Protection against necrotic enteritis in broiler chickens by co-administration of Enterococcus faecalis EF32 and recombinant Lactobacillus plantarum strain co-expressing FimA, FimB, NetB and Fba.

Necrotic enteritis (NE), caused by Clostridium perfringens(C. perfringen) type G carrying the NetB gene, represents a major infectious disease threatening the global poultry industry. Following the ban on in-feed antibiotic growth promoters, the incidence of NE has increased markedly, highlighting the urgent need for safe and effective prevention and control strategies. To address this challenge, we developed a novel probiotic-based oral immunization strategy.We constructed a recombinant Lactobacillus plantarum (L. plantarum) NC8 strain surface-displaying the FimB antigen [NC8(pYL515)] and orally co-immunized broiler chickens with the previously developed NC8(pYL590) strain co-expressing FimA, FBA, and NetB, together with Enterococcus faecalis EF32, to evaluate their protective efficacy against NE in broilers. In vitro, EF32 demonstrated a strong ability to promote the maturation of chicken bone marrow-derived DCs, as evidenced by the upregulation of CD83, CD86, CCL5, and CCR7 expression. Prior to challenge, the combination of live EF32 and multi-antigen recombinant strains established an enhanced systemic immunity (significantly elevated IgG), mucosal immunity (early activation of IgM and marked increase in sIgA) and cellular immunity (enhanced splenic lymphocyte proliferation and increased expression and secretion of IFN-γ and IL-4). In addition, the co-administration also significantly upregulated the mRNA levels of intestinal CCR7, CCR9, and CD86. Finally, in a broiler challenge model, this combined immunization strategy significantly alleviated intestinal lesions and mitigated growth suppression. Notably, live EF32 bacteria, as a key component exerting synergistic effects in this combined immunization strategy. Our findings demonstrate that combined immunization with recombinant L. plantarum co-expressing multiple antigens and live EF32 synergistically enhances the immunogenicity and protective efficacy of an oral vaccine, offering a promising antibiotic-free strategy for NE control.