Serologic analyses of cottontail rabbits for antibodies to Borrelia burgdorferi.

An enzyme-linked immunosorbent assay was developed to detect antibodies to Borrelia burgdorferi in cottontail rabbits captured in Millbrook, N.Y., and New York, N.Y. Five antigenically variable strains of B. burgdorferi were analyzed to determine the variability of serologic test results. In analyses of 79 serum samples, seropositivity ranged from 56% for a strain cultured from kidney tissues of a cottontail rabbit to 68% for a strain isolated from a larva of Ixodes dentatus, a tick that parasitized a cottontail rabbit. There were false-positive results when reference rabbit antisera to B. hermsii and Treponema pallidum were screened against B. burgdorferi. Cross-reactivity with antisera to Leptospira interrogans serovars was less pronounced. Western blot (immunoblot) analyses revealed reactivities of test sera to two or more surface or subsurface proteins of B. burgdorferi with approximate molecular masses of 18, 25 to 27, 34, 36, 41, and 59 kilodaltons. Cottontail rabbits respond immunologically to B. burgdorferi, but the observed variations in serologic test results should not be a limitation in field and laboratory investigations of Lyme borreliosis.     

Comparison of whole-cell antibodies and an antigenic flagellar epitope of Borrelia burgdorferi in serologic tests for diagnosis of Lyme borreliosis.

A recombinant protein (p41-G) of an antigenic region of flagellin was used in a standard and amplified enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Comparable sensitivities (88 to 94%) were noted when sera from 17 persons who had erythema migrans and antibodies to whole-cell B. burgdorferi were tested against the p41-G antigen. In tests of a second study group of 36 persons who had erythema migrans but no detectable antibodies to whole-cell B. burgdorferi, 3 (8%) were positive when the p41-G antigen was used. Assay specificity likewise increased when the p41-G fragment was included in an ELISA with human sera containing treponemal antibodies. Recombinant flagellar proteins of B. burgdorferi, such as the p41-G antigen, can be used in an ELISA and may help confirm Lyme borreliosis during early stages of infection and improve specificity.     

Serosurvey for antibodies to Borrelia burgdorferi in Danish dogs

To estimate the regional presence of B. burgdorferi we performed a serosurvey for antibodies to B. burgdorferi in 205 healthy dogs from representative areas of Denmark. Blood samples were collected from November 1986 to March 1987. Twenty dogs bred for research purposes served as negative controls. IgG-antibodies to the B. burgdorferi strain DK-ECM 1 were measured by indirect immunofluorescens assay. Antibody titers ranged from nonreactive to 640. In 33 dogs (16.1%) titres were greater than or equal to 80, indicating exposure to B. burgdorferi. There were no significant differences in the prevalence rate of seropositive dogs due to place of residence, sex or age. The results indicate that dogs are exposed to tickborne B. burgdorferi in most areas of Denmark.     

Serologic analysis of white-tailed deer sera for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and western immunoblotting.

White-tailed deer serum samples were collected in the Minneapolis-St. Paul, Minn., metropolitan area during the fall and winter months from 1989 to 1992 and analyzed for antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. Ninety-eight percent of the serum samples were collected from regions where currently the vector tick, Ixodes dammini, is nonexistent. Antibodies to B. burgdorferi were detected in 2.2% of 508 samples by enzyme-linked immunosorbent assay, and their presence was confirmed by Western immunoblot analysis. Western immunoblotting yielded mean numbers of reactive bands of 0.1 and 6.0 for samples that were negative and positive for antibodies by enzyme-linked immunosorbent assay, respectively. The molecular weights of the antigens in many of the reactive bands from positive samples were similar to the molecular weights of antigens reactive with samples from humans with Lyme borreliosis. An antibody response to the major outer surface proteins A and B was not detected. Serologic analysis of deer sera may provide a valuable method for surveillance programs designed to monitor the spread of B. burgdorferi in nature.     

Serologic proteome analysis of Borrelia burgdorferi membrane-associated proteins

Lyme disease, a global health concern, is caused by infection with Borrelia burgdorferi, B. afzelii, or B. garinii. The spirochete responsible for the disease in the United States is B. burgdorferi and is spread by the bite of an infected Ixodes tick. We utilized multiple two-dimensional gel techniques combined with proteomics to reveal the full humoral immune response of mice and Lyme patients to membrane-associated proteins isolated from Borrelia burgdorferi. Our studies indicated that a subset of immunogenic membrane-associated proteins (some new and some previously identified) was recognized by mice experimentally infected with Borrelia burgdorferi either by low-dose needle inoculation or by tick infestation. Moreover, the majority of these immunogenic membrane-associated proteins were recognized by sera from patients diagnosed with early-disseminated Lyme disease. These included RevA, ErpA, ErpP, DbpA, BmpA, FtsZ, ErpB, LA7, OppA I, OppA II, OppA IV, FlhF, BBA64, BBA66, and BB0323. Some immunogens (i.e., BBI36/38) were more reactive with sera from mice than Lyme patients, while additional membrane proteins (i.e., FlaB, P66, LA7, and Hsp90) were recognized more strongly with sera from patients diagnosed with early-localized, early-disseminated, or late (chronic)-stage Lyme disease. We were able to examine the humoral response in Lyme patients in a temporal fashion and to identify the majority of immunoreactive proteins as the disease progresses from early to late stages. This serologic proteome analysis enabled the identification of novel membrane-associated proteins that may serve as new diagnostic markers and, more importantly, as second-generation vaccine candidates for protection against Lyme disease.     

Mapping the major antigenic domains of the native flagellar antigen of Borrelia burgdorferi.

Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a monoclonal antibody raised by immunization of mice with purified flagella and pooled sera from patients with multiple erythema migrans, late Lyme borreliosis, or secondary syphilis were analyzed by Western blots (immunoblots) of peptides transferred to Immobilon polyvinylidene difluoride filters. The major epitope binding one murine monoclonal antibody (158) was localized to a carboxy-terminal domain that includes residues 300 to 336. The dominant epitopes binding human polyclonal antibodies are in the central portion of the molecule (residues 182 to 218) that is not conserved compared with other bacterial flagellins. Additional reactive epitopes were identified in the amino-terminal domain of the protein. Sera from patients with syphilis bound strongly to the amino-terminal conserved domain, providing a structural basis for cross-reactivity seen in standard enzyme-linked immunosorbent assays, but not to the central part of the molecule. Specific and cross-reactive antigenic determinants need to be considered in the design of improved immunodiagnostics for spirochetal diseases.     

Antigenically variable Borrelia burgdorferi isolated from cottontail rabbits and Ixodes dentatus in rural and urban areas.

Spirochetes were isolated from 71 subadult Ixodes dentatus removed from cottontail rabbits captured in Millbrook, N.Y., and in New York, N.Y. Spirochetes were also cultured from kidney tissues of six rabbits. While all isolates reacted with monoclonal antibody H9724, which identifies the spirochetes as borreliae, more than half did not bind with antibody H5332 and even fewer reacted with H3TS, both of which were produced to outer surface protein A of Borrelia burgdorferi. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of three isolates differed from one another and from all previously characterized B. burgdorferi strains from humans, ticks, and wildlife in North America. The 12 periplasmic flagella that originated subterminally from each pointed end of a rabbit Borellia isolate contrasted with the 11 or fewer flagella for B. burgdorferi reported previously from North America. Although DNA homology and restriction endonuclease analysis also revealed differences among a rabbit kidney isolate, an I. dentatus isolate, and B. burgdorferi B31, similarities were sufficient to lead us to conclude that the borreliae in rabbits and I. dentatus are B. burgdorferi. Enzyme-linked immunosorbent assay titers of sera from humans with diagnosed Lyme disease to rabbit tick B. burgdorferi were often similar to one another and to those recorded for a reference B. burgdorferi strain.     

Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi.

A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease. Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins. The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266). However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein. The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease. The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen.     

Prognostic B-cell epitopes on the flagellar protein of Borrelia burgdorferi.

Overlapping decapeptides based on the flagellin sequence of Borrelia burgdorferi B31 (G. S. Gassmann, M. Kramer, U. B. G?bel, and R. Wallich, Nucleic Acids Res. 17:3590, 1989) were used to identify immunologically reactive regions of flagellin. Five serum specimens from patients with late manifestations of Lyme disease and borrelia-specific monoclonal antibody H9724 reacted with an epitope in the central region of the flagellar protein (amino acids 205 to 226), which is heterologous to the amino acid sequences of other bacterial flagellins. This epitope was not recognized by human sera with antibodies to Treponema pallidum, sera of healthy individuals, or sera from patients with stage I or II of Lyme disease.     

Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.     

Serologic analyses of Peromyscus leucopus, a rodent reservoir for Borrelia burgdorferi, in northeastern United States.

An enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent-antibody test were used to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme disease, in Peromyscus leucopus (white-footed mouse). Of the 661 mice captured in Connecticut, Rhode Island, and New York during 1980 and 1983 to 1987, 166 (25.1%) had antibodies to B. burgdorferi by ELISA. Comparative analyses of 210 serum specimens, collected in areas where Lyme disease is endemic, revealed a threefold difference in sensitivity between the ELISA (38.1% positive) and the indirect fluorescent-antibody method (12.4%). Although prevalence of seropositive P. leucopus was highest during June, elevated amounts of antibody (1:1,280 to 1:2,560) were detected in mice that harbored spirochetes during all seasons. Being reservoirs for B. burgdorferi, these rodents are suitable for monitoring spirochete infections at foci and should be included in field evaluations of control programs aimed at suppressing Lyme disease.     

Protein and antigenic analysis of Borrelia burgdorferi isolated in northern Italy: computerized analysis of phenotypic characteristics.

Four Borrelia burgdorferi strains isolated in the same restricted geographic area share different protein patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The use of polyclonal rabbit antisera and a battery of monoclonal antibodies directed toward the immunodominant proteins OspA, OspB, and pC also revealed different epitope distributions and specificities of these antigens on the strains examined. For the first time, a computerized analysis of these phenotypic characters was done mainly by cluster analysis. The computerized analysis revealed the levels of similarities among the strains and indicated, on a quantitative basis, that one of them is much closer to the American strain B31 than to the other strains.     

Selection of an escape variant of Borrelia burgdorferi by use of bactericidal monoclonal antibodies to OspB.

Two immunoglobulin G (IgG) monoclonal antibodies (MAbs) to outer surface protein B (CB2 and CB6), affinity purified from mouse ascitic fluid, exhibited concentration-dependent inhibitory and bactericidal properties against Borrelia burgdorferi after a 24-h incubation period in spirochete medium. Fab fragments derived from these MAbs showed the same effects, indicating that they were not caused by agglutination of the organisms by the intact MAbs. The inhibition of spirochete growth in cultures containing MAbs was also detected by spectrophotometric analysis of the media. CB2 did not inhibit the growth of Borrelia hermsii or the BEP4 strain of B. burgdorferi, neither of which is recognized by the MAb. Affinity-purified IgG from hybridoma supernatants had similar effects on B. burgdorferi as the ascitic-fluid-derived IgG did, indicating that the inhibitory and bactericidal properties were not due to nonspecific toxic contaminants. The bactericidal properties of the MAbs were not complement dependent as there was none in the serum-free system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of B. burgdorferi organisms surviving after exposure to CB2 revealed an escape variant which failed to express OspB. The continued presence of OspA in these escape variants indicates that the lack of OspB was not due to the loss of the plasmid which contains the genes for both of these proteins.     

Temporal analysis of the antigenic composition of Borrelia burgdorferi during infection in rabbit skin

The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.     

Borrelia burgdorferi in an urban environment: white-tailed deer with infected ticks and antibodies.

Ticks and blood samples were collected from white-tailed deer (Odocoileus virginianus) in forests located in an insular, urban area of Bridgeport, Conn., and in rural south central Connecticut during 1992 and 1993. Immature and adult Ixodes scapularis ticks were tested for Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, by indirect fluorescent-antibody staining methods. Deer sera were analyzed for antibodies to this bacterium by an enzyme-linked immunosorbent assay. Infected ticks parasitized deer in Bridgeport from May through December; the prevalence of infection varied from 1.1% of 93 larvae to 28.1% of 114 adult females. The percentages of infected males (10.5% of 380 ticks) and females (13.7% of 328 ticks) were relatively lower in south central Connecticut. In antibody tests, the prevalence of seropositive specimens collected in Bridgeport (61% of 146 serum specimens) was more than twofold greater than that of specimens obtained in south central Connecticut (26.7% of 116 serum specimens). Foci for Lyme borreliosis can occur in forested, urban settings as well as in rural areas if there are ticks, rodents, birds, and large mammals present. Human exposure to ticks in such sites should be considered as a possible source of B. burgdorferi infection.     

Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent.

A simple procedure for extraction of plasmid-enriched DNA from borreliae was used in a plasmid analysis of 13 strains of the Lyme disease agent, Borrelia burgdorferi. The extracted DNA was subjected to low-percentage agarose gel electrophoresis and examined either directly by ethidium bromide staining or after hybridization of the plasmids in situ with a DNA probe for the gene encoding the major outer membrane protein OspA. Each isolate had four to seven discernible plasmids of various sizes. Only 2 of the 13 strains had the same plasmid profile. The ospA gene probe hybridized to large plasmids to strains from both North America and Europe. A strain which had been passaged many times was found to have lost two of the six plasmids originally present. These findings indicate the potential usefulness of plasmid analysis as a strain-typing procedure and for identifying possible plasmid-conferred virulence factors.     

Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease.

The isolation of Borrelia burgdorferi flagella and an enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G (IgG) and IgM to the B. burgdorferi flagellum are described. The diagnostic performance of the flagellum ELISA for serodiagnosis of Lyme disease was compared with the performance of a traditional whole cell B. burgdorferi sonic extract ELISA. We examined sera and cerebrospinal fluid (CSF) from 56 patients with lymphocytic meningoradiculitis (Bannwarth's syndrome), the most frequent secondary-stage manifestation of Lyme disease in Europe. Two hundred healthy individuals and patients with aseptic meningitis, encephalitis, Guillain-Barré syndrome, and syphilis served as controls. The flagellum ELISA was significantly more sensitive than the sonic extract ELISA. The diagnostic sensitivities were increased from 41.1 to 76.8% (P less than 0.01) for IgG and from 35.7 to 67.9% (P less than 0.05) for IgM detection in serum. The increase in sensitivity was most pronounced in patients with a short duration of disease (less than 20 days after onset). The diagnostic specificity increased for IgG detection but was almost unaltered for IgM. The flagellum ELISA did not improve the diagnostic sensitivity of measuring antibodies to borreliae in CSF, most likely owing to the low level of unspecific antibodies in CSF compared with serum. The cross-reactivity of sera and CSF from patients with syphilis decreased significantly. The flagellum antigen of B. burgdorferi shows no strain variation, is easy to purify in sufficient quantity, and is therefore a suitable reference antigen for routine serodiagnosis of Lyme disease.     

An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.     

Interactions between extracellular Borrelia burgdorferi proteins and non-Borrelia-directed immunoglobulin M antibodies.

Previous work showed that outer surface protein A (OspA) and OspB of Borrelia burgdorferi may occur within an extracellular multiprotein complex, which was resolved by electrophoresis as an 83-kDa major extracellular protein band. To characterize the 83-kDa band, we sequenced the N terminus of the predominant peptide in the band and examined the interaction between the associated proteins. Peptide sequence and amino acid composition comparisons showed identity with the heavy chain of immunoglobulin M (IgM). Reduction sensitivity experiments and the recognition of the band by antibodies specific for rabbit mu chain indicated that the multiprotein complex contained pentameric IgM. Immunoelectron microscopy showed that anti-mu chain antibodies and monoclonal antibodies to OspA and OspB bound to extracellular amorphous material surrounding cells. Furthermore, the Osps coprecipitated with either nonspecific polyclonal rabbit IgM antibodies or with murine monoclonal anti-human serum albumin IgM antibodies, using insoluble anti-mu chain antibody conjugates. Although the apparent 83-kDa complex was stable under conditions of chelation and concentrated salts, it was disrupted by treatment with neuraminidase. These results indicate that extracellular B. burgdorferi proteins, including OspA and OspB, interact with IgM. The association is apparently not a classic antibody-antigen interaction but may result from other mechanisms.     

Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment.

In specific-pathogen-free dogs experimentally infected with Borrelia burgdorferi by tick exposure, treatment with high doses of amoxicillin or doxycycline for 30 days diminished but failed to eliminate persistent infection. Although joint disease was prevented or cured in five of five amoxicillin- and five of six doxycycline-treated dogs, skin punch biopsies and multiple tissues from necropsy samples remained PCR positive and B. burgdorferi was isolated from one amoxicillin- and two doxycycline-treated dogs following antibiotic treatment. In contrast, B. burgdorferi was isolated from six of six untreated infected control dogs and joint lesions were found in four of these six dogs. Serum antibody levels to B. burgdorferi in all dogs declined after antibiotic treatment. Negative antibody levels were reached in four of six doxycycline- and four of six amoxicillin-treated dogs. However, in dogs that were kept in isolation for 6 months after antibiotic treatment was discontinued, antibody levels began to rise again, presumably in response to proliferation of the surviving pool of spirochetes. Antibody levels in untreated infected control dogs remained high.