Meanwhile, the ETEC cells were harvested by centrifugation at 3,000 × g for 10 min at 4°C after being incubated at 37°C for 12 h with vigorous shaking. Next, the cell pellet was washed with sterile physiological saline twice. The cells were then diluted to 1 × 109 cfu mL−1 in PBS for the infectious challenge.