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Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.