Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127)

Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.     

Organization of ribosomal RNA genes in Borrelia burgdorferi sensu lato isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.     

Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes

We developed a rapid and reliable method for the identification Borrelia burgdorferi sensu lato species in ticks. We used the DNA sequence polymorphism of the spacer region between 5S and 23S rRNA genes, which has been shown to be able to discriminate between eight genomic groups of B. burgdorferi sensu lato (D. Postic, M. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). Spacer DNA was amplified by PCR and was then hybridized to five membrane-bound oligonucleotides. The oligonucleotides were specific for B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and group VS116. A probe which reacted with all genomic groups of B. burgdorferi sensu lato was also used. Ninety-six ticks collected in the field were destructed by bead beating, and the supernatant was used directly in a PCR. B. burgdorferi sensu lato DNA was detected in 6 of 57 adult ticks (11%) and 9 of 39 nymphs (23%). B. garinii was found in three nymphs and four adults, three nymphs carried B. afzelii, and one adult and one nymph carried group VS116. Double infections with B. afzelii and group VS116 were found in two nymphs and one adult. Thus, our method can simultaneously identify three genomic groups of B. burgdorferi sensu lato in ticks collected in the field. This technique provides new ways to study the association of genomic groups present in ticks and the risk of Lyme borreliosis.     

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.     

Lyme disease Borrelia species in northeastern China resemble those isolated from far eastern Russia and Japan

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.     

Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates

A total of 365 isolates of Borrelia burgdorferi sensu lato from 12 major administrative territories of Russia (from St. Petersburg in the west to South Sakhalin in the east) and from the Czech Republic, Estonia, Lithuania, Byelorussia, Moldavia, Ukraine and Kirghizia were identified by analysis of restriction polymorphism of ribosomal rrf-rrl spacer amplicons. The isolates were obtained mainly from ixodes persulcatus and I. ricinus ticks. Other sources included small mammals, human patients and I. trianguliceps ticks. The results showed that B. garinii (two variants) together with B. afzelii circulated throughout the territories studied. The distribution of the variant NT29 of the species B. garinii, the most frequently isolated, was associated with that of I. persulcatus ticks. B. burgdorferi sensu stricto, and the species B. valaisiana and B. lusitaniae (formerly the genomospecies VS116 and PotiB2, respectively) were isolated only from I. ricinus ticks in the western part of the studied territories. None of these three species were found in 327 isolates from Russia where I. persulcatus is the most frequently distributed vector. This work also provides evidence for a high incidence of mixed Borrelia infections within vectors and hosts (9.3% of isolates were mixtures of Borrelia species). A detailed analysis of Borrelia species distribution over the territories studied is presented.     

Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.     

Molecular analysis of sequence heterogeneity among genes encoding decorin binding proteins A and B of Borrelia burgdorferi sensu lato

Immunization of mice with Borrelia burgdorferi decorin binding protein A (DbpA), one of two gene products of the dbpBA locus, has been shown recently to confer protection against challenge. Hyperimmune DbpA antiserum killed a large number of B. burgdorferi sensu lato isolates of diverse phylogeny and origin, suggesting conservation of the protective epitope(s). In order to evaluate the heterogeneity of DbpA and DbpB and to facilitate defining the conserved epitope(s) of these antigens, the sequences of the dbpA genes from 29 B. burgdorferi sensu lato isolates and of the dbpB genes from 15 B. burgdorferi sensu lato isolates were determined. The predicted DbpA sequences were fairly heterogeneous among the isolates (58.3 to 100% similarity), but DbpA sequences with the highest similarity tended to group into species previously defined by well-characterized chromosomal markers. In contrast, the predicted DbpB sequences were highly conserved (96.3 to 100% similarity). Substantial diversity in DbpA sequence was seen among isolates previously shown to be killed by antiserum against a single DbpA, suggesting that one or more conserved protective epitopes are composed of noncontiguous amino acids. The observation of individual dbpA alleles with sequence elements characteristic of more than one B. burgdorferi sensu lato species was consistent with a role for genetic recombination in the generation of dbpA diversity.     

Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice

A Borrelia burgdorferi N40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, of B. burgdorferi 297 and B31. N40-dbpA DNA hybridized with B. burgdorferi N40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting from B. burgdorferi sensu stricto N40 and B31, and from B. burgdorferi sensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than against B. burgdorferi N40 recombinant P39, OspC, or OspA. Sera from mice infected with B. burgdorferi sensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected with B. burgdorferi N40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with cultured B. burgdorferi N40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout the B. burgdorferi sensu lato family, but they are likely to be heterogeneous.     

Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.     

Nursing intervention to increase hope and quality of life in newly diagnosed cancer patients

A DNA library of strain HB19 from Borrelia burgdorferi sensu stricto, an agent of Lyme borreliosis, was constructed in the cosmid pLA2917. Genes involved in initiation of DNA replication and resolution of recombination intermediates (Holliday junctions) were found on a 23-kbp region up to 0.7 kbp of the "left" extremity of the linear chromosome in representative species of B. burgdorferi sensu lato. The potential ruvB gene, located at 22 kbp from the left telomere, was identified by the similarity of its deduced amino acid sequence to RuvB (helicases) of other bacteria. B. burgdorferi ruvB is part of an operon which comprises the homologues of ruvA, queA and pfbB. Expression of the B. burgdorferi ruvB and ruvA genes renders a wild-type Escherichia coli sensitive to UV light and mitomycin, indicative of negative complementation. priA, which encodes the potential recognition factor for the primosome assembly site, was found at 15 kbp from the left telomere. RuvB and PriA sequences have motifs characteristic of helicases: a DExH box and an ATP binding site.     

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.     

Genospecies identification and characterization of Lyme disease spirochetes of genospecies Borrelia burgdorferi sensu lato isolated from rodents in Taiwan

Lyme disease spirochetes of the genospecies Borrelia burgdorferi sensu lato were identified and characterized for the first time in Taiwan. Seven isolates, designated TWKM1 to TWKM7, were purified from the ear tissues of three species of rodents captured from seven localities of Taiwan. The immunological characteristics of these Taiwan isolates were compared with those of other genospecies of Lyme disease spirochetes by analyzing the protein profiles and reactivities with B. burgdorferi-specific monoclonal antibodies (MAbs). The genospecies of these Taiwan isolates were also identified by the similarities in their plasmid profiles and differential reactivities with genospecies-specific PCR primers. Although two distinct protein profiles were observed among the seven Taiwan isolates, the MAb reactivities against the outer surface proteins of B. burgdorferi of all of these isolates were consistent with those of B. burgdorferi sensu lato. The similarities of the plasmid profiles also confirmed the identities of these Taiwan isolates. PCR analysis indicated that all of these Taiwan isolates were genetically related to the genospecies B. burgdorferi sensu stricto. These results demonstrate the first identification of Lyme disease spirochetes in Taiwan and also highlight the increasing demand for defining the reservoirs and vector ticks of B. burgdorferi. A serosurvey for Lyme disease infection in the human population of Taiwan may also be required.     

Clinical guidelines and the law: advice, guidance or regulation?

A broth microdilution assay was performed to determine the minimal inhibitory concentrations (MICs) of 25 antimicrobial agents for two strains of Borrelia (B.) burgdorferi sensu lato and one strain of B. hermsii. The method comprised BSK II medium lacking gelatin and an incubation period of 72 hours. To investigate the influence of reading mode and density of inoculum on MIC values, microscopical as well as macroscopical MIC reading was performed using standardized final inocula of 10(6) and 10(7) borreliae/ml. Data were processed by two-way analysis of variance. In the microdilution assay, MIC values were significantly influenced either by the inoculum density or reading mode. However, using clearly defined criteria for macroscopical endpoint determination, MICs from macroscopical and microscopical reading were found to be in close agreement. B. burgdorferi sensu lato strains tested were highly susceptible to azithromycin, erythromycin, mezlocillin, piperacillin as well as ceftriaxone, with MICs ranging from < or = 0.016 to 0.125 microgram/ml. B. hermsii was highly susceptible to azithromycin and erythromycin. In comparison to B. hermsii, the beta-lactam antibiotics revealed a significantly higher activity and gentamicin, ofloxacin, and rifampin revealed a significantly lower activity against B. burgdorferi sensu lato strains. To further investigate interactions between BSK II medium, incubation time, and antibiotic efficacy, an agar diffusion bioassay was performed. Out of seven antibiotics tested, the activities of mezlocillin, penicillin G, and piperacillin were significantly influenced by BSK II medium and incubation period and showed a marked decrease of on average 84.0% within 72 hours of incubation.     

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.     

Integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

Individual pharmacodynamics assessed by antilymphocyte action predicts clinical cyclosporine efficacy in psoriasis

The vector competence of 2 tick species, Ixodes ricinus (L.) and Ixodes scapularis Say, was determined and compared for 3 genospecies of Borrelia burgdorferi. The 3 genospecies of B. burgdorferi used in the following experiments were Borrelia burgdorferi sensu stricto (B-31 and B-31.D1 clone), Borrelia afzelii (strain Pgau. C3), and Borrelia garinii (strain VS286 and VSBP). Spirochetes from all 5 strains were inoculated intradermally into outbred mice; larval ticks of both species were subsequently fed on those mice and replete larvae were assayed for infection by culture in BSK-H media every 7 d for 4 wk. Infection frequencies in I. scapularis exposed to the 5 strains were as follows: B-31 (90%), B-31.D1 (83%), Pgau.C3 (87%), VS286 (10%), and VSBP (5%). The comparable infection frequencies for I. ricinus were B-31 (3%), B-31.D1 (3%), Pgau.C3 (90%), VS286 (5%), and VSBP (3%). Resultant nymphal I. scapularis successfully transmitted B-31, B-31,D1, Pgau.C3, and VS286 to outbred mice. I. ricinus nymphs transmitted Pgau.C3 and VS286. Both species failed to transmit strain VSBP.     

Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.     

Screening and chemoprophylaxis for tuberculosis infection in college populations

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragment length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe.     

Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein: implications for taxonomy and for use of OspC protein as a diagnostic antigen

The nucleotide sequences of the ospC gene from five Danish human Borrelia burgdorferi isolates representing all three B. burgdorferi genospecies (B. burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461) and from the American type strain B31 were determined and compared with the published ospC sequence from the German B. burgdorferi isolate PKo (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). The ospC gene was present in all isolates, regardless of the presence or absence of its product, OspC. The deduced amino acid sequences of OspC from the seven isolates were aligned and revealed pairwise sequence identities ranging from 60.5 to 100%. Differences were scattered throughout the amino acid sequences. A phylogenetic tree was constructed and revealed three distinct phenotypic groups OspCI to OspCIII corresponding to the three delineated genospecies. Immunoblot analysis revealed that the seven OspC proteins tested have both common and specific epitopes. There is significant epitope diversity, since even polyclonal antisera showed serotype-restricted specificity. Therefore, a serodiagnostic assay for Lyme borreliosis utilizing OspC as a test antigen should include all three OspC phenotypes in order to obtain a species-wide sensitivity.