Detection of borreliacidal antibodies in dogs after challenge with Borrelia burgdorferi-infected ixodes scapularis ticks.

Detection of borreliacidal antibodies is an accurate serodiagnostic test for confirmation of Lyme disease in humans. In this study, 13 pathogen-free beagles, 12 to 26 weeks old, were infected with Borrelia burgdorferi by tick challenge. Dogs were monitored for clinical signs and symptoms of Lyme disease along with borreliacidal antibody production against B. burgdorferi sensu stricto isolates 297 and 50772. Ten (77%) dogs developed lameness in one or more legs within 210 days after attachment of Ixodes scapularis ticks. Eight (80%) of the lame animals had concurrent fever of > or =38 degrees C. Spirochetes were also recovered from the skin and joints of 12 (92%) dogs, but rarely from other organs. Borreliacidal antibodies against B. burgdorferi isolate 297 were detected in only four (31%) dogs, and the levels of killing antibodies remained low for the duration of the infection. In contrast, borreliacidal antibodies against B. burgdorferi isolate 50772 were detected in 13 (100%) dogs within 21 days of infection. Furthermore, the borreliacidal antibody levels correlated with the severity of B. burgdorferi infection. Detection of borreliacidal antibodies, especially against B. burgdorferi isolate 50772, is also a reliable serodiagnostic test for detection of Lyme disease in dogs.

Production of borreliacidal antibody to outer surface protein A in vitro and modulation by interleukin-4.

Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide, but not recombinant OspA. Anti-OspA borreliacidal antibody was detected in supernatants of cultures of lymph node cells obtained on day 7 after vaccination, peaked on day 17, and rapidly declined. The borreliacidal activity was attributable to immunoglobulin G1 (IgG1), IgG2a, and IgG2b antibodies. When lymph node cells were treated with interleukin-4 (IL-4), production of borreliacidal antibody was inhibited but was unaffected by treatment with anti-IL-4 antibodies. These results suggest that other cytokines, but not IL-4, are mainly responsible for production of the secondary borreliacidal antibody response.

Evaluation of whole-cell and OspC enzyme-linked immunosorbent assays for discrimination of early lyme borreliosis from OspA vaccination.

A recombinant Lyme borreliosis vaccine consisting of outer surface protein A (OspA) is commercially available for vaccination of humans against infection with Borrelia burgdorferi. Vaccination with OspA induces an antibody response that makes serologic interpretation of infection with B. burgdorferi difficult, especially by screening tests based on whole-cell preparations of B. burgdorferi. We show that an enzyme-linked immunosorbent assay with B. burgdorferi sensu stricto 50772, which lacks the plasmid encoding OspA and OspB, or a full-length recombinant OspC protein can identify patients infected with B. burgdorferi. We found that 69 and 65% of serum samples from patients with case-defined early Lyme borreliosis had anti-B. burgdorferi sensu stricto 50772 and anti-OspC reactivities, respectively. In addition, little or no reactivity was detected with sera obtained from individuals vaccinated with OspA. Unfortunately, 51 and 33% of sera from healthy patients and sera from patients with other illnesses were also reactive against B. burgdorferi sensu stricto 50772 and OspC, respectively. Although these assays can discriminate B. burgdorferi infection from vaccination with OspA, their lack of specificity highlights the necessity for confirming equivocal or positive reactivities with more specific serodiagnostic tests.

Detection of borreliacidal antibodies in Lyme borreliosis patient sera containing antimicrobial agents.

The borreliacidal-antibody test has been used for the serological detection and confirmation of Lyme borreliosis. However, the presence of antimicrobial agents in serum can confound the accurate detection of borreliacidal antibodies. In this study, we developed a Bacillus subtilis agar diffusion bioassay to detect small concentrations of antimicrobial agents in serum. We also used XAD-16, a nonionic polymeric resin, to adsorb and remove high concentrations of amoxicillin, cefotaxime, ceftriaxone, cefuroxime, doxycycline, and erythromycin without significantly affecting even small concentrations of immunoglobulin M (IgM) or IgG borreliacidal antibodies. High concentrations of penicillin could also be removed by adding 1 U of penicillinase without significantly influencing the levels of borreliacidal antibodies. These simple procedures greatly enhance the clinical utility of the borreliacidal-antibody test.

Borreliacidal antibody production against outer surface protein C of Borrelia burgdorferi.

Early Lyme borreliosis sera with significant titers of anti-outer surface protein C (OspC) borreliacidal antibodies were identified. Human anti-OspC borreliacidal antibodies could be either IgM or IgG. Significant concentrations of borreliacidal activity were detected after vaccination of mice with OspC. Detection of anti-OspC borreliacidal activity was dependent on surface expression of OspC by Borrelia burgdorferi isolate 50772. The ability of OspC to induce borreliacidal antibodies in vivo and after vaccination offers another possible explanation for the ability of vaccination with OspC to protect against infection with B. burgdorferi. Furthermore, detection of anti-OspC borreliacidal antibodies, especially IgM antibodies, in early Lyme borreliosis sera provides additional evidence that borreliacidal antibody detection may be useful for the serodiagnosis of early Lyme borreliosis.

Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine.

Significant borreliacidal antibody was induced in volunteers and hamsters 60 days after primary and secondary vaccination with high concentrations of recombinant outer surface protein A (rOspA). However, the borreliacidal antibody response waned rapidly. Only 1 person had detectable cidal activity 180 days after vaccination. Similarly, the borreliacidal antibody response waned rapidly in hamsters by week 10 of vaccination. By contrast, the total anti-rOspA antibody response remained elevated in volunteers and hamsters. When isolates of Borrelia burgdorferi sensu lato were incubated in sera from vaccinated humans or hamsters, only the vaccine-specific isolate was killed. These results were confirmed by challenging rOspA-vaccinated hamsters with different isolates of B. burgdorferi sensu lato. The results showed that monitoring total rOspA antibody is inappropriate for evaluating the efficacy of an rOspA vaccine. The rOspA vaccine must be improved to yield comprehensive protection and maintain sustained levels of protective borreliacidal antibodies.

Sensitivity and specificity of the borreliacidal-antibody test during early Lyme disease: a "gold standard"?

The serodiagnosis of early Lyme disease has been plagued with problems of sensitivity and specificity. We found that the flow-cytometric borreliacidal-antibody test had a sensitivity of 72% for the detection of patients with early Lyme disease. By contrast, the sensitivity of the enzyme immunofluorescence assay was 28%. The enhanced sensitivity of the borreliacidal-antibody test was due to the use of Borrelia burgdorferi 50772, which lacks OspA and OspB. When B. burgdorferi 297, which expresses both OspA and OspB, was used, the sensitivity of the borreliacidal-antibody test was 15%. Our results also showed that the borreliacidal-antibody test was specific. No borreliacidal activity was detected in normal sera or in sera from patients with mononucleosis, rheumatoid factor, or syphilis. These results demonstrate that the flow-cytometric borreliacidal-antibody test may be the laboratory "gold standard" for the serodiagnosis of Lyme disease.

Development of destructive arthritis in vaccinated hamsters challenged with Borrelia burgdorferi.

We present the first direct evidence that adverse effects, particularly severe destructive arthritis, can develop in vaccinated hamsters after challenge with Borrelia burgdorferi sensu lato isolates. Hamsters were vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant. A severe destructive arthritis was readily evoked in vaccinated hamsters challenged with the homologous B. burgdorferi sensu stricto isolate C-1-11 before high levels of protective borreliacidal antibody developed. Once high levels of C-1-11 borreliacidal antibody developed, hamsters were protected from homologous challenge and development of arthritis. Vaccinated hamsters, however, still developed severe destructive arthritis when challenged with other isolates of the three genomic groups of B. burgdorferi sensu lato (B. burgdorferi sensu stricto isolate 297, Borrelia garinii isolate LV4, and Borrelia afzelii isolate BV1) despite high levels of C-1-11 specific borreliacidal antibody. Vaccines that contained whole spirochetes in adjuvant induced destructive arthritis, but this effect was not dependent on the isolate of B. burgdorferi sensu lato or the type of adjuvant. These studies demonstrate that caution is necessary when employing whole spirochetes in adjuvant for vaccination to prevent Lyme borreliosis. Additional studies are needed to identify the antigen(s) responsible for the induction and activation of arthritis and to define the immune mechanisms involved.

Detection of borreliacidal antibodies by flow cytometry. An accurate, highly specific serodiagnostic test for Lyme disease.

BACKGROUND: Borreliacidal antibodies can be detected in serum samples from patients with early or late Lyme disease symptoms. When these serum samples are incubated with Borrelia burgdorferi and complement, spirochetes are rapidly killed. Detection of these antibodies can be used as a serodiagnostic test. METHODS: Individual serum samples containing IgM or IgG borreliacidal antibodies were Used to develop a method for detection using flow cytometry. An additional 10 case-defined Lyme disease serum samples and 10 normal serum samples were used to confirm appropriate flow cytometric parameters. To determine specificity, 157 normal serum samples and 104 potential cross-reactive serum samples were tested for borreliacidal activity and antibodies to B burgdorferi using indirect fluorescent antibody or enzyme immunoassay. RESULTS: Flow cytometry can be used to detect borreliacidal activity within 16 to 24 hours after incubation of B burgdorferi organisms. Lyme disease serum, and complement. Significant borreliacidal activity was detected in all Lyme disease serum samples. The percentages of positive normal serum samples were comparable (6% to 10%) using all three assays. In addition, the indirect fluorescent antibody and enzyme immunoassay identified 41 (39%) and 47 (45%) potential cross-reactive serum samples as positive, respectively. In contrast, significant borreliacidal activity was not detected in any potential cross-reactive serum samples. CONCLUSION: Detection of borreliacidal antibody, unlike indirect fluorescent antibody and enzyme immunoassay, is an accurate, highly specific serodiagnostic test for detection of Lyme disease.

Ability of canine Lyme disease vaccine to protect hamsters against infection with several isolates of Borrelia burgdorferi.

We used flow cytometry to determine levels of borreliacidal antibodies in hamsters after vaccination with a commercially available canine Lyme disease vaccine. In addition, we evaluated the ability of vaccinated hamsters to resist infection with several isolates of Borrelia burgdorferi. Borreliacidal antibodies could be detected 1 week after a primary vaccination, peaked at weeks 3 to 5, and then rapidly declined. One week after a booster vaccination, borreliacidal activity was detected at a dilution of 1:10,240, and it decreased fourfold by week 10 after the booster vaccination. Vaccinated hamsters were protected against infection with < or = 10(6) B. burgdorferi 297 organisms during the peak borreliacidal response (5 weeks after primary vaccination or 2 weeks after booster vaccination). However, hamsters were not fully protected from development of Lyme arthritis when the titer of borreliacidal antibodies was < 1:5,120. In addition, no significant borreliacidal activity was induced against B. burgdorferi C-1-11, LV4, or BV1, which belong to three other seroprotective groups. These studies demonstrate that vaccination with the canine Lyme disease vaccine induces protective antibodies against B. burgdorferi 297. However, significant levels of borreliacidal antibodies are not produced until 5 weeks after vaccination, and protection is short-lived. In addition, no borreliacidal activity was induced against other isolates of B. burgdorferi. Because of this, the incorporation of multiple isolates or protein subunits may be necessary to increase the effectiveness of future vaccines.

Lyme disease: laboratory diagnosis and serologic testing.

Although identified less than 20 years ago, Lyme disease has proved to be the most common tick-borne disease in the United States: some 10,000 cases were reported in 1992. In some cases the disease may be transitory and of little consequence but in others it may become chronic and severely disabling. Accurate diagnosis is, therefore, of great importance but, as this article shows, laboratory testing techniques still need improvement.

Recovery of Borrelia burgdorferi by filtration.

Bacterial contamination frequently interferes with successful recovery of the Lyme spirochete from cultures of tissue from Borrelia burgdorferi-infected humans, rodents, or ticks. We used 0.20- and 0.45-microns-pore-size syringe-tip filters to recover spirochetes from cultures contaminated with other bacteria. Low concentrations (1 to 10/ml) of B. burgdorferi organisms could be recovered from cultures seeded with 1 x 10(8) to 4 x 10(8) Staphylococcus aureus, Streptococcus faecium, Escherichia coli, or Bacillus subtilis organisms per ml. We also used this technique to recover B. burgdorferi from contaminated environmental and clinical cultures of B. burgdorferi. We conclude the filtration is an efficient method for recovering Lyme spirochetes from contaminated samples and increasing the number of successful isolations of B. burgdorferi.

In vitro susceptibilities of Borrelia burgdorferi to five oral cephalosporins and ceftriaxone.

We determined the in vitro susceptibilities of eight Borrelia burgdorferi isolates to five oral cephalosporins. MICs for B. burgdorferi 297 were 23 micrograms/ml (cephalexin), 45 micrograms/ml (cefadroxil), 91 micrograms/ml (cefaclor), 0.13 microgram/ml (cefuroxime), 0.8 microgram/ml (cefixime), and 0.02 microgram/ml (ceftriaxone). When B. burgdorferi isolates were exposed to concentrations twice the MIC of cefuroxime, cefixime, or ceftriaxone, at least 72 h of incubation was required to kill 99% of the organisms.

Survey for Ixodes spp. and Borrelia burgdorferi in southeastern Wisconsin and northeastern Illinois.

Forested areas adjacent to Milwaukee, Wis., and Chicago, Ill., were investigated for rodents and ticks infected with Borrelia burgdorferi, the causative agent of Lyme disease. White-footed mice (Peromyscus leucopus or Peromyscus maniculatus), meadow voles (Microtus pennsylvanicus), and eastern chipmunks (Tamias striatus) were captured; and specimens from these animals were cultured for B. burgdorferi to define whether the midwestern Lyme disease area currently encompasses these large metropolitan centers. During 1988, B. burgdorferi was successfully cultured from the tissues of two M. pennyslvanicus voles captured from the Chicago area. However, no Ixodes spp. ticks were captured. None of 274 animals captured from sites I3 and 12 additional sites in Wisconsin and Illinois during the summer of 1989 were infected with B. burgdorferi or Ixodes spp. In addition, no ticks were recovered when the underbrush in 11 contiguous areas was flagged. Apparently, B. burgdorferi is rarely found in these areas because of the absence of the appropriate tick vectors. Further studies are needed to monitor the dispersal of B. burgdorferi-infected Ixodes dammini into this heavily populated midwestern region.