FIG 4.

Sera from immunized animals with individual vaccine formulations reduced adherence and pedestal formation on mouse primary intestinal epithelial cells (IECs). (A) Mouse primary IECs were infected at an MOI of 10 with EHEC O157:H7 previously incubated in the presence or absence of naive (adjuvant-only) or immune sera for 1 h at 37°C. After infection, cells were washed to remove nonadherent bacteria and fixed and stained with rhodamine phalloidin and DAPI for IECs and an EHEC-specific primary antibody conjugated to FITC. (B) The percentage of adhered bacteria was calculated 1 h after infection, where cells were washed, detached, and processed for CFU enumeration. (C) Recognition of EHEC O157:H7 by immune sera was visualized using immune or naive sera from vaccinated animals, followed by a secondary goat anti-mouse IgG antibody conjugated to Alexa488. All adherence data are expressed as means ± SEM of results from at least three independent experiments using sera from n = 8 mice per group. Significant differences were determined via one-way ANOVA followed by Tukey’s post hoc test (**, P < 0.01; ns, not significant).