Serum samples from the immunized mice or the control mice were examined for in vitro antibody activities against bacterial adherence as previously described . Briefly, ETEC bacteria expressing each CFA adhesin (3.5 × 106 CFUs; MOI of five bacteria per cell) pre-treated with 10% mannose were mixed with 20 μl serum from the immunized or the control mice and incubated on a shaker (50 rpm) for 1 h at room temperature. The bacteria/serum mixture (brought to 300 μl with PBS) was added to each well of a 24-well tissue culture plate which contains Caco-2 cells (ATCC, #HTB-37TM, 7 × 105 in confluent monolayer; in 700 μl cell culture medium) and incubated in a CO2 incubator (5% CO2) for 1 h at 37°C. After washes with PBS to remove non-adherent ETEC or E. coli bacteria, Caco-2 cells were dislodged with 0.5% triton X-100 (300 μl per well). Adherent ETEC or E. coli bacteria were collected by centrifugation (15,000 g for 10 min), suspended in 1 ml PBS, serially diluted, and plated on LB plates. Bacteria (CFUs) were counted after overnight growth at 37°C.