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.     

Vaccination with the ospA- and ospB-Negative Borrelia burgdorferi Strain 50772 Provides Significant Protection against Canine Lyme Disease

Beagles received placebo or ospA- and ospB-negative Borrelia burgdorferi before a tick challenge. A total of 28 (41%) ticks and skin biopsy specimens from each control dog (n = 10) contained B. burgdorferi. In contrast, 12 (19%) ticks recovered from the vaccine recipients (n = 10) were infected (P = 0.0077), and 5 dogs yielded spirochetes from the skin biopsy specimens (P = 0.0325). In addition, 9 (90%) placebo recipients and 4 (40%) vaccine recipients developed joint abnormalities (P = 0.0573). Therefore, vaccination with the ospA- and ospB-negative spirochete provided significant protection against Lyme disease.     

The usefulness of ‘in vivo’ antigens in the diagnosis of human Lyme borreliosis

A total of 161 positive sera of the IgM class against Borrelia burgdorferi s.l. and 149 positive sera of the IgG class obtained from screening tests with Polish forestry workers (ELISA recombinant Borrelia IgM and IgG, Biomedica, Austria) were tested with IgM and IgG immunoblot -Borrelia LINE (Genzyme Virotech GmbH, Germany). Of the IgM ELISA-positive sera, 72% were positive using Borrelia LINE IgM. IgM antibodies against particular antigens in Borrelia LINE tests were as follows: OspC (70%), VlsE (1.8%), P39 (40%), EBV (0.6%). All sera which were positive for IgG using ELISA were also found to be positive for IgG in Borrelia LINE IgG. IgG antibodies against individual antigens using Borrelia Line tests were as follows: VlsE (100%), p39 (95%), p83 (67.5%), iv1-BBA36 (42.5%), iv2-BBO323 (72.5%), iv3-Crasp3 (50%), iv4-p4 (12.5%). Antibodies against VlsE in the IgM class undergo quick seroconversion to the IgG class (demonstrated by Borrelia LINE tests) in an ongoing infection despite a persistingly positive result for the IgM class obtained using ELISA with recombinant antigens. The presence of numerous antibodies indicates the varied immunological response against B. burgdorferi antigens. A broad range of ‘in vivo’ IgG class antigens may be very helpful to confirm B. burgdorferi infection using Borrelia LINE tests.     

The expanding Lyme Borrelia complex—clinical significance of genomic species?

Ten years after the discovery of spirochaetes as agents of Lyme disease in 1982 in the USA, three genomic species had diverged from the phenotypically heterogeneous strains of Borrelia burgdorferi isolated in North America and Europe: Borrelia afzelii, B. burgdorferi sensu stricto (further B. burgdorferi), and Borrelia garinii. Whereas B. burgdorferi remained the only human pathogen in North America, all three species are aetiological agents of Lyme borreliosis in Europe. Another seven genospecies were described in the 1990s, including species from Asia (Borrelia japonica, Borrelia turdi, and B. tanukii), North America (Borrelia andersonii), Europe (Borrelia lusitaniae and Borrelia valaisiana), and from Europe and Asia (Borrelia bissettii). Another eight species were delineated in the years up to 2010: Borrelia sinica (Asia), Borrelia spielmanii (Europe), Borrelia yangtze (Asia), Borrelia californiensis, Borrelia americana, Borrelia carolinensis (North America), Borrelia bavariensis (Europe), and Borrelia kurtenbachii (North America). Of these 18 genomic species B. afzelii, B. burgdorferi and B. garinii are the confirmed agents of localized, disseminated and chronic manifestations of Lyme borreliosis, whereas B. spielmanii has been detected in early skin disease, and B. bissettii and B. valaisiana have been detected in specimens from single cases of Lyme borreliosis. The clinical role of B. lusitaniae remains to be substantiated.     

Evaluation of RevA,a Fibronectin-Binding Protein of Borrelia burgdorferi,as a Potential Vaccine Candidate for Lyme Disease

Previous studies indicated that the Lyme disease spirochete Borrelia burgdorferi expresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected with B. burgdorferi mounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidal in vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged with B. burgdorferi by inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive for B. burgdorferi. Vaccinated animals also appeared to have similar levels of B. burgdorferi DNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect against Borrelia burgdorferi infection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.     

Prevalence and incidence of antibodies to Borrelia burgdorferi and to tick-borne encephalitis virus in agricultural and forestry workers from Tuscany, Italy

The ticks Ixodes persulcatus and Ixodes ricinus are the main vectors of both Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus in Eurasia. Borrelia burgdorferi is the cause of Lyme borreliosis, and TBE is a biphasic meningoencephalitis induced by an arbovirus belonging to the flavivirus family. The principal aims of the current investigation were (i) to determine the frequency of serological evidence of Borrelia burgdorferi sensu lato and TBE infections in healthy agricultural and forestry workers, (ii) to determine the incidence of seroconversion for antibodies against Borrelia burgdorferi sensu lato and TBE virus in Tuscan workers during a 1-year survey; and (iii) to assess the occupational risk for agricultural and forestry activities in a defined area (Tuscany, Italy). A total of 412 blood samples were taken from agricultural and forestry workers, and information on age, duration of employment, and history of tick bites was collected in a questionnaire to establish the risk factors for the diseases. Three hundred sixty-five blood donors from the same region served as controls. To estimate the rate of seroconversion, 176 of the agricultural and forestry workers were tested 1 year later. IgG and IgM antibodies against Borrelia burgdorferi sensu lato and TBE virus were detected in serum by an enzyme-linked immunosorbent assay and confirmed by Western blot analysis for Borrelia burgdorferi and by a test for inhibition of hemagglutination for TBE. Antibodies against Borrelia burgdorferi were more frequent among the workers than in the control group (7.8% vs. 4.9% in the IgG-IgM enzyme-linked immunosorbent assay and 7.03% vs. 3.56% in the confirmatory test). No seropositivity was observed for TBE virus. Eighteen of 176 subjects who underwent a second blood test developed specific antibodies against Borrelia burgdorferi within 1 year.     

Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks in Austria

Unfed ticks of all instars (Ixodes ricinus, n=853; Haemaphysalis concinna, n=11) collected in all nine federal states of Austria were individually examined for the presence of Borrelia burgdorferi sensu lato (s.l.) using PCR. The mean overall infection rate was 14.4%. Infection rates were 24.5% in adult ticks, 16.1% in nymphs, and 1.6% in larvae. Four genospecies were detected, including B. valaisiana which was detected for the first time in Austria. The most common B. burgdorferi s.l. genospecies was B. garinii (66.9%), followed by B. valaisiana (13.7%), B. afzelii (11.3%), and B. burgdorferi sensu stricto (s.s.) (6.5%). Two specimens (1.6%) could not be identified to the genospecies level. Geographically, the highest infection rates were detected in the federal state of Vorarlberg (33.3%), B. garinii and B. afzelii being the most prevalent genospecies. B. valaisiana occurred most often in the federal state of Lower Austria, and B. burgdorferi s.s. was focally distributed in the Tyrol, in the surroundings of Imst.     

Borrelia burgdorferi BmpA, BmpB, and BmpD Proteins Are Expressed in Human Infection and Contribute to P39 Immunoblot Reactivity in Patients with Lyme Disease

The Bmp proteins are a paralogous family of chromosomally encoded Borrelia burgdorferi lipoproteins. They have similar predicted immunogenicities and similar electrophoretic mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P39 reactivity against Borrelia burgdorferi lysate in immunoblots of Lyme disease patients has long been identified with reactivity to BmpA, but responses to other Bmp proteins have not been examined. To determine if patients with Lyme disease developed such responses, immunoglobulin G (IgG) anti-Bmp reactivity in patient and control sera was studied by using soluble recombinant Bmp (rBmp) proteins expressed in Escherichia coli. Although some patient sera contained IgG immunoblot and immunodot reactivities against all four Bmp proteins, analysis of IgG anti-Bmp fine specificity by a competitive enzyme-linked immunosorbent assay with graded doses of soluble homologous and heterologous rBmp proteins showed that only the responses to BmpA, BmpB, and BmpD were specific. This suggests that at least three of the four Bmp proteins are expressed by B. burgdorferi in infected patients and that specific antibodies to them are likely to be present in the P39 band in some patients.     

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi.     

Prevalence of Borrelia burgdorferi sensu lato in the selected Ixodes ricinus (Acari: Ixodidae) population in Weilburg forests,Hesse, Germany

We studied the presence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in woodland areas in the vicinity of Weilburg (Hesse, Germany). Ticks (n = 13347) were collected in five localities in 1996 and were fixed in 70% ethanol. Out of them, 4404 specimens were analysed individually (n = 406) and in 796 pools of 2-10 specimens (n = 3998) by the polymerase chain reaction (PCR). The calculated minimal infection rate of individual and pooled ticks was 0% in larvae (n = 280), 6.9 % in nymphs (n = 3600), 13.9 % in males (n = 280) and 20.9 % in females (n = 244). Prevalence of B. burgdorferi s. 1. in I. ricinus was determined with respect to the abundance and seasonal activity of the ticks.     

Prevalence of Four Species of Borrelia burgdorferi Sensu Lato and Coinfection with Anaplasma phagocytophila in Ixodes ricinus Ticks in Central Germany

A total of 305 Ixodes ricinus ticks collected from three areas of Thuringia in central Germany were investigated for infection with Borrelia burgdorferi sensu lato species and Anaplasma phagocytophila. Overall, 11.1% were infected with Borrelia burgdorferi and 2.3% with Anaplasma phagocytophila. Adult ticks showed a significantly higher rate of infection with both borreliae and Anaplasma phagocytophila. Borrelia garinii (55.9%) was detected most frequently, followed by Borrelia burgdorferi sensu stricto (32.4%), Borrelia afzelii (17.6%), and Borrelia valaisiana (5.9%). Four ticks had dual infection with Borrelia garinii and Borrelia burgdorferi sensu stricto. Two of the Borrelia-positive ticks were coinfected with Anaplasma phagocytophila.     

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.     

Gamma Interferon Inhibits Production of Anti-OspA Borreliacidal Antibody In Vitro

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-γ). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-γ was not affected by the time of treatment. In addition, treatment with rIFN-γ inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-γ marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-γ does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.     

Specificity of Infection-Induced Immunity among Borrelia burgdorferi Sensu Lato Species

The specificity of infection-induced immunity in mice infected with cultured or host-adapted Borrelia burgdorferi sensu lato, the agent of Lyme disease, was examined. Sera obtained from mice following infection with high and low doses of cultured B. burgdorferi sensu stricto, transplantation of infected tissue (host-adapted spirochetes), or tick-borne inoculation all showed protective activity in passive immunization assays. Infection and disease were similar in mice infected with cultured spirochetes or by transplantation. Thus, the adaptive form of inoculated spirochetes did not influence the immune response during active infection. Mice infected with B. burgdorferi sensu stricto and then cured of infection with an antibiotic during early or late stages of infection were resistant to challenge with high doses of homologous cultured spirochetes for up to 1 year. In contrast, actively immune mice infected with different Borrelia species (B. burgdorferi sensu lato, B. burgdorferi sensu stricto cN40, Borrelia afzelii PKo, and Borrelia garinii PBi) and then treated with an antibiotic were resistant to challenge with cultured homologous but not heterologous spirochetes. Similar results were achieved for actively immune mice challenged by transplantation and by passive immunization with sera from mice infected with each of the Borrelia species and then challenged with cultured spirochetes. Arthritis and carditis in mice that had immunizing infections with B. afzelii and B. garinii and then challenged by transplantation with B. burgdorferi sensu stricto were equivalent in prevalence and severity to those in nonimmune recipient mice. These results indicate that protective immunity and disease-modulating immunity that develop during active infection are universal among species related to B. burgdorferi sensu lato but are species specific.     

Detection of Immune Complexes Is Not Independent of Detection of Antibodies in Lyme Disease Patients and Does Not Confirm Active Infection with Borrelia burgdorferi

The Borrelia burgdorferi-specific immune complex (IC) test, which uses polyethylene glycol (PEG) precipitation to isolate ICs from serum, has been used as a research test in the laboratory diagnosis of early Lyme disease (LD) and has been proposed as a marker of active infection. We examined whether B. burgdorferi-specific antibodies were present within PEG-precipitated ICs (PEG-ICs) in patients with LD, posttreatment Lyme disease syndrome, and controls, including individuals who received the outer surface protein A (OspA) vaccine. Using a B. burgdorferi whole-cell enzyme-linked immunosorbent assay (ELISA), we obtained positive PEG-IC results not only in patients with a history of LD, but also in individuals vaccinated with OspA vaccine. The frequency of positive PEG-IC ELISAs in OspA vaccinees was significantly higher with ELISA-reactive than with ELISA-negative unprocessed serum samples (P = 0.001), demonstrating dependency between the tests. Similar results were found using samples from rhesus macaques infected with B. burgdorferi, uninfected macaques vaccinated with OspA, and controls. Therefore, testing for the presence of antibodies against B. burgdorferi in PEG-IC preparations is not more likely to reflect active infection than testing in unprocessed serum and should not be used in individuals who received the OspA vaccine.     

Foci of tick-borne diseases in Southwest Germany

Presently known tick-borne diseases in Germany include Lyme borreliosis, tick-borne encephalitis (TBE-virus, western subtype), Q-fever, babesiosis and presumably ehrlichiosis. Blood samples of 4,368 forestry workers in the State of Baden-Wuerttemberg (B-W), southwestern Germany, were tested for the presence of antibodies against Borrelia burgdorferi sensu lato, TBE-virus and Ehrlichia spp. (genogroup E. phagocytophila). Furthermore 12,327 ticks (Ixodes ricinus) collected in various areas of B-W were analysed by PCR and genotyping for the prevalence of pathogen RNA and DNA. The human seroprevalence rates of antibodies to B. burgdorferi sensu lato ranged from 18 % to 52 %, for TBE-virus from 0 % to 43 % and for Ehrlichia spp. from 5 % to 16 % in various counties of the State. The foci of B. burgdorferi and TBE-virus as indicated by antibody prevalence in humans are only partly overlapping with each other. The highest rates of TBE-virus antibodies are in concordance with available clinical data. However antibody prevalence up to 27 % in areas with no reports of clinical cases was found, suggesting that TBE occurs throughout the State of B-W, The prevalence of Ehrlichia spp. antibodies suggests that ehrlichiosis plays a role as a tick-borne disease in Germany, but as long as no clinical data are available, this will remain unclear.Investigations of ticks for TBE-virus (n = 9,189) by nested PCR showed prevalence rates from 0% to 2.3% and for Ehrlichia spp. (n = 1,963) from 2.6% to 3.1%. Examination of ticks (n = 3,138) for the presence of B. burgdorferi sensu lato DNA was performed by PCR and revealed prevalence rates from 13.9% up to 24%. Furthermore 1,106 samples positive for B. burgdorferi sensu lato were used for genotyping. B. afzelii DNA was found in 407 ticks (36.8%), followed by B. garinii (21.9 %), B. valaisiana (13.7%), and B. burgdorferi sensu stricto (9.9 %). Double infection was found in 6.4% and triple infection in 0.8% of the ticks. 10.5% of the positive samples could not be classified.Prevention of tick-borne diseases has to focus on behavioural intervention to reduce individual tick exposure by proper behaviour in the environment, as a large-scale control of the tick population seems impossible and thus reduction of Lyme borreliosis and TBE through tick control is unlikely. Vaccination against TBE-virus should not only be recommended for high endemic areas but also for persons with a high individual risk.     

Species-Specific Plasmid Sequences for PCR Identification of the Three Species of Borrelia burgdorferi Sensu Lato Involved in Lyme Disease

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.     

Heat Shock Protein 70 of the Agent of Human Granulocytic Ehrlichiosis Binds to Borrelia burgdorferi Antibodies

We describe a patient with human granulocytic ehrlichiosis (HGE), a diagnosis confirmed by PCR and immunoblot analysis. Unexpectedly, immunoglobulin G (IgG) directed towards an 80-kDa ehrlichial antigen (without detectable IgM) was present in the patient’s serum in the first week of illness. Lyme disease immunoblots were reactive for IgG (but not IgM), a result indicative of prior exposure to the Lyme disease spirochete. Amino-terminal sequencing revealed that the 80-kDa ehrlichial antigen was an HSP-70 homolog similar to Borrelia burgdorferi HSP-70. We conclude that antibodies against B. burgdorferi HSP-70 may cross-react with the ehrlichial heat shock protein and that this possibility must be considered when serologic test results for HGE and Lyme disease are interpreted.     

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.     

Human Exposure to a Granulocytic Ehrlichia and Other Tick-Borne Agents in Connecticut

Indirect fluorescent-antibody (IFA) staining methods with Ehrlichia equi (MRK or BDS strains) and Western blot analyses containing a human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain) were used to confirm probable human cases of infection in Connecticut during 1995 and 1996. Also included were other tests for Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis (HME), Babesia microti, and Borrelia burgdorferi. Thirty-three (8.8%) of 375 patients who had fever accompanied by marked leukopenia or thrombocytopenia were serologically confirmed as having HGE. Western blot analyses of a subset of positive sera confirmed the results of the IFA staining methods for 15 (78.9%) of 19 seropositive specimens obtained from different persons. There was frequent detection of antibodies to a 44-kDa protein of the HGE agent. Serologic testing also revealed possible cases of Lyme borreliosis (n = 142), babesiosis (n = 41), and HME (n = 21). Forty-seven (26.1%) of 180 patients had antibodies to two or more tick-borne agents. Therefore, when one of these diseases is clinically suspected or diagnosed, clinicians should consider the possibility of other current or past tick-borne infections.