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Outer surface protein (Osp) C immune pressure during persistent infection with Borrelia burgdorferi was examined in relation to genetic variation of ospC. Mice were infected with clonal B. burgdorferi sensu stricto (s.s.) N40 or B. afzelii PKo and then were hyperimmunized with homologous recombinant OspC or with decorin-binding protein A (DbpA) (controls). After 6 months, B. burgdorferi isolates were subjected to restriction enzyme analysis of the amplified ospC genes and were found to have no differences among 9 B. burgdorferi s.s. N40 and 9 B. afzelii PKo isolates from OspC hyperimmune mice or among 10 B. burgdorferi s.s. N40 and 10 B. afzelii PKo isolates from DbpA hyperimmune mice, compared with input inocula. Comparison of gene sequences among 4 B. burgdorferi s.s. N40 and 9 B. afzelii PKo isolates from OspC-immunized mice revealed no ospC variation from input inocula. Variation in ospC among B. burgdorferi isolates and species during chronic infection is not likely to be an important mechanism for immune evasion.