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Background: The risk of acquiring Lyme disease has been evaluated by xenodiagnostic procedures with laboratory strains of Borrelia burgdorferi and laboratory-reared Ixodes ticks, or by clinical trials in which diagnosis was based on clinical findings, culture, or serologic tests. Objective: Our purpose was to determine the risk of infection from tick bites in a natural setting in which wild strains of B. burgdorferi were involved, by a biopsy culture technique. Methods: Skin biopsy specimens were obtained from Ixodes scapularis tick bite sites, processed, and examined for the presence of B. burgdorferi . Results: B. burgdorferi was cultivated from only 2 of 48 skin biopsy specimens. In both instances duration of tick attachment was approximately 24 hours. Conclusion: In a hyperendemic region for Lyme disease the risk of infection after a deer tick bite appears to be low, particularly if the tick has been attached for less than 24 hours.

Background: We recently demonstrated that cultivation of Borrelia burgdorferi from skin biopsy specimens obtained from erythema migrans lesions was an efficacious procedure to confirm the diagnosis of Lyme disease. Objective: Our purpose was to investigate the efficacy of our microbiologic technique on blood samples obtained from patients with Lyme disease and erythema migrans. Methods: Whole blood samples were obtained from 52 patients with erythema migrans and early localized or early disseminated Lyme disease and placed into polystyrene tubes containing 6 ml of modified Barbour-Stoenner-Kelly medium, processed, and examined for B. burgdorferi by dark-field microscopy. Results: B. burgdorferi was cultured from the blood of two of our 52 patients (4%). Clinically, both of these patients were considered to have early localized Lyme disease. Conclusion: The culture of B. burgdorferi from the blood of patients with early Lyme disease does not appear to be an efficacious procedure to confirm the diagnosis of Lyme disease. However, the demonstration of spirochetemia in patients with erythema migrans without any extracutaneous evidence of disseminated illness does have therapeutic significance.

A mutant of virulent Borrelia burgdorferi 297 was apparently selected for by long-term storage at 5 degrees C. This mutant was found to lack the plasmid which encodes outer surface protein A (OspA) and OspB. In addition to the loss of the OspA and OspB proteins, the mutant lacked two lipoproteins, of 20 and 7.5 kDa, that were observed in the wild type. Since the mutant was not recovered from the tissues or blood of hamsters injected with the mutant, the mutant was determined to be noninfectious. Hamsters vaccinated with noninfectious mutant 297 were protected completely from challenge with virulent wild-type 297 spirochetes. Prechallenge sera from hamsters vaccinated with mutant 297 lacked antibodies to OspA and OspB, while those from hamsters vaccinated with virulent wild-type 297 or avirulent 297 exhibited antibodies to these proteins. Hamsters vaccinated with virulent wild-type 297 or mutant 297 elicited antibodies to OspC and a 39-kDa protein (P39), whereas hamsters vaccinated with avirulent 297 lacked these antibodies. These results suggest that OspC and/or P39 are important for the development of a protective immune response. Study of this mutant may elucidate factors important to the development of a Lyme disease vaccine.

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila . Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused by Ehrlichia chaffeensis , Rickettsia rickettsii , and Coxiella burnetti , no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples from R. rickettsii -infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests for Borrelia burgdorferi . Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease ( n = 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure to Ehrlichia . Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.

The correlation of plasmid profiles with infectivity was investigated by using five clones of Borrelia burgdorferi sensu stricto strain B31 (ATCC 35210). Plasmid profiles were determined by pulsed-field and two-dimensional gel electrophoresis. The 50% infectious dose (ID50) in hamsters was determined. The ID50 of the clone that possessed a full complement of eight linear and three circular plasmids was 10(3) cells. The loss of the 27.5- and 40-kb linear plasmids did not decrease the infectivity of these cells. Rather, the loss of the 27.5-kb linear plasmid was associated with a more disseminated infection. A moderate decrease of the ID50 from 10(3) to 10(5) cells correlated with the loss of the 9.0-kb circular plasmid and the 27.5-kb linear plasmid. A major loss of infectivity (ID50 > 10(3) cells) occurred with cells that lost the 24.7- and 27.5-kb linear plasmids and the 9.0-kb circular plasmid. A 3.0-kb HindIII fragment of the 24.7-kb linear plasmid was used as a probe to determine the presence of the homologous sequences in the three genospecies of Lyme disease spirochetes. An analysis of 21 infectious strains of B. burgdorferi sensu stricto, B. garinii, and B. afzelii revealed a consistent association of infectivity with strains possessing a linear plasmid (size range, 24 to 36 kb) that hybridized with the HindIII fragment. Western immunoblotting with hamster antisera against infectious B31 clone C-3 revealed two proteins with molecular masses of 28 and 43 kDa that were absent in the noninfectious B31 clone C-1. Additionally, a 14-kDa protein was absent in C-1 but present in infectious clone C-9 as shown by two-dimensional polyacrylamide gel electrophoresis.

Background: Borrelia burgdorferi has been cultivated from clinically normal skin (previous erythema migrans sites) after antibiotic therapy for Lyme disease. Objective: We investigated the possibility of similar findings in 13 of our patients with antibiotic-treated Lyme disease from whom B. burgdorferi was cultivated from their erythema migrans lesions before antibiotic therapy. Methods: After treatment with doxycycline or a combination of amoxicillin and probenecid, skin biopsy specimens were obtained from clinically normal skin adjacent to the previous biopsy sites and cultured. Results: B. burgdorferi was not cultivated from these post treatment biopsy sites. Conclusion: The failure of B. burgdorferi to survive in the former erythema migrans sites of our antibiotic-treated patients, as well as their favorable clinical response, supports the use of doxycycline or combined amoxicillin and probenecid in the treatment of early Lyme disease but does not preclude the survival of the organism in other tissues.