Charles S. Pavia

Isolation of borrelia burgdorferi from the blood of seven patients with lyme disease

PURPOSE Borrelia burgdorferi, the etiologic agent of Lyme disease, has rarely been successfully cultured from blood. We report on seven patients from Westchester County, New York, with B. burgdorferi bacteremia diagnosed between April 1987 and August 1987. PATIENTS AND METHODS One hundred thirty-two attempts to isolate spirochetes were made on blood specimens obtained from 104 patients. Twenty-two of these specimens were obtained from nine patients who had recently been bitten by Ixodes ticks but who were asymptomatic. Heparinized blood or serum specimens (0.2 to 0.4 mL) were inoculated onto 6 mL of modified Barbour-Stoenner-Kelly medium. Lyme serology was performed by enzyme-linked immunosorbent polyvalent, IgM, and IgG assays, fluorescent immunoassay, and microhemagglutination. RESULTS Four of the seven patients had erythema migrans, two had facial nerve palsy, and one had a flu-like syndrome without rash. These patients represented 21% (four of 19) of all patients with the characteristic skin lesion who had blood cultures for B. burgdorferi, and 40% (two of five) of all those with facial nerve palsy. Serologic testing was frequently nonreactive; two patients had no detectable antibody on multiple sera by five different assays. All patients improved with antibiotic treatment, and had negative subsequent blood cultures, but five of seven had persistent complaints after completion of therapy. CONCLUSION Culturing blood for B. burgdorferi may be useful in confirming the diagnosis of Lyme disease in selected patients. Use of spirochete blood cultures may facilitate a better understanding of the pathogenesis and natural history of Lyme disease.

Impact of the saponin adjuvant QS-21 and aluminium hydroxide on the immunogenicity of recombinant OspA and OspB of Borrelia burgdorferi

The impact of the adjuvants QS-21 and aluminium hydroxide (alum) on the immunogenicity of recombinant outer surface proteins A (OspA) and B (OspB) of Borrelia burgdorferi was investigated. Both non-acylated OspA and OspB derived from strain B31 were expressed in Escherichia coli and purified by reversible citraconylation and anion-exchange chromatography. Antisera to OspA or OspB were prepared in mice with antigens formulated with QS-21 or alum, and evaluated for specific immunoglobulin G isotypes, agglutination and borreliacidal activity. QS-21 significantly enhanced IgG2a and IgG2b antibody responses to OspA and OspB, and IgG1 response to OspA when compared with the formulation containing antigen alone. In contrast, alum significantly inhibited the induction of IgG2a and IgG2b responses to OspA. Alum had no significant effect on IgG1 response to OspA, or IgG2a and IgG2b responses to OspB, but significantly enhanced IgG1 antibody response to OspB. Antisera to OspA or OspB formulated by QS-21 possessed higher titres of agglutinating antibody than antisera to OspA or OspB alone. Borreliacidal activity was eight- to 64-fold higher in antisera to OspA formulated with QS-21 than in antisera to OspA formulated with or without alum. These antisera were highly borreliacidal to New York strain B31, a California isolate CA-2-87, German isolate Fr, and Swedish isolate G25. Antisera to OspB formulated with QS-21 were highly borreliacidal to strains B31 and Fr, but not to CA-2-87 and G25. Antisera to OspB formulated with alum were borreliacidal only to B31. Thus, OspA was superior to OspB and QS-21 superior to alum at eliciting functional antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymerase chain reaction amplification of culture supernatants for rapid detection of Borrelia burgdorferi

A combination of culture and polymerase chain reaction (PCR) amplification was employed to detectBorrelia burgdorferi in in vitro cultures of skin biopsy specimens. Spirochetes were observed by microscopic examination in 56 % (20/36) of the cultures from patients with erythema migrans who had not received prior antibiotic treatment. No growth ofBorrelia burgdorferi was detected in control cultures or those from patients who had received antibiotics. PCR analysis of culture supernatants agreed with microscopic evaluation in 50/51 evaluable cultures tested (both positive and negative). At two weeks of incubationBorrelia burgdorferi could be detected by PCR in 19/20 cultures (95 %) compared to 14/20 (70 %) by visual inspection. This study indicates that a combined culture-PCR test for detection ofBorrelia burgdorferi is more rapid and specific than culture alone.

Immune response to the Lyme spirochete Borrelia burgdorferi affected by ethanol consumption.

Rats fed excessive amounts of ethanol developed marked hematologic and immunologic changes. These included a reversal of the normal lymphocyte to granulocyte ratio in the peripheral blood, lower spleen and lymph node weights and a greatly reduced capacity to express normal cell mediated immune functions, based on poor lymphocyte reactivity in vivo, and in vitro to T and B cell mitogens and borrelial antigens shortly after primary immunization with the bacterial spirochete, Borrelia burgdorferi. Further evidence for impaired immune function caused by ethanol was based on little or no antibody response against Borrelia in rats following in vivo sensitization with B. burgdorferi incorporated in complete Freunds adjuvant. These findings provide substantial direct evidence strengthening the notion that high levels of ethanol ingestion adversely affect the host immune system and can interfere with the immune response to microorganisms.

Suppression of cellular immunity by lyme disease spirochete Borrelia burgdorferi

Modulation ofin vitro cellular immune responses by the spirocheteBorrelia burgdorferi, the bacteria that causes Lyme disease, was demonstrated. When cultured in the presence of sonicatedBorrelia preparation (Bb), the mitogen- or antigen-stimulated proliferative responses of normal lymphocytes were consistently lowered as compared with control values. The degree of the proliferation reduction was directly proportional to both Bb quantity and length of exposure in culture. Bb caused the greatest reduction in both Con A and specific antigen such as BCG-stimulated proliferation, where an almost 100% reduction in proliferation could be achieved. Bb also reduced PHA- or PWM-stimulated PBL proliferation, with the PWM proliferation being the least affected. Also, IL2 production was significantly reduced from Bb-exposed lymphocytes. The entry of Bb-exposed lymphocytes into the proliferating phases of the cell cycle was also shown to be blocked. This regulatory activity was determined to be Bb proteins, sensitive to trypsin treatment and estimated to be 62–100 kDa in molecular weight. Therefore, the presence of spirochetes or their antigens in the hosts may block lymphocyte proliferation and activation, underscoring an important pathogenic mechanism of Lyme disease.