Fig 5. B. burgdorferi expresses distinct levels of CspA at different stages of enzootic cycle.

C3H/HeN mice were infected with 10⁵ B. burgdorferi strain B31-5A15. At 14 days post infection, the uninfected I. scapularis larval ticks were allowed to feed on these mice to repletion. After the replete larvae molted into nymphs, these B. burgdorferi-infected nymphs were allowed to feed on naïve C3H/HeN mice for different period of time or to repletion. The mice were euthanized at 7 or 14 days post feeding (“7dpf” or “14dpf”) to collect the inoculation site of skin (for mice at 7 and 14 days post feeding), ears, tibiotarsus joints, bladder, and heart (for mice at 14 days post feeding). RNA was extracted from replete larvae (“larvae replete”), flat nymphs (“nymph flat”), fed nymphs at 24 hours post feeding (“24hpf”) or replete nymphs (“nymph replete”) as well as in vitro cultured B. burgdorferi strain B313 (“B313”, negative control) or B31-5A15 (“B31-5A15”, positive control) in BSKII medium. (A) RNA was also extracted from mouse tissues including tick biting site (“inoc. site”), ears, tibiotarsus joints, bladder, and heart at 7 and/or 14 days post feeding. The extracted RNA was then used to determine the expression levels of cspA and constitutive expressed genes flaB and recA using qRT-PCR (see Materials and methods). The expression levels of (Top panel; negative control) recA and (Bottom panel) cspA are presented by normalizing the expression levels of flaB (negative control) (see Materials and methods). Each bar represents the mean of five independent determinations ± SEM (“#”). Significant differences (P < 0.05 by one-way ANOVA with post hoc Bonferroni correction) in the normalized expression levels of cspA between two conditions relative to each other. (B and C) The bacteria isolated from ticks or in vitro cultured B. burgdorferi strains were applied to flow cytometry. (B) Representative histograms of flow cytometry analysis showing the production levels of CspA in B. burgdorferi in the indicated environment. (C) The production of FlaB (Top panel; negative control) and CspA (Bottom panel) are presented as “ΔMFI”, the mean fluorescence index obtained from each of these strains subtracting that obtained from the strains stained only by the secondary antibody. Each bar represents the mean of three independent determinations ± SEM. Significant differences (P < 0.05 by one-way ANOVA with post hoc Bonferroni correction) in the production of CspA relative to the B313 cultured in vitro (“*”) or between two conditions relative to each other (“#”).