B. burgdorferi strain B31-5A15 (“B31-5A15”), B31-5A4NP1ΔcspA harboring the vector pBSV2G (“ΔcspA/Vector”), or this cspA mutant strain producing CspAB31 (“ΔcspA/pCspAB31”), CspAPKo (“ΔcspA/pCspAPKo”), CspAZQ1 (“ΔcspA/pCspAZQ1”), or CspAB31L246D (“ΔcspA/pCspAB31L246D”), or B313 carrying the vector pBSV2G (“B313/Vector”, negative control) was incubated with serum from human, mouse, or horse with a final concentration of 20%. The bacteria were stained with a guinea pig anti-C3 polyclonal IgG, a mouse anti-C5b-9 monoclonal antibody aE11 (for spirochetes incubated with human or horse serum), or a rabbit anti-C5b-9 polyclonal IgG (for spirochetes incubated with mouse serum) followed by an Alexa 647-conjugated goat anti-guinea pig IgG or goat anti-mouse IgG, or goat anti-rabbit IgG prior to being applied to flow cytometry analysis. Representative histograms of flow cytometry analysis showing the deposition levels of mouse (A) C3b or (C) MAC on the surface of indicated B. burgdorferi strains. The deposition levels of (B) C3b or (D) MAC of indicated animals on the surface of B. burgdorferi were measured by flow cytometry and presented as mean fluorescence index (MFI). 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 deposition levels of C3b or MAC relative to the B313/Vector (“*”) or between two strains relative to each other (“#”).