| Summary: | <i>Clostridium perfringens</i> (<i>C. perfringens</i>) is a bacterium that commonly causes zoonotic disease. The pathogenicity of <i>C. perfringens</i> is a result of the combined action of α, β, and ε exotoxins. In this study, <i>Lactobacillus crispatus</i> (pPG-T7g10/<i>L. crispatus</i>) expressing the main toxoids of <i>C. perfringens</i>, α, ε, β1, and β2, with EGFP-labeling, was constructed, and the protective effect was estimated in chickens. The α-β2-ε-β1 toxoid was constitutively expressed for confirmation by laser confocal microscopy and western blotting, and its immunogenicity was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical assays. After booster immunization, the probiotic vaccine group showed significantly higher levels (<i>p</i> < 0.05) of specific secretory IgA (sIgA) and IgY antibodies in the serum and intestinal mucus. Furthermore, the levels of cytokines, including interferon (IFN)-γ, interleukin (lL)-2, IL-4, IL-10, IL-12, and IL-17, and the proliferation of spleen lymphocytes in chickens orally immunized with pPG-E-α-β2-ε-β1/<i>L. crispatus</i> increased significantly. Histopathological observations showed that the intestinal pathological changes in chickens immunized with pPG-E-α-β2ε-β1/<i>L. crispatus</i> were significantly alleviated. These data reveal that the probiotic vaccine could stimulate mucosal, cellular, and humoral immunity and provide an active defense against the toxins of <i>C. perfringens</i>, suggesting a promising candidate for oral vaccines against <i>C. perfringens.</i>
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