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By using real-time quantitative PCR, the population dynamics and gene transcription of Borrelia burgdorferi were examined in ticks and skin of mice during acquisition of the infection from mice by ticks and during transmission of the infection from ticks to mice. Population dynamics were determined by using a flaB DNA target. A quantitative analysis of flaB, ospA, ospC, dbpA, and arp transcription was also performed. The results revealed that both uninfected larval and nymphal Ixodes scapularis ticks acquired B. burgdorferi as early as 1 day after attachment and that the sizes of spirochete populations within ticks increased during feeding. In addition, all gene targets revealed that there was RNA transcription during feeding. Similar events occurred within infected nymphal ticks feeding on uninfected hosts. Transmission from infected nymphal ticks to mice could be detected within 1 day after attachment. Analysis of skin during the first 3 days after attachment of infected ticks revealed rising numbers of spirochetes but minimal gene transcription. In contrast, the skin of mice with established infections revealed static populations of spirochetes and active but stable transcription of flaB, ospC, dbpA, and arp. There were consistent reductions in the number of spirochetes in the skin at the tick attachment sites compared to the number of spirochetes in the skin at nontick sites, but there were no differences in gene expression between tick and nontick skin sites. Evidence of ospA transcription in skin could be found 1 day after tick attachment but not thereafter.