Genetic Heterogeneity of Borrelia burgdorferi Sensu Lato in the Southern United States Based on Restriction Fragment Length Polymorphism and Sequence Analysis

Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.     

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.     

A bactericidal antibody to Borrelia burgdorferi is directed against a variable region of the OspB protein.

Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.     

Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains.

The relationship between plasmid profiles and genospecies of the Lyme disease borreliae was investigated by using 40 strains from diverse biological and geographical sources. The genospecies of the strains were determined by examination of rRNA gene restriction patterns with cDNA probes complementary to the 16S and 23S rRNAs of Escherichia coli. Plasmid profiles were obtained by pulsed-field gel electrophoresis. The number of plasmids per strain and the size of these plasmids ranged from 4 to 10 and from 13.3 to 57.7 kb, respectively. The strains all contained a single large plasmid of 50 to 57.7 kb, with the exception of two Borrelia garinii strains that contained two or three of the large plasmids. The large plasmids of Borrelia burgdorferi sensu stricto strains ranged in size from 51.4 to 52.7 kb and were consistently smaller than the 54.0- to 57.7-kb plasmids present in B. garinii and Borrelia afzelii. The exceptions of this observation were the two B. garinii strains with multiple large plasmids; in this case the large plasmids were 50.6 to 53 kb. Although a large degree of heterogeneity in the sizes and frequencies of occurrence of smaller plasmids was observed, there were some differences among the three genospecies. The differences in plasmids were further studied by using two BamHI DNA fragments from a 28.7-kb plasmid of B. burgdorferi sensu stricto 297 as probes. Both probes hybridized with the 27- to 29-kb plasmids of B. burgdorferi sensu stricto strains. In contrast, two patterns of hybridization were observed with B. garinii and B. afzelii.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diversity of the outer surface protein C gene of southern Borrelia isolates and its possible epidemiological,clinical, and pathogenetic implications

The ospC genes of 20 southern Borrelia strains were sequenced. The strains consisted of B. burgdorferi sensu stricto, B. andersonii, B. bissettii, one undescribed genospecies, MI-8, and one probably new Borrelia species, TXW-1. A high degree of similarity exists between B. burgdorferi sensu stricto and B. bissettii and between B. bissettii and B. andersonii. Lateral transfers of the ospC gene probably occurred between B. burgdorferi sensu stricto and B. bissettii and between B. bissettii and B. andersonii. Internal gene recombination appears to occur among them. The highest degree of genetic diversity among them was observed in the two variable domains (V1 and V2), semivariable domain (SV), and the species-specific epitopes (between amino acids 28 and 31). Differences in ospC sequences among southern strains reflect diversity at the strain and genospecies levels. MI-8, which was recognized as an undescribed genospecies in our previous reports, remains distinguishable in our current analysis of ospC genes and is distinct from B. burgdorferi sensu stricto. Interestingly, another undescribed southern isolate, TXW-1, was not amplified under various PCR conditions. Compared to European B. burgdorferi sensu stricto strains, American B. burgdorferi sensu stricto strains show greater genetic heterogeneity. Southern B. burgdorferi sensu stricto, B. andersonii, and B. bissettii isolates were intermixed with each other in the phylogenetic trees. In the derived trees in our work, at least one southeastern strain of B. burgdorferi, MI-2, most closely aligns with a so-called invasive cluster that possesses many proven human-invasive strains. Transmission experiments show that MI-2 and the strains in this group of southern spirochetes are able to infect mice and hamsters and that the typical vector of Lyme disease, Ixodes scapularis, can acquire the spirochetes from infected mammals. Currently, strain MI-2 appears to be the only southern isolate among the 20 we analyzed that clusters with an OspC invasive group and thus might be invasive for humans.     

Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal

To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu lato isolated from Ixodes ricinus ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of B. burgdorferi sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu lato from this region, 55 I. ricinus and 27 Hyalomma marginatum questing adults, collected during the spring of 1998 from a sylvatic habitat south of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in I. ricinus ticks and 7% in H. marginatum ticks were detected by a nested PCR that targets the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the I. ricinus-derived amplicons showed that the sequences in the majority of samples were similar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of rrf-rrl spacer sequences revealed a diverse population of B. lusitaniae in questing adult I. ricinus ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were present in the local I. ricinus population. We hypothesize that B. lusitaniae has a narrow ecological niche that involves host species restricted to the Mediterranean Basin.     

DNA microarray assessment of putative Borrelia burgdorferi lipoprotein genes

A DNA microarray containing fragments of 137 Borrelia burgdorferi B31 putative lipoprotein genes was used to examine Lyme disease spirochetes. DNA from B. burgdorferi sensu stricto B31, 297, and N40; Borrelia garinii IP90; and Borrelia afzelii P/Gau was fluorescently labeled and hybridized to the microarray, demonstrating the degree to which the individual putative lipoprotein genes were conserved among the genospecies. These data show that a DNA microarray can globally examine the genes encoding B. burgdorferi lipoproteins.     

Genetic analysis of the outer surface protein C gene of Lyme disease spirochaetes (Borrelia burgdorferi sensu lato) isolated from rodents in Taiwan

The outer surface protein C gene (ospC) of Lyme disease spirochaetes (Borrelia burgdorferi sensu lato) was analysed for the first time in Taiwan. The genetic identities of these Taiwan isolates (TWKM1-7) were determined by restriction fragment length polymorphism (RFLP) analysis and sequence similarities of the PCR-amplified ospC gene amplicons. After cleavage by nuclease Dral, differential fragment patterns of PCR-amplified ospC DNA in relation to different genospecies of Lyme disease spirochaetes were observed and all of these Taiwan isolates were genetically affiliated to the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis on the sequence similarity of these Taiwan isolates revealed a highly homogeneous genotype, ranging from 99.3% to 100%, within the genospecies of B. burgdorferi sensu stricto and was distinguished from other genospecies of Borrelia isolates. The sequence similarity analysis also revealed the high sequence variability of the ospC gene among Borrelia strains that belong to the same genospecies but were isolated from different biological and geographical sources. Thus, these results provide the first investigation on the genetic identity of the ospC gene of these Taiwan isolates and show that these Taiwan isolates were closely related genetically to the genospecies of B. burgdorferi sensu stricto.     

In vitro susceptibility testing of four antibiotics against Borrelia burgdorferi: a comparison of results for the three genospecies Borrelia afzelii,Borrelia garinii,and Borrelia burgdorferi sensu stricto

MICs and minimal bactericidal concentrations (MBCs) were evaluated for the four antibiotics azithromycin, amoxicillin, ceftriaxone, and doxycycline against the three main genospecies of Borrelia burgdorferi sensu lato. In MBC testing, statistically significant differences between the genospecies could be found in 7 out of 12 comparative evaluations (P < 0.05).     

中国莱姆病螺旋体的核糖体基因分型研究

目的 莱姆病螺旋体的基因型和临床表现、疫苗菌株和抗原菌株的选择存在密切的关系，所以对中国菌株进行分子流行病学研究，可为莱姆病的防治提供科学依据。方法 5S－23S rRNA基因间隔区RFLP分析和16＋23S rRNA基因RFLP分析。结果 中国菌株至少被分为3个基因型（B.burgdorferi sensu stricto、B.garinii和B.afzelii),B.garinii和B.afzelii占优势，B.burgdorferi sensu stricto少见。少数菌株用上述方法尚不能明确其分类地位，需进一步研究，中国很可4能存在世界上独特的新基因型。结论 中国菌株的基因型明显不同于北美菌株，而同欧洲菌株比较接近，5S－23S rRNA基因间隔区RFLP分析方法简便、快速、准确，是理想的基因型分类方法，可作为国内菌株基因型鉴定的常规方法。     

Protection elicited by native outer membrane protein Oms66 (p66) against host-adapted Borrelia burgdorferi: conformational nature of bactericidal epitopes

Oms66 is a Borrelia burgdorferi outer membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection with B. burgdorferi (IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using "host-adapted" B. burgdorferi B31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.     

Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs.

Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs. Adsorption studies confirmed that the antibodies were specific for B. burgdorferi. Experimentally exposed dogs were asymptomatic. Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease. Naturally exposed dogs were from four geographic regions of the country. No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country. The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity to fewer and different protein bands. Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not. Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for the observed differences.     

Borrelia isolates in Northern Colorado identified as Borrelia bissettii

Previous work described Borrelia burgdorferi sensu lato group DN127 as a new genospecies, Borrelia bissettii, and prompted the present study to identify the Borrelia spp. that exist in northern Colorado. To determine the genospecies present, we analyzed two specific intergenic spacer regions located between the 5S and 23S and the 16S and 23S ribosomal genes. Phylogenetic analysis of the derived sequences clearly demonstrated that these isolates, originating from rodents captured in the foothills of northern Colorado, diverged from B. burgdorferi sensu stricto by 5 to 5.5% and were members of the new genospecies B. bissettii.     

Identification and characterization of 31 isolates of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> (Spirochaetales,Spirochaetaceae) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes ricinus (L., 1758) ticks. The aim of our study was to identify spirochaete isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their characterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infraorder Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.     

Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi.

Twenty-two specific-pathogen-free beagles were vaccinated with recombinant OspA (ospA gene derived from Borrelia burgdorferi B31) alone or with adjuvant (QuilA, Montanide ISA25, or aluminum hydroxide) at 6 weeks of age. Thirteen dogs were used as nonvaccinated controls with or without adjuvant. Three dogs were kept as contact controls and received neither vaccine nor challenge. Six weeks or 6 months after the first vaccination, the vaccinated (20 of 22) and nonvaccinated dogs (13) were challenged by exposure to adult ticks (Ixodes scapularis) naturally that were infected with B. burgdorferi (tick infection rate, > or = 60%) and that were collected from Westchester County, N.Y. Protection from infection was evaluated by culture for B. burgdorferi from skin biopsies taken near the sites of tick bites. Skin biopsies were taken at monthly intervals for 3 months. B. burgdorferi was not isolated from any of the vaccinated dogs. In contrast, 12 of 13 control dogs challenged by exposure to the ticks were culture positive. The histopathology of the joint capsules 3 months after the challenge was used to evaluate protection from arthritis. Eight of 13 control dogs showed synovitis in single or multiple joints, while only 1 of the 22 vaccinated dogs had a single focus of mild inflammation in a single joint. At the time of the challenge, the vaccinated dogs had antibody to B. burgdorferi, which was demonstrable by kinetic enzyme-linked immunosorbent assay, Western blotting (immunoblotting), and a serum growth inhibition assay. The vaccinal antibody declined gradually after the challenge, especially in dogs vaccinated with OspA without adjuvants. Antibodies in the challenge control dogs were only detectable by 4 to 6 weeks after the challenge and remained at high levels until the termination of the study. Contact control dogs showed no antibody responses or histopathologic lesions and were culture negative. By Western blot analysis, antibodies to OspA first appeared in the sera of vaccinated dogs 3 weeks after the first vaccination. The absence of additional bands after the challenge suggests that infection in vaccinated dogs was blocked. Results from this study show that vaccination with recombinant OspA protected dogs against infection and disease after an experimental challenge with B. burgdorferi by exposure to ticks.     

Differences of two Borrelia burgdorferi strains in complement activation and serum resistance.

Complement activation and serum resistance of the Borrelia burgdorferi strains B31 (American strain) and PKo (European strain) were compared. In 25% (v/v) normal human serum (NHS) free of B. burgdorferi-specific antibodies the cells of the PKo strain were high activators of complement as indicated by rapid and strong C9 consumption, by deposition of up to 336763 C9 molecules per cell and by the formation of the terminal complement complex on the cell surface. By comparison, complement activation by the B31 strain was low with 5.4-fold less C9 deposited per cell. The addition of B. burgdorferi-specific antibodies to NHS either as purified IgG or heat-inactivated patient sera, had no influence on the results with both strains. After an incubation period of 2h at 37 degrees C in 25% (v/v) NHS most cells of the PKo strain had lost their viability as indicated by cell immobilization and failure to multiply in subcultures. In addition, extensive cell fragmentation and bleb formation were observed in the electron microscope. In contrast, the B31 strain remained alive and morphologically intact after the same incubation with NHS. We conclude from our results that complement activation and serum resistance are properties which differ considerably between isolated strains of B. burgdorferi.     

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.     

Comparative Analysis and Immunological Characterization of the Borrelia Bdr Protein Family

Multiple circular and linear plasmids of Lyme disease and relapsing fever Borrelia spirochetes carry genes for members of the Bdr (Borrelia direct repeat) protein family. To define their common and divergent attributes, we first comprehensively compared the known homologs. Bdr proteins with predicted sizes ranging from 10.7 to 30. 6 kDa formed five homology groups, based on variable numbers of short direct repeats in a central domain and diverse N- and C-terminal domains. In a further characterization, Western blots were probed with rabbit antisera raised against either of two purified recombinant Bdr proteins from Borrelia burgdorferi B31. The results showed that antibodies cross-react and several Bdr paralogs 19.5 to 30.5 kDa in size are expressed by cultured strain B31 in a temperature-independent manner. In situ proteolysis, immunofluorescence, and growth inhibition assays indicated that Bdr proteins are not surface exposed. Distinct patterns of cross-reacting proteins of 17.5 to 33 kDa were also detected in other B. burgdorferi, Borrelia garinii, and Borrelia afzelii strains as well as in relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae. Last, we examined whether these proteins are antibody targets during Lyme disease. Analysis of 47 Lyme disease patient sera by immunoblotting and enzyme-linked immunosorbent assays showed that 24 (51%) and 20 (43%), respectively, had detectable antibodies to one or more of the Bdr proteins. Together, these data indicate that Bdr proteins constitute a family of cross-reactive Borrelia proteins which are expressed in the course of Lyme disease and in vitro.     

Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients.

Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis.     

Antigenic properties of Borrelia burgdorferi isolated from Ixodes ovatus and Ixodes persulcatus in Hokkaido, Japan.

Spirochete strains HP3 and HO14, isolated from Ixodes persulcatus and I. ovatus in Hokkaido in 1989, were the first isolates of Borrelia burgdorferi, the etiological agent of Lyme disease, to be recognized in Japan. Antigenic properties of the Japanese strains were compared with those of the strains isolated in the United States (B31 and 297) and Europe (IRS, P/Gau, P/Bi, 2/B45, and 3/B56) by Western blotting (immunoblotting), by using monoclonal antibodies (MAbs) against strains B31 and P/Bi. The Japanese strains reacted with MAb U40 against the 41-kDa antigen. MAb E34a31 against Osp A reacted with all the strains tested except for strain HP3. Furthermore, MAb U31b against Osp A reacted with all the American and European strains but did not react with the Japanese strains. When MAbs against Osp B were used, MAb E34b reacted only with European strains and MAb U34b reacted only with the American strains. However, neither showed reactivity to two Japanese strains. MAb E60 against 60-kDa antigen reacted with all the U.S. and European strains and strain HP3 but did not react with Japanese strain HO14. These results indicate that the antigenicity of the Japanese strains isolated from two species of ixodid ticks is different from that of the strains isolated in the United States and Europe. It is suggested that the Japanese strains are much more suitable than the U.S. or European strains as the antigen source for the serodiagnosis of Lyme disease in Japan.