Abstract
ABSTRACTClostridium perfringens is a common opportunistic pathogen endangering livestock and poultry breeds. Here, using enhanced green fluorescent protein as screening marker, a recombinant lactobacillus tetravalent vaccine constitutively expressing α, ε, β1, and β2 toxoids of C. perfringens was developed, and its immunogenicity in mice was investigated via oral administration. This probiotic vaccine could effectively induce antigen-specific secretory IgA (sIgA)-based mucosal and IgG-based humoral immune responses, and significantly high levels (p< 0.05) of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and IFN-γ were produced in immunized mice. Moreover, lymphoproliferation and percentage of CD4+ and CD8+ T cells significantly increased in mice of the probiotic vaccine group. Challenge experiments were performed in mice with C. perfringens toxinotypes A, C, and D crude toxins to evaluate protection efficiency of the probiotic vaccine, using a commercial inactivated C. perfringens vaccine made by C. perfringens toxinotypes A, C, and D as vaccine control. We observed 80% protection rate in the probiotic vaccine group, which was higher than commercial vaccine group, whereas all mice in control groups died and obvious histopathological changes were observed in liver, spleen, kidney, and intestines of mice. Significantly, we compared the immunogenicity and protection efficiency of lactobacillus constitutive expression system and lactobacillus inducible expression system, and results showed that lactobacillus constitutive expression system has obvious advantages. Our study clearly demonstrated that the probiotics vaccine could effectively induce mucosal, humoral, and cellular immunity, and provide effective protection against C. perfringens toxins, suggesting a promising strategy for the development of oral vaccine against C. perfringens.
Highlights
Clostridium perfringens (C. perfringens), a Grampositive, spore-forming anaerobic bacillus that is widely distributed in natural environments and can usually be found in the gastrointestinal tract of livestock and poultry as an opportunistic pathogen, is associated with human food-borne diseases as well as animal diseases [1,2], and exotoxins are its main virulence factors
The expression of the enhanced green fluorescent protein (EGFP) screening marker by pPG-ΔE-α-β2-ε-β1/L. casei 393 cultured in normal MRS medium was identified by ultra-high resolution microscopy and flow cytometric analysis, and the results showed that significant green fluorescence was observed on the pPG-Δ-E-α-β2-εβ1/L. casei 393 (Figure 2(a): A), but not on the pPG-T7g10PPT/L. casei 393 (Figure 2(a): B)
In order to confirm the constitutive expression of the fusion protein, the pPG-Δ-E-α-β2-ε-β1/L. casei 393 was grown in basic MRS broth supplemented with different carbon sources, and the immunoblot results showed that the α-β2-ε-β1 fusion protein of C. perfringens was constitutively expressed by the pPG-Δ-E-α-β2-ε-β1/L. casei 393 (Figure 2C), which can be recognized by mouse anti-α, β2, ε, and β1 toxoid monoclonal antibody, respectively
Summary
Clostridium perfringens (C. perfringens), a Grampositive, spore-forming anaerobic bacillus that is widely distributed in natural environments and can usually be found in the gastrointestinal tract of livestock and poultry as an opportunistic pathogen, is associated with human food-borne diseases as well as animal diseases [1,2], and exotoxins are its main virulence factors. The α-toxin with phospholipase C and sphingomyelinase activities is a major pathogenic factor influencing C. perfringens infections that are responsible for gas gangrene in sheep [5], and humans [6], which can be produced by all toxinotypes of C. perfringens but is the only toxin produced by toxinotype A [7,8]. C. perfringens can cause various diseases in different animals, including gas gangrene, acute enterotoxemia, and enteritis syndrome. The development of an effective vaccine against C. perfringens toxins is of great importance
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