ELISA-based measurement of antibody responses and PCR-based detection profiles can distinguish between active infection and early clearance of Borrelia burgdorferi

Abstract

Borrelia burgdorferi is a spirochetal bacterium that causes Lyme disease. These studies address whether current research methods using either ELISA to detect seroconversion to B. burgdorferi antigens or PCR quantification of bacterial DNA within tissues can accurately distinguish between a productive infection versus a B. burgdorferi exposure that is rapidly cleared by the innate responses. Mice receiving even minimal doses of live B. burgdorferi produced significantly more B. burgdorferi-specific IgM and IgG than groups receiving large inocula of heat-killed bacteria. Additionally, sera from mice injected with varied doses of killed B. burgdorferi recognized unique borrelial antigens compared to mice infected with live B. burgdorferi. Intradermal injection of killed B. burgdorferi resulted in rapid DNA clearance from skin, whereas DNA was consistently detected in skin inoculated with viable B. burgdorferi. These data indicate that both ELISA-based serological analyses and PCR-based methods of assessing B. burgdorferi infection clearly distinguish between an established infection with live bacteria and exposure to large numbers of bacteria that are promptly cleared by the innate responses.

Figure 1

Comparison of Bb-specific antibody levels produced during active infection versus exposure to high numbers of killed bacteria. (a) Groups of B6 mice were injected with a single dose of live or heat-killed Bb, and serum was collected at either 2 or 4 weeks after infection; in some cases the killed Bb inoculum included an equal volume of complete Freund's adjuvant (CFA). The doses injected ranged from 1000 (“1e ³”) to 5 × 10⁷ (“5e ⁷”) killed bacteria, and 250 to 5 × 10⁴ (“5e ⁴”) live Bb. Individual sera were assessed for Bb-specific IgM content at 2 weeks after infection and for IgG content at 4 weeks after infection by ELISA analyses. Each circle represents the serum value for an individual animal, and the number beside the bar indicates the average value for that group. *indicates values that are significantly different from control mice (Un); **indicates values that are significantly different from mice injected with killed bacteria. (b) The sera assessed for IgG content in (a) were also assessed for the levels of the indicated individual IgG isotypes using similar ELISA techniques.

Figure 2

Antibodies produced against live and killed spirochetes recognize unique Bb antigens. Sera were collected from individual mice 4 weeks after injection with a single dose of either BSK II medium (lanes 2-3), 5 × 10⁷ killed Bb (lanes 4–9), 250 live Bb (lanes 10–12), or 5 × 10⁴ live Bb (lanes 14–16); one group initially received 10⁵ killed Bb + CFA, with a boost 3 weeks later with 10⁵ killed Bb + IFA, and sera collected 3 weeks later (lane 13). All sera were diluted either 1 : 25 (lanes 2-3 and 7–9) or 1 : 500 before using to immunoblot membranes containing electrophoresed Bb. A mAb specific for Bb OspA, H5332 [49], was included as a marker for OspA reactivity (lane 1).

Figure 3

DNA from killed Bb is rapidly cleared from infection sites and is undetectable by PCR. Groups of mice were injected intradermally with 10⁴ of either live or heat-killed Bb. Mice were sacrificed at the indicated times after injection, and a 6 mm skin sample encompassing the injection site was excised for DNA preparation. The relative Bb levels were assessed by real-time PCR using recA primers and normalized relative to the murine nid content. Each circle represents Bb recA levels relative to 1000 nid copies for an individual animal, and the number beside the bar indicates the average value for that group. These data reflect the combined results of two separate experiments.