burgdorferi sensu lato species obtained from sequence databases. The phylogenetic analysis revealed that the genomic species PotiB2 strains clustered in a separate lineage, which was consistent with data from previous DNA-DNA hybridization experiments (D. Postic, M. V. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). A PCR-restriction fragment length polymorphism analysis was used to identify genomic species PotiB2 and to differentiate it from B. burgdorferi sensu lato species. Moreover, signature nucleotide positions were identified for each B. burgdorferi sensu lato species. In accordance with DNA relatedness values, our findings suggest that genomic species PotiB2 can be more clearly defined and identified, and we propose that it should be referred to as a new species, Borrelia lusitaniae. The type strain is PotiB2.

Genetic and phenotypic analysis of Borrelia valaisiana sp.nov. (Borrelia Genomic Groups VS116 and M19)

To clarify the taxonomic status of two recently described Borrelia genomic groups, groups VS116 and M19, three group VS116 strains and eight group M19 strains isolated from Ixodes ricinus ticks in Switzerland, The Netherlands, and the United Kingdom were characterized. PCR-restriction fragment length polymorphism (RFLP) analysis of the 5S-23S intergenic spacer amplicon, rRNA gene restriction analysis, 16S rRNA gene sequence analysis, randomly amplified polymorphic DNA (RAPD) fingerprinting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with monoclonal antibodies were used for genetic and phenotypic analysis. The PCR-RFLP and RAPD patterns of three group VS116 strains and eight group M19 strains were identical but differed from those of Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and Borrelia japonica. DNAs from all group VS116 and M19 strains yielded three fragments (6.9, 3.2, and 1.4 kb) and four fragments (2.1, 1.2, 0.8, and 0.6 kb) after digestion with EcoRV and HindIII, respectively, hybridizing with an Escherichia coli 16S + 23S cDNA probe. The SDS-PAGE protein profiles of group VS116 and M19 strains were heterogeneous. Phylogenetic analysis of the partial 16S rRNA gene sequences showed that group VS116 and M19 spirochetes were members of a Borrelia species distinct from previously characterized members of the genus Borrelia. Based on our present study and data from previous DNA-DNA hybridizations, a new Borrelia species, Borrelia valaisiana sp.nov., in the B. burgdorferi complex, is proposed. Strain VS116 is the type strain of this new species.

Western blot analysis of sera from lyme borreliosis patients according to the genomic species of the Borrelia strains used as antigens

Sera of 52 Lyme borreliosis patients classified according to their clinical features were analysed by Western blot using as antigensBorrelia strains belonging to three recently described genomic species. The antibody response was demonstrated to be homologous within each genospecies. Serum reactivity was studied for each of the type strainsBorrelia burgdorferi sensu stricto (strain B31T),Borrelia garinii (strain 20047T) and group VS461. Seven of 15 sera (46.6 %) of patients with menin-goradiculitis showed preferential reactivity withBorrelia garinii (strain 20047T), all of 8 sera (100 %) of patients with acrodermatitis chronica atrophicans showed preferential reactivity with group VS461 (strain VS461) and 8 of 16 sera (50 %) of patients with arthritis showed preferential reactivity withBorrelia burgdorferi sensu stricto (strain B31T). The presence of a strong response to OspA and OspB proteins ofBorrelia burgdorferi sensu stricto was found only in this last group of patients. These results suggest that there are clinical implications of the recently described modifications in the taxonomy ofBorrelia burgdorferi.

An Avian Reservoir (Turdus merula) of the Lyme Borreliosis Spirochetes

The reservoir competence of passerine birds for the Lyme borreliosis spirochetes was studied in an enzootic focus in Switzerland. Skin aspirates and skin biopsies were used to isolate Borrelia spirochetes from Turdus species. B. burgdorferi sensu lato was isolated and/or PCR-detected in BSK medium containing skin biopsy or skin aspirate from 5 blackbirds (T. merula) and one song thrush (T. philomelos). Seven isolates were obtained from 3 different blackbirds. Either B. garinii or Borrelia from the genomic group VS116 was found in bird skin samples. Mixed infection occurred in 2 cases. Tick xenodiagnosis was used to determine whether blackbirds transmitted Borrelia to ticks. Five xenodiagnoses were performed on 3 different blackbirds. Borrelia DNA was detected in BSK medium inoculated with xenodiagnostic ticks from all the passerines tested. Isolates cultured from xenodiagnostic ticks were obtained from 2 blackbirds. Isolates belonged to group VS116 (n = 10) and to B. garinii (n = 1). Our study has shown that Turdus sp. are infected by B. garinii and by Borrelia from group VS116 and that blackbirds are implicated as reservoirs for these 2 genomic groups of Borrelia, as they transmit living borreliae to ticks. An association seems to exist between birds and Borrelia VS116, and to a lesser extent, B. garinii, similar to the association existing between small rodents and B. afzelii. Our observations emphasize the fact that different enzootic cycles maintain Lyme borreliosis spirochetes in nature.

Phylogenesis of relapsing fever Borrelia spp.

The phylogenetic relationships of 20 relapsing fever (RF) Borrelia spp. were estimated on the basis of the sequences of rrs genes. Complete sequences were aligned and compared with previously published sequences, and the similarity values were found to be 97.7 to 99.9%. Phylogenetic trees were constructed by using the three neighbor-joining, maximum-parsimony, and maximum-likelihood methods. The results of the comparative phylogenetic analysis divided the RF Borrelia spp. into three major clusters. One cluster included Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis, and Borrelia hispanica. Another cluster comprised tow main branches with Borrelia coriaceae, Borrelia lonestari, and Borrelia miyamotoi on one side and Borrelia parkeri, Borrelia turicatae, and Borrelia hermsii on the other side. Borrelia anserina constituted the third cluster. The phylogenetic position of Borrelia persica was more uncertain. These results suggested that the taxonomy of these spirochetes should be revised. To overcome the problems of culturing the spirochetes, RF Borrelia primers were defined. Following PCR amplification of the rrs gene, restriction length fragment polymorphism could be used to distinguish between RF Borrelia strains.

Distinct levels of genetic diversity of Borrelia burgdorferi are associated with different aspects of pathogenicity

Different species of pathogenic Borrelia show different symptoms and tick vector specificity. Even within regions where only one species is found, Lyme disease progresses very differently from one patient to another. Since Borrelia shows very little recombination either within or between species, alleles of a gene can be used to mark clones. The ospC gene is highly variable within each species and can be used to define groups of related clones. It has been previously shown that only four out of seventeen ospC groups of Borrelia burgdorferi sensu stricto cause invasive forms of the disease. Other groups cause erythema migrans, a skin rash at the site of the tick bite, but not invasive disease, while still other groups seem to be nonpathogenic to humans. In this study we extend the analysis of the ospC gene to the other pathogenic species, Borrelia garinii and Borrelia afzelii. Only two groups in B. afzelii and four groups in B. garinii cause invasive disease. Thus, only ten out of the 58 defined ospC groups cause invasive and presumably chronic Lyme disease.

Identification of Borrelia burgdorferi sensu lato species in Europe

Characterisation at the species level of 142 Borrelia isolates obtained from ticks, humans and rodents in Western Europe was carried out and their geographical distribution 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 samples, 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 vegetation and from I. ricinus engorged on birds.