In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) F41 infections, we have developed a surface antigen display system using poly-␥-glutamate synthetase A (PgsA) as an anchoring matrix. The recombinant fusion proteins comprised of PgsA and fimbrial protein of F41 were stably expressed in Lactobacillus casei 525. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy, and flow cytometry. Oral inoculation of recombinant L. casei 525 into specific-pathogen-free BALB/c mice resulted in significant mucosal immunoglobulin A (IgA) titers that remained elevated for >16 weeks. High levels of IgG responses in sera specific for F41 fimbriae were also induced, with prominent IgG1 titers as well as IgG2a and IgG2b titers. The helper T-cell (Th) response was Th2-cell dominant, as evidenced by increased mucosal and systemic interleukin-4-producing T cells and a concomitant elevation of serum IgG1 antibody responses. More than 80% of the mice were protected against challenge with a 2 ؋ 10 4 -fold 50% lethal dose of standard-type F41 (C83919). The induced antibodies were important for eliciting a protective immune response against F41 infection. These results indicated that the use of recombinant L. casei 525 could be a valuable strategy for future vaccine development for ETEC.Enterotoxigenic Escherichia coli (ETEC) strains colonize the small intestine, secrete enterotoxins, and cause diarrhea. Colonization is facilitated by pili (fimbriae). Pili facilitate the adherence of ETEC to intestinal mucosa (27). Pilus adhesins that are known to be important in ETEC infections of neonatal animals are K88, K99, 987P, FY, and F41 (26,28,29,38). F41 is less prevalent than K88, K99, or 987P and is usually accompanied by K99 (25). There is, however, strong suggestive evidence that F41 can mediate colonization by adhesion. Variants of a K99-and F41-positive porcine ETEC strain that have lost the K99 gene (29) and still carry the gene for and produce F41 are still virulent for newborn pigs (13).The previously conventional vaccine variability in levels of protective immunity may have been due to the lack of stimulation of appropriate mucosal immunity, since these vaccines were delivered parenterally. Mucosal immunization has proven to be an effective approach against the colonization of pathogens and their further spread to the systemic circulation (15, 34). Therefore, it is necessary to develop efficient and safe antigen vectors that will be able to trigger mucosal and systemic immune responses. One promising approach relies on the use of live bacterial vehicles (22). For mucosal immunization, lactic acid bacteria (LAB) are more attractive as delivery vehicles than other live vaccine vectors (e.g., Shigella, Salmonella, and Listeria spp.) (1,3,20,21) because LAB are considered safe, they exhibit adjuvant properties, and they are weakly immunogenic (7,9,10,12,23,41). In addition, extracellularly accessible antigens expressed on the s...