Ultrasonic inactivation of Escherichia coli followed by irradiation was found to be the most efficient method for preparation of an effective vaccine against colibacillosis. Challenge experiments revealed that this vaccine provided the best protection compared with other methods of inactivation: heat, formaldehyde, and irradiation. Preparing the ultrasonicated vaccine from O2:K1 strain increased its range and also supported adequate protection against homologous strain O78:K80. The degree of protection conferred by the vaccine was positively correlated with the antibody titer against E. coli as measured on day of challenge. Low antibody titers detected 5 days post-vaccination resulted in only 20% protection. High antibody titers detected at 8 and 15 days post-vaccination correlated with a low number of chicks with lesions. In each challenged group, the live chicks that did not develop lesions had higher antibody titers than chicks with lesions, revealing a correlation between numbers of chicks with lesions and antibody titers as measured by enzyme-linked immunosorbent assay.