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Previous studies with different Borrelia burgdorferi sensu stricto (s.s.) strains revealed that temperature as well as cocultivation with tick cells modulates the expression of outer surface proteins (Osp) A and C. We investigated the effects of temperature and of interaction with tick cells in culture on the expression of OspA and OspC of the B. afzelii clones cPKo97 and cPKo345 in comparison to the B. burgdorferi s.s. strain N40. To follow the dynamics of Osp expression of single borreliae we used indirect immunofluorescence microscopy with double staining of OspA and OspC. Clone PKo345 always showed expression of only OspA, regardless the conditions it was subjected to. Sequencing of the ospC gene disclosed a insertion leading to a stop codon after base 222 and inability to produce OspC. In cPKo97 and N40 OspC is down-regulated at lower temperatures and up-regulated at higher temperatures, which was especially pronounced on cocultivation with tick cells. Borreliae adherent to tick cells showed greater OspA expression compared to the nonadherent ones, an indication that OspA might play a role as adhesin for tick cells. Interestingly, cPKo97 and N40 displayed different patterns of Osp expression: cPKo97 simultaneously presents OspA and OspC on single borreliae, while N40 has either OspA or OspC on single cells. Adaptation of OspC expression in cPKo97 seems to occur by up- or down-regulation of this protein on single borreliae, as shown by alternating intensities of OspC expression at different temperatures. In contrast, N40 seem to consist of two subsets of borreliae one expressing only OspA and the other only OspC, and change in temperature results in growth benefit for one of these subtypes. Our findings indicate that, regarding OspA and OspC expression, response to temperature and cocultivation with tick cells of B. afzelii is comparable to B. burgdorferi s.s., but the mode of regulation seems phenotypically different. Further European isolates should be investigated for OspA and OspC regulation, especially in the face of vaccine development for the European situation.