MM-3 was a live vaccine strain candidate for protecting neonatal piglets from diarrhea. Designed in the 1980s, a high degree of protection from colibacillosis was afforded to piglets in a challenge study and field trials. However MM-3 had a drawback of carrying the antibiotic resistance gene (chloramphenicol acetyltransferase gene, cat). The introduction of a host-plasmid balanced lethal system into the vaccine was a good idea to solve the problem. The lambda-Red recombination system was adopted in this study to realize the replacement of cat by aspartate-semialdehyde dehydrogenase gene (asd) in the plasmid pMM085. The new plasmid named pMMASD was introduced into an Escherichia coli strain chi6097 and Salmonella typhimurium chi4072 where the asd gene had been knocked out in their chromosomes. Cultured in an Erlenmeyer flask, expression levels of two antigens K88ac fimbriae and heat-labile enterotoxin B subunit (LTB) in cell lysate were similar among MM-3, chi4072(pMMASD) and chi6097(pMMASD). However, chi4072(pMMASD) possessed the more effective secretion mechanism to transport LTB enterotoxin into culture liquid. The relatively higher stability of pMMASD in Salmonella typhimurium chi4072 than that of pMM085 in MM-3 was determined both in vitro in the absence of selective pressure, and in vivo following oral inoculation. Oral immunization of BALB/c mice with chi4072(pMMASD) or chi6097(pMMASD) was sufficient to elicit IgA responses in mucosal tissues as well as systemic IgG antibody responses to the K88 fimbriae, while MM-3 failed to elicit specific antibody responses to K88 fimbriae in mucosal tissues. Among three live strains, only chi4072(pMMASD) could develop strong humoral responses against LTB enterotoxin. The results suggest that chi4072(pMMASD) is expected to be a promising live vaccine strain.