Apodemus species mice are reservoir hosts of Borrelia garinii OspA serotype 4 in Switzerland

Among Borrelia burgdorferi sensu lato isolates, seven outer surface protein A (OspA) serotypes have been described: serotypes 1 and 2 correspond to B. burgdorferi sensu stricto and Borrelia afzelii, respectively, and serotypes 3 to 7 correspond to Borrelia garinii. In Europe, serotype 4 has never been isolated from Ixodes ricinus ticks until recently, although this serotype has been frequently isolated from cerebrospinal fluid from patients. In Europe, B. afzelii and B. burgdorferi sensu stricto were found associated with rodents and B. garinii was found associated with birds. In this study, the reservoir role of Apodemus mice for B. garinii OspA serotype 4 was demonstrated by xenodiagnosis. Apodemus mice are the first identified reservoir hosts for B. garinii OspA serotype 4.     

Analysis of Borrelia burgdorferi sensu lato isolated from cerebrospinal fluid

Involvement of the nervous system in Lyme borreliosis may occur with or without erythema migrans and it may present with a variety of neurological symptoms. In this study we analysed phenotypic and genotypic characteristics of 40 Borrelia strains isolated from cerebrospinal fluid (CSF) of 38 Slovenian patients with different clinical manifestations of Lyme borreliosis. In seven of the patients, Borreliae were also isolated from skin lesions. Species identification and plasmid profiles were determined by pulsed-field gel electrophoresis and protein profiles by SDS-PAGE. MluI digestion profiles of Borrelia burgdorferi sensu lato DNA showed that 25 (62.5%) isolates were B. garinii, 14 (35%) B. afzelii, and one (2.5%) B. burgdorferi sensu stricto. All strains, except one, possessed a large plasmid and a varying number of smaller plasmids. Three (7.5%) isolates exhibited an unusual plasmid profile, with a large plasmid dimer or three copies of the large plasmid. In protein analyses, all strains expressed OspA protein. OspB was present significantly more often in B. afzelii than B. garinii strains (p=0.0000), while OspC was more often present in B. garinii than B. afzelii strains (p=0.0052). In the seven patients with Borreliae isolated also from the skin, the CSF and skin isolates were identical, either B. garinii (six patients) or B. afzelii (one patient). Species and plasmid heterogeneity as well as antigen diversity could play a role in the pathogenesis of the infection. When combined with our own earlier data, the results suggest species-related organotropism.     

Genotypic and phenotypic characterisation of Borrelia burgdorferi sensu lato strains isolated from human blood

Lyme borreliosis often presents initially with erythema migrans. Borreliae may disseminate from the primary skin lesion, and different organs and systems could be affected. Borrelia strains were isolated from blood of 70 patients with Lyme borreliosis, including 10 patients from whom borreliae were also isolated from skin. The aim of the present study was to characterise the isolates with regard to their phenotypic and genotypic characteristics. Borreliae were cultivated in MKP medium. Species identification and plasmid profiles were determined by pulsed-field gel electrophoresis (PFGE) and protein profiles by SDS-PAGE. Digestion of Borrelia burgdorferi sensu lato DNA showed 63 (90%) B. afzelii Mla1 and 7 (10%) B. garinii Mlg2. No B. burgdorferi sensu stricto were isolated. Borreliae were isolated from both skin and blood of 10 patients, nine pairs of isolates were identical: seven B. afzelii and two B. garinii. B. afzelii was isolated from the skin and B. garinii from blood of the tenth patient. All but one isolate possessed at least one large plasmid and varying numbers of smaller plasmids. Eight (11.4%) of 70 isolates possessed an unusual plasmid profile (2 of 63 B. afzelii and 6 of 7 B. garinii). Borreliae differed in their protein profiles. OspA and OspB proteins were expressed by all B. afzelii isolates; 85.7% of B. garinii isolates expressed OspA and 71.4% expressed OspB. OspC was expressed by 65% of B. afzelii isolates and all B. garinii isolates. The ratios of B. afzelii and B. garinii isolated from blood and skin were similar. These results do not support the hypothesis that B. garinii has a higher propensity for haematogenous dissemination than B. afzelii. Antigen diversity as well as species and plasmid heterogeneity could play a role in the pathogenesis of the infection, suggesting distinctive strain organotropism.     

Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain

Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic.     

Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland

Objective: To evaluate by species-specific immunoblots the association of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii with neuroborreliosis in Switzerland.
Methods: Borrelia strains isolated from the cerebrospinal fluid (CSF) of three children with neuroborreliosis were typed by phenotypic and genotypic analysis. The serologic reactions (IgG) of these three patients as well as those of 28 patients, including one of these three children, with confirmed neuroborreliosis were characterized and scored by immunoblots on the three individual Borrelia species antigens. Twenty patients with typical erythema migrans served as a control group.
Results: Phenotypic and genotypic analysis confirmed that all three CSF isolates were B. garinii. In the 28 patients with neuroborreliosis, the comparatively strongest reactions were as follows: 18 to B. garinii , three to B. burgdorferi sensu stricto and two to B. afzelii ; five were inconclusive. In the control group (erythema migrans), the comparatively strongest reactions were as follows: six B. garinii , one to B. burgdorferi sensu stricto and five to B. afzelii ; eight were indeterminate.
Conclusions: Typing of these three CSF isolates and characterization by immunoblots of the antibody reactions of patients with neuroborreliosis give additional evidence of the association of B. garinii and neuroborreliosis. Our serologic results suggest that B. burgdorferi sensu stricto and B. afzelii are also responsible for some neuroborreliosis cases in Switzerland. Our immunoblots and the scoring system proved particularly useful for the serologic typing of patients with late Lyme borreliosis.     

Isolation and characterization of Borrelia burgdorferi sensu lato strains in an area of Italy where Lyme borreliosis is endemic

Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to "San Martino" Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.     

Abilities of OspA proteins from different seroprotective groups of Borrelia burgdorferi to protect hamsters from infection.

The ability of vaccination with recombinant OspA from six seroprotective groups of Borrelia burgdorferi sensu lato to induce protection against infection with homologous and other Lyme spirochetes was examined in hamsters. Antisera generated against the OspA proteins of B. burgdorferi sensu stricto S-1-10 and C-1-11 (seroprotective groups 1 and 2, respectively), Borrelia afzelii BV1 (seroprotective group 4), and Borrelia garinii LV4 (seroprotective group 5) were able to kill the homologous spirochete in vitro but not other isolates. Surprisingly, antisera against B. afzelii PKo (seroprotective group 6) and B. burgdorferi sensu lato LV5 (seroprotective group 3) OspA proteins were unable to kill the homologous organism, although LV5 OspA antisera killed the heterologous isolates S-1-10 and LV4. In vivo vaccination studies supported the in vitro findings, confirming that vaccination with a single OspA protein does not provide complete protection against challenge with all Lyme disease spirochetes. In addition, OspA antibodies from some isolates may not protect against the homologous isolate. The induction of protective antibodies against other B. burgdorferi proteins may be necessary to insure a comprehensive Lyme disease vaccine.     

Heterogeneity of BmpA (P39) among European isolates of Borrelia burgdorferi sensu lato and influence of interspecies variability on serodiagnosis.

The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed. The bmpA sequences of 12 isolates representing all three species of B. burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced. The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B. burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity). The interspecies identities ranged from 86 to 92%. Cluster analysis of BmpA reflected the subdivision of B. burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B. garinii strains. The BmpA protein of each species of B. burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies. Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B. afzelii and B. garinii. A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B. burgdorferi sensu stricto but no or only weak reactivity with BmpA of B. garinii and B. afzelii, respectively. Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100%. IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%). With the BmpA proteins of B. afzelii and B. garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B. burgdorferi sensu stricto. Therefore, we recommend that recombinant BmpA of B. afzelii or B. garinii should be used solely, or in addition to B. burgdorferi sensu stricto BmpA, in serodiagnostic tests for Lyme borreliosis in Europe.     

Phenotypic analysis of outer surface protein C (OspC) of Borrelia burgdorferi sensu lato by monoclonal antibodies: relationship to genospecies and OspA serotype.

Molecular analyses of the genes encoding OspC, a major immunodominant protein of Borrelia burgdorferi sensu lato, revealed a considerable degree of heterogeneity. In the present study, we investigated whether a similar heterogeneity of the OspC phenotype can be shown by analysis with monoclonal antibodies (MAbs). Thirteen OspC-specific MAbs (L22 MAbs) were produced by immunizing mice with either different combinations of whole-cell antigens or recombinantly expressed OspCs cloned from strains belonging to different Borrelia spp. Ten of them differed in their reactivities with various strains. Western blot (immunoblot) analyses of 38 B. burgdorferi sensu lato strains resulted in 13 different reactivity patterns. These 13 different patterns were observed among only six different OspA serotypes, indicating that OspC is more heterogeneous than OspA. Patterns 1 to 4 were present only in B. burgdorferi sensu stricto, patterns 5 to 7 were present only in Borrelia afzelii, and patterns 9 to 13 were present only in Borrelia garinii. Pattern 8 was observed among B. afzelii and B. garinii strains but not among B. burgdorferi sensu stricto strains. One L22 MAb (2B8) recognized a common OspC-specific epitope of all 38 B. burgdorferi sensu lato strains analyzed, and another one (22C11) recognized a common epitope of OspC from both B. afzelii and B. garinii and was not reactive with OspC from B. burgdorferi sensu stricto. Western blot and sequence analysis of truncated OspCs located the 22C11 epitope as well as a species-specific sequence motif between amino acids 20 and 35.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diversity among Borrelia strains determined by single-strand conformation polymorphism analysis of the ospC gene and its association with invasiveness

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three Borrelia species: Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac.). Previous research based on analysis of ospC sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted ospC groups by testing human clinical strains isolated from disseminated forms. To screen for ospC genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known ospC sequences from 44 different strains were first tested, revealing that each ospC group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose ospC sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of B. afzelii, seven of B. garinii, and one of B. burgdorferi sensu stricto. This study confirmed that invasive strains are not distributed among all 69 ospC groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of B. burgdorferi.     

Characterization of Finnish Borrelia burgdorferi sensu lato isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with monoclonal antibodies.

Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS-PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis.     

Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. Valaisiana in humans

An immunoglobulin G immunoblot was developed with antigenic extracts of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana genospecies and was reacted with sera from patients with neuroborreliosis, acrodermatitis, and Lyme arthritis. A detailed analysis of the reactivities of the protein bands was performed, and a two-step scoring procedure was selected to determine the preferential reactivity of sera to one particular genospecies. The discriminative potential of 5 proteins (12-kDa, 16-kDa, 18-kDa, OspA, and 66-kDa proteins) was used as a rapid first-step scoring method, followed by scoring of 14 additional protein bands if necessary. The advantage of this procedure is the low percentage of serum samples with inconclusive results for one of the four species (10% for patients with neuroborreliosis, 6% for patients with acrodermatitis chronica atrophicans, and 6% for patients with Lyme arthritis). Among 31 serum samples from patients with neuroborreliosis, 16 were more reactive to B. garinii, 7 were more reactive to B. afzelii, 3 were more reactive to B. valaisiana, and 2 were more reactive to B. burgdorferi sensu stricto. Of 31 serum samples from patients with acrodermatitis, 26 showed a higher level of reactivity to B. afzelii. Of 34 serum samples from patients with Lyme arthritis, 21 were more reactive to B. burgdorferi sensu stricto, 10 were more reactive to B. afzelii, and 1 was more reactive to B. valaisiana. Our results suggest an organotropism of Borrelia species and provide some evidence of a pathogenic potential of B. valaisiana in humans.     

Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.

A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus Ixodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with MluI were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only fragments greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgdorferi sensu stricto isolates were recognized by a band at 135 kbp, each of the 12 Borrelia garinii isolates by two bands (220 and 80 kbp), and each of the 20 Borrelia afzelii isolates by three bands (460, 320, and 90 kbp). Whereas differences in the PFGE patterns among B. burgdorferi sensu stricto isolates and B. garinii isolates were noted, B. afzelii isolates were all similar. Identification of isolates by PFGE correlates with their belonging to a given species within B. burgdorferi sensu lato.     

Characterisation of Borrelia burgdorferi sensu lato strains isolated from patients with skin manifestations of Lyme borreliosis residing in Slovenia

Lyme borreliosis is the most prevalent tick-borne infection in Slovenia. Skin disorders are the most frequent clinical manifestations. The aim of the present study was to assess the phenotypic and genotypic diversity of a large number of human Borrelia burgdorferi sensu lato isolates and to evaluate any association between the isolates and different clinical manifestations. All 103 strains tested were from patients suffering from the skin disorders of Lyme borreliosis. Skin biopsies, cerebrospinal fluid and blood samples from patients were inoculated into modified Kelly Pettenkofer medium. Protein profiles were determined by SDS-PAGE and species identification and plasmid profiles by pulsed-field gel electrophoresis. MluI digestion profiles showed that 87 (84.5%) isolates belonged to B. afzelii, 15 (14.5%) to B. garinii and 1 (1%) to B. burgdorferi sensu stricto. The number of plasmids in each strain varied from three to seven, and the plasmid size ranged from 15 to 65 kb. Four isolates of B. garinii possessed multiple large plasmids and four isolates had a large plasmid dimer (three B. afzelii and one B. garinii). Isolates showed qualitative and quantitative differences in protein expression. The study found differences in the expression of OspB and OspC proteins between B. afzelii and B. garinii strains. OspB was expressed significantly more often by B. afzelii (78 of 87, 89.6%) than by B. garinii (4 of 15, 26.6%) isolates, while OspC protein was expressed significantly more often by B. garinii (14 of 15, 93.3%) than by B. afzelii (51 of 87, 58.6%) isolates. In Slovenia, B. afzelii causes the majority of skin lesions. The isolates investigated showed plasmid and protein diversity. Heterogeneity of the spirochaetes may be important for virulence, and may have implications for pathogenesis and therapy of the infection. Differences in immunodominant proteins also have an important impact on serological testing and vaccine development.     

Differential immune response to the variable surface loop antigen of P66 of Borrelia burgdorferi sensu lato species in geographically diverse populations of lyme borreliosis patients

We have studied the immune response to a variable surface-exposed loop region of the P66 outer membrane protein from Borrelia burgdorferi sensu lato by using an enzyme immunoassay. Lyme borreliosis populations found in North America and Sweden were preferentially more seroreactive to P66 from their respective regional species, namely, B. burgdorferi sensu stricto and B. garinii and B. afzelii, respectively.     

Direct molecular typing of Borrelia burgdorferi sensu lato species in synovial samples from patients with lyme arthritis

Since Lyme arthritis was first described in the United States, it has now been reported in many countries of Europe. However, very few strains of the causative bacterium, Borrelia burgdorferi, have been isolated from synovial samples. For this reason, different molecular direct typing methods were developed recently to assess which species could be involved in Lyme arthritis in Europe. We developed a simple oligonucleotide typing method with PCR fragments from the flagellin gene of B. burgdorferi sensu lato, which is able to differentiate seven different Borrelia species. Among 10 consecutive PCR-positive patients with Lyme arthritis from the northeastern France, two species were identified in synovial samples: B. burgdorferi sensu stricto in 9 cases and B. garinii in 1 case. Conversely, all B. burgdorferi sensu lato species detected in 10 consecutive PCR-positive biopsies from a second set of patients with erythema migrans from the same geographical area were identified as either B. afzelii or B. garinii (P < 0.001). These results indicate that B. burgdorferi sensu stricto is the principal but not the only Borrelia species involved in Lyme arthritis in northeastern France.     

Molecular Typing of Borrelia burgdorferi Sensu Lato: Taxonomic, Epidemiological, and Clinical Implications

Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the LB-related spirochetes and their potential association with distinct clinical syndromes. These methods include serotyping, multilocus enzyme electrophoresis, DNA-DNA reassociation analysis, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis, plasmid fingerprinting, randomly amplified polymorphic DNA fingerprinting analysis, species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and sequence analysis of 16S rRNA and other conserved genes. On the basis of DNA-DNA reassociation analysis, 10 different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, and Borrelia bissettii sp. nov. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well known to be responsible for causing human disease. Different Borrelia species have been associated with distinct clinical manifestations of LB. In addition, Borrelia species are differentially distributed worldwide and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomic status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especiallly correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail.     

Identification of three clinically relevant Borrelia burgdorferi sensu lato genospecies by PCR-restriction fragment length polymorphism analysis of 16S-23S ribosomal DNA spacer amplicons

We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.     

Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots).

The performance of Western blots (immunoblots) prepared with eight strains of Borrelia burgdorferi representing B. burgdorferi sensu stricto, B. garinii, and B. afzelii genospecies was tested with a panel of sera with various clinical presentations collected from eight geographic regions. European sera were generally more reactive to blots prepared with B. garinii or B. afzelii strain antigens, in particular B. garinii 20047 and B. afzelii VS461. North American sera were more reactive with B. burgdorferi sensu stricto strains. Our observation of significant differences in the levels of reactivity of some sera on Western blots of certain strains is potentially important for the development and implementation of generic interpretive criteria. Preferential reactivity of sera from patients with nerve and/or palsy symptoms to B. garinii strains and with cutaneous disease to B. afzelii strains was observed. On the basis of our results, we have concluded that strain 20047 is the best strain to use for the development of a generic Lyme borreliosis Western blot for Europe.     

Diverse Lyme disease spirochetes bind integrin alpha IIb beta 3 on human platelets.

Lyme disease is a chronic, multisystemic infection caused by Borrelia burgdorferi sensu lato. An infectious strain of B. burgdorferi was previously shown to bind to human platelets via the integrin alpha IIb beta 3. In this study, a diverse group of Lyme disease spirochetes was tested for platelet- and alpha IIb beta 3-binding activity. This collection included representatives of each of the three species that cause Lyme disease, B. burgdorferi (sensu stricto), B. garinii, and B. afzelii. Strains were characterized for infectivity in mouse models or were low-passage isolates from human patients. Each of the 11 infectious strains bound to platelets immobilized in microtiter wells and in suspension. Binding to platelets in suspension was specifically inhibited by a blocking anti-alpha IIb beta 3 antibody, and representatives of each species bound to purified alpha IIb beta 3. The strains that did not bind alpha IIb beta 3 or platelets were all noninfectious. No obvious relationship was observed between binding to platelets and expression of the bacterial outer surface protein OspA, OspB, or OspC, as assessed by immunoblotting. These results demonstrate that integrin alpha IIb beta 3-binding activity is widespread among the Borrelia species that cause Lyme disease and are consistent with a role for alpha IIb beta 3 binding in the transmission and/or pathogenesis of Lyme disease.