Construction of Recombinant Lactiplantibacillus plantarum NC8 Expressing ETEC FaeC and LngC and Preliminary Evaluation of Immune Responses in an ETEC-Challenged Piglet Model

Enterotoxigenic Escherichia coli (ETEC) is a major pathogen responsible for diarrhea and mortality in piglets, causing substantial economic losses to the swine industry. Its pathogenicity relies on the synergistic action of colonization factors and enterotoxins, which mediate intestinal colonization and induce secretory diarrhea. Commercial vaccines against ETEC remain limited, and their protective efficacy in piglets is often unsatisfactory. Consequently, probiotics have attracted increasing attention as mucosal immunomodulatory agents for the prevention and control of ETEC infection. In this study, ETEC, the minor subunit located at the tip of the fimbriae, FaeC, and the fimbrial assembly accessory protein, LngC, were individually inserted into Lactiplantibacillus plantarum NC8 using the anchoring expression vector pPG612.1, successfully generating the recombinant strains NC8/pPG612.1-FaeC and NC8/pPG612.1-LngC. Western blot analysis confirmed that both recombinant strains correctly expressed the target proteins upon lactose induction, and their growth curves exhibited no significant differences from those of the wild-type strain, indicating that genetic modification did not compromise bacterial proliferation. To evaluate the immune efficacy of the recombinant strains, thirty-six piglets were randomly assigned to six groups: PBS control, ETEC challenge, empty vector, LngC immunization, FaeC immunization, and combined immunization (MIX). After seven oral gavage immunizations, piglets were challenged with ETEC and euthanized on day 7 post-challenge for sample collection. The results showed that immunized piglets resumed body weight gain by day 4 after challenge, whereas the ETEC group exhibited sustained weight loss. Histopathological examination revealed relatively intact jejunal villus architecture and markedly less severe mucosal damage in immunized groups compared with the ETEC-challenged group. RT-qPCR analysis of intestinal tissues and ELISA analysis of serum showed that immunization with the recombinant strains significantly decreased the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-8) in intestinal tissues and their protein levels in serum, while increasing both the mRNA expression and protein levels of the anti-inflammatory cytokine IL-10. Flow cytometric analysis revealed that immunization with the recombinant strains significantly increased the frequencies of CD4+IFN-γ+ and CD4+IL-4+T cells and CD21+ B cells in MLNs, LPLs, and spleen compared with the control groups. The increased CD4+IFN-γ+ T-cell population indicated enhanced Th1-mediated cellular immunity, whereas the increased CD4+IL-4+ T-cell population suggested activation of Th2-associated humoral immunity. The expansion of CD21+B cells further indicated enhanced B-cell activation. Collectively, these findings suggest that the recombinant strains induced coordinated mucosal and systemic immune responses. Additionally, the secretory immunoglobulin A (sIgA) concentration in the jejunal contents was markedly increased, suggesting that the intestinal mucosal immune system was effectively activated. Collectively, these findings indicate that NC8/pPG612.1-FaeC and NC8/pPG612.1-LngC can elicit robust mucosal immune responses and alleviate diarrhea induced by enterotoxigenic Escherichia coli (ETEC) in piglets, providing a scientific basis for the development of oral probiotic-based vaccines against ETEC infection in swine.