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.     

DbpA, but Not OspA, Is Expressed by Borrelia burgdorferi during Spirochetemia and Is a Target for Protective Antibodies

DbpA is a target for antibodies that protect mice against infection by cultured Borrelia burgdorferi. Infected mice exhibit early and sustained humoral responses to DbpA and DbpB, suggesting that these proteins are expressed in vivo. Many antigens expressed in mammals by B. burgdorferi are repressed in vitro at lower growth temperatures, and we have now extended these observations to include DbpA and DbpB. To confirm that the protective antigen DbpA is expressed in vivo and to address the question of its accessibility to antibodies during infection, we examined B. burgdorferi in blood samples from mice following cutaneous inoculation. B. burgdorferi was visualized by dark-field microscopy in plasma samples from spirochetemic mice, and an indirect immunofluorescence assay showed that these spirochetes were DbpA positive and OspA negative. We developed an ex vivo borreliacidal assay to show that hyperimmune antiserum against DbpA, but not OspA, killed these plasma-derived spirochetes, demonstrating that DbpA is accessible to antibodies during this phase of infection. Blood transferred from spirochetemic donor mice readily established B. burgdorferi infection in naive recipient mice or mice hyperimmunized with OspA, while mice hyperimmunized with DbpA showed significant protection against challenge with host-adapted spirochetes. Antiserum from persistently infected mice had borreliacidal activity against both cultured and plasma-derived spirochetes, and adsorption of this serum with DbpA substantially depleted this killing activity. Our observations show that immunization with DbpA blocks B. burgdorferi dissemination from the site of cutaneous inoculation and suggest that DbpA antibodies may contribute to control of persistent infection.     

Experimental infection of dogs with<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu stricto using<Emphasis Type="Italic"> Ixodes scapularis</Emphasis> ticks artificially infected by capillary feeding

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.     

Antibiotic Treatment of Animals Infected with Borrelia burgdorferi

Summary: Despite resolution of the objective manifestations of Lyme disease after antibiotic treatment, a minority of patients have fatigue, musculoskeletal pain, and/or difficulties with concentration or short-term memory of uncertain etiology; these are called post-Lyme disease symptoms or, in more severe cases, post-Lyme disease syndrome or “chronic Lyme disease.” Several recent studies in which Borrelia burgdorferi-infected animals were treated with antibiotic therapy have demonstrated the presence of PCR positivity for B. burgdorferi DNA in the absence of culture positivity. In mice that were treated with antibiotic therapy, residual spirochetes could be taken up by ticks during a blood meal and could be transmitted to SCID mice. These spirochetes are attenuated; their presence is not associated with either inflammation or disease. In this review the methodology and findings of these studies are critically analyzed, and the significance of the results with regard to human Lyme disease is evaluated, with special emphasis on whether these studies provide useful insights into post-Lyme disease syndrome. A serious methodological concern is the failure to consider the pharmacokinetic-pharmacodynamic properties of the antibiotic in choosing the dosage regimen used. We conclude that there is no scientific evidence to support the hypothesis that such spirochetes, should they exist in humans, are the cause of post-Lyme disease syndrome.     

Salivary gland extract from engorged Ixodes ricinus (Acari: Ixodidae) stimulates in vitro growth of Borrelia burgdorferi sensu lato

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK‐H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Rate of infection ofIxodes ricinus ticks withBorrelia burgdorferi sensu stricto,Borrelia garinii,Borrelia afzelii and group VS116 in an endemic focus of Lyme disease in Italy

A study to evaluate the natural rate of infection ofIxodes ricinus withBorrelia burgdorferi sensu lato was carried out in an endemic focus of Lyme disease in the Trieste area in northern Italy. Two-hundred and twenty-seven ticks collected in ten different stations were tested individually for the presence of the spirochetes using polymerase chain reaction techniques able to identify bothBorrelia burgdorferi sensu lato and the four genospecies (Borrelia burgdorferi sensu stricto,Borrelia garinii, Borrelia afzelii and group VS116). Multiple infection of individual ticks was found. The infection rate ranged from 0–70%. Infection ofIxodes ricinus withBorrelia burgdorferi group VS116 was found for the first time in Italy in both a high and a low endemic focus of Lyme disease.     

Recruitment of macrophages and polymorphonuclear leukocytes in L:yme carditis

Lyme arthritis, caused by the spirochete Borrelia burgdorferi, can be recurrent or prolonged, whereas Lyme carditis is mostly nonrecurring. A prominent difference between arthritis and carditis is the differential representation of phagocytes in these lesions: polymorphonuclear leukocytes (PMN) are more prevalent in the joint, and macrophages predominate in the heart lesion. We have previously shown differential efficiency of B. burgdorferi clearance by PMN and macrophages, and we now investigate whether these functional differences at the cellular level may contribute to the observed differences in organ-specific pathogenesis. When we infected mice lacking the neutrophil chemokine receptor (CXCR2^−/− mice) with spirochetes, we detected fewer PMN in joints and less-severe arthritis. Here we have investigated the effects of the absence of the macrophage chemokine receptor CCR2 on the development and resolution of Lyme carditis in resistant (C57BL/6J [B6]) and sensitive (C3H/HeJ [C3H]) strains of mice. In B6 CCR2^−/− mice, although inflammation in hearts is mild, we detected an increased burden of B. burgdorferi compared to that in wild-type (WT) mice, suggesting reduced clearance in the absence of macrophages. In contrast, C3H CCR2^−/− mice have severe inflammation but a decreased B. burgdorferi burden compared to that in WT C3H mice both at peak disease and during resolution. Histopathologic examination of infected hearts revealed that infected C3H CCR2^−/− animals have an increased presence of PMN, suggesting compensatory mechanisms of B. burgdorferi clearance in the hearts of infected C3H CCR2^−/− mice. The more efficient clearance of B. burgdorferi from hearts by CCR2^−/− versus WT C3H mice suggests a natural defect in the recruitment or function of macrophages in C3H mice, which may contribute to the sensitivity of this strain to B. burgdorferi infection.     

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0–11%), 10.8% (2–43%) and 17.4% (3–58%) for larvae (n = 5699), nymphs (n = 48 804) and adults (n = 41 666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17–20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed. Received: 18 August 1997 / Accepted: 12 September 1997     

Cytokines (IL-4 and IFN-γ) and antibodies (IgE and IgG2a) produced in mice infected with Borrelia burgdorferi sensu stricto via nymphs of Ixodes ricinus ticks or syringe inoculations

Mice were tolerant to tick bites during three infestations with nymphs of Ixodes ricinus infected with Borrelia burgdorferi sensu stricto. To determine whether tick bites influence the immune response against B. burgdorferi, we examined the production of cytokines IL-4 and IFN-γ by lymph node cells of BALB/c mice and IL-4 deficient BALB/c mice after tick inoculation versus syringe inoculation of B. burgdorferi. We also measured IgG2a anti-borrelial antibodies and total IgE in these mice. Results showed that BALB/c mice developed a Th2 immune response against B. burgdorferi after tick inoculation and a mixed Th1/Th2 response after syringe inoculation of B. burgdorferi. IL-4 deficient mice produced a Th1 immune response in both cases. IL-4 produced following tick bites greatly decreased the production of anti-borrelial IgG2a antibodies by comparison with the production of anti-borrelial IgG2a antibodies produced following syringe injection of B. burgdorferi. Received: 23 November 1999 / Accepted: 9 December 1999     

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.     

Adenoviral delivery of interleukin-10 fails to attenuate experimental Lyme disease

Production of interleukin-10 (IL-10) by C57BL/6 mice following infection with Borrelia burgdorferi has been proposed as a mechanism whereby resistance to the development of experimental Lyme arthritis is maintained. In the current study, we sought to determine the role of IL-10 during infection of arthritis- and carditis-susceptible C3H mice. Infection of C3H IL-10^−/− mice led to increased joint swelling and arthritis severity scores over those of wild-type C3H mice. Measurement of B. burgdorferi numbers in joints or disseminated tissues indicated a more efficient clearance of spirochetes in the absence of IL-10, similar to that reported in C57BL/6 IL-10^−/− mice. However, in contrast to previous in vitro work, infection of C3H IL-10^−/− mice led to decreased in vivo expression of the cytokines KC, IL-1β, IL-4, and IL-12p70 in the infected joints. Finally, adenoviral expression of IL-10 in the infected joints of C3H mice was unable to modulate the development of severe Lyme arthritis and had no effect on spirochete clearance or Borrelia-specific antibody production. Development of Lyme carditis appeared to be independent of modulation by IL-10. These results suggest that IL-10 limits the development of joint inflammation in both arthritis-resistant and -susceptible mouse strains infected with B. burgdorferi and that increased IL-10 production cannot rescue genetic susceptibility to development of pathology in this model.     

Bacterial Heterogeneity Is a Requirement for Host Superinfection by the Lyme Disease Spirochete

In nature, mixed Borrelia burgdorferi infections are common and possibly can be acquired by either superinfection or coinfection. Superinfection by heterologous B. burgdorferi strains has been established experimentally, although the ability of homologous B. burgdorferi clones to superinfect a host has not been studied in detail. Information regarding any potential immune barriers to secondary infection also currently is unavailable. In the present study, the ability to superinfect various mouse models by homologous wild-type clones was examined and compared to superinfection by heterologous strains. To assess the ability of homologous B. burgdorferi clones to successfully superinfect a mouse host, primary- and secondary-infecting spirochetes were recovered via in vitro cultivation of collected blood or tissue samples. This was accomplished by generating two different antibiotic-resistant versions of the wild-type B31-A3 clone in order to distinguish superinfecting B. burgdorferi from primary-infecting spirochetes. The data demonstrate an inability of homologous B. burgdorferi to superinfect immunocompetent mice as opposed to heterologous strains. Attempts to superinfect different types of immunodeficient mice with homologous B. burgdorferi indicate that the murine innate immune system represents a major barrier to intrastrain superinfection. Consequently, the possibility of innate immunity as a driving force for B. burgdorferi heterogeneity during the enzootic cycle is discussed.     

Influence of Outer Surface Protein A Antibody on Borrelia burgdorferi within Feeding Ticks

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick’s salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 μg/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 μg/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induce ospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.     

Persistent atrioventricular block in Lyme borreliosis

Summary Cardiac manifestations are reported in 0.3%–4.0% of European patients withBorrelia burgdorferi (B.b.) infection. Usually symptoms disappear within 6 weeks. We report a case with persistent impairment of atrioventricular (AV) conduction. Diagnosis was confirmed by demonstration of IgM antibodies and increase of IgG antibody titers against B.b. in serum, by isolation of the spirochete from skin biopsy material and by the typical clinical combination of erythema migrans, Bannwarth syndrome (meningoradiculitis), and complete heart block. Despite immediate antibiotic therapy with ceftriaxone, first degree AV block and second degree block Wenckebach with atrial pacing at 100 beats/minute persisted for 2 years. We conclude, that Lyme carditis can cause long-standing or irreversible AV conduction defects despite adequate and early antimicrobial therapy.Abbreviations AV atrioventricular - AVt atrioventricular time - B. burgdorferi Borrelia burgdorferi - CSF cerebrospinal fluid - ECG electrocardiogram - ESR erythrocyte sedimentation rate - FRG Federal Republic of Germany - I. dammini/ricinus Ixodes dammini/ricinus - IgG immunoglobulin G - IgM immunoglobulin M     

Detection of Active Infection in Nonhuman Primates with Lyme Neuroborreliosis: Comparison of PCR, Culture, and a Bioassay

Ideally a diagnosis of infection of the central nervous system (CNS) is made by culture of the etiologic pathogen, but Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis (LNB), is rarely cultured from the cerebrospinal fluid (CSF). PCR and measurement of specific antibody in the CSF also have their limitations. The role of available assays for LNB has not been studied carefully in a comparative investigation. There is a need to assess the reliability of assays and to increase the ability to document active infection in the CNS. The recent development of the nonhuman primate (NHP) model of LNB allowed us to address this need in a faithful model of human LNB. In this study we compared the abilities of PCR and culture to detect the presence of spirochetes in the CSF and brain tissue of infected NHPs and related these measures of infection to the development of anti-B. burgdorferi antibody. We also tested a bioassay, the mouse infectivity test (MIT), in this model. Fourteen of 16 CSFs from four NHPs were positive by at least one of these techniques. Detection of spirochetes in the CSF by PCR, the MIT, and culture was inversely related to the concomitant presence of anti-B. burgdorferi antibody intrathecally. The performance of any particular test was associated with the strength of the host immune response. In early CNS infection, when anti-B. burgdorferi antibody had not yet appeared, or in immunocompromised hosts, the MIT compared favorably to culture and PCR for infected NHPs; antibody in the CSF was the most useful assay for immunocompetent NHPs.     

New Colorimetric Microdilution Method for In Vitro Susceptibility Testing of Borrelia burgdorferi Against Antimicrobial Substances

 A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, ≤0.125 μg/ml), ceftriaxone (MIC range, ≤0.015–0.06 μg/ml), and azithromycin (MIC range, ≤0.015–0.06 μg/ml). Whereas tobramycin (MIC range, 8–64 μg/ml) exhibited little activity, spectinomycin (MIC range, 0.25–2 μg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P<0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.     

Characterization of a 30-kDa Borrelia burgdorferi substrate-binding protein homologue

A Borrelia burgdorferi chromosomal gene encodes a 30-kDa antigen (P30) that has considerable homology with periplasmic substrate-binding proteins of Gram-negative bacteria, and is recognized by antibodies in sera from a subset of patients with Lyme disease and from B. burgdorferi-infected mice. The p30 gene is 801 nucleotides in length and P30 contains 267 amino acids, with predicted molecular mass of 30 kDa. The P30 amino acid region 36–258 has homology to conserved domains of the oligopeptide permease A of Gram-negative bacteria. Immunofluorescence studies using murine anti-P30 serum suggest that P30 is on the outer surface of B. burgdorferi. P30 expression could be detected in representatives of all 3 subspecies of B. burgdorferi sensu lato, but not in all of the tested strains. Antibodies to P30 were detected in sera of 18 out of 82 patients (22%) with Lyme disease, including individuals with early- or late-stage infection. Although antibodies to P30 are present in the sera of C3H/HeN mice infected with B. burgdorferi for at least 90 days, immunization with recombinant P30 does not protect mice from infection. We conclude that P30 is a putative substrate-binding protein of B. burgdorferi and is immunologically recognized in human and murine Lyme borreliosis.     

Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Lyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection of Borrelia burgdorferi infection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinant in vivo-expressed B. burgdorferi antigens for objective detection of a host immune response to B. burgdorferi infection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinant B. burgdorferi in vivo-expressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (C_q) value for each sample minus the mean background C_q plus 3 standard deviations (ΔC_q) being 4 to 10, whereas ΔC_q was 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation = 0.895, P < 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using the B. burgdorferi VlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes to B. burgdorferi infection.