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A single injection of serum from C3H mice at 90 days after intradermal inoculation with 10(4) Borrelia burgdorferi spirochetes protected naive mice when administered subcutaneously at -18 h relative to intradermal challenge inoculation with 10(4) B. burgdorferi spirochetes. When immune serum was given at intervals (-18, 0, +24, +48, and +96 h) relative to intradermal challenge with 10(4) B. burgdorferi spirochetes, it was protective if given before or at the time of challenge but not at later times. Protection with 90-day serum given at -18 h was effective at dilutions up to 1:32, but not 1:64, on the basis of culture or disease at either 5 or 15 days after challenge. Passive immunizing activity was also present in sera from mice at 21 days after intradermal challenge with 10(4), 10(2), or 10(1) spirochetes, indicating that the immunizing component was not dose dependent and probably not related to antibody to outer surface protein A. Passive immunizing titers of sera from mice at days 1, 15, 30, 90, 180, and 360 after intradermal B. burgdorferi inoculation appeared as early as day 15, were highest on day 30, and then declined progressively on days 90, 180, and 360. Immunizing titers of sera from mice at 360 days after intradermal B. burgdorferi inoculation were identical in passively immunized mice challenged with the original inoculum or with B. burgdorferi isolated at 360 days after inoculation, suggesting that there was no antigenic discrimination between the original inoculum and late isolates. These results suggest that protective antibody is produced early in the course of B. burgdorferi infection and is unrelated to antibody to outer surface protein A. In addition, the decline of protective serum titers over time despite persistent infection suggests that the antigens eliciting the protective response are either modified or suppressed, but antigenic modification could not be demonstrated.