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The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.