Immunization of sheep with a recombinant vaccine containing immunogenic nontoxic domains of Clostridium perfringens alpha and beta toxins.

Clostridium perfringens (types A and C) can cause several diseases by secreting alpha (CPA) and beta (CPB) exotoxins in the gastrointestinal tract. Although vaccination is the main measure of immunization against C. perfringens, available vaccines have limitations in terms of productivity and safety. Thus, recombinant vaccines are an important, more effective, practical, and safer strategy in the immunization of animals. In this study, we evaluated the immunization of sheep with recombinant Escherichia coli bacterins expressing CPA and CPB complete proteins (co-administered), the immunogenic nontoxic domains rCPA-C247-370 and rCPB-C143-311 co-administered or fused as a bivalent chimera (rCPBcAc). For this, in silico analysis was performed to design rCPBcAc, considering the stability of the mRNA (-278.80 kcal/mol), the degree of antigenicity (0.7557), the epitopes of the B cell ligand, and different physicochemical characteristics. All proteins were expressed in vitro. In vivo, animals vaccinated with the co-administered antigens rCPA + rCPB and rCPA-C+ rCPB-C (200 µg each) had mean CPA and CPB neutralizing antitoxin titers of 4, 10, 4.8, and 14.4 IU/mL, respectively, while those vaccinated with 200 µg of rCPBcAc chimera (approximately 100 µg of each antigen) had titers of <4 and 12 IU/mL of CPA and CPB antitoxins, respectively, 56 days after the administration of the first dose. In addition, the chimera was considered to be immunogenic for inducing antitoxin titers using the half dose. In this study, we presented a new recombinant antigen potentially applicable for vaccines against the CPA and CPB toxins for preventing diseases caused by Clostridium perfringens.

Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools.

Beta toxins (CPB) produced by Clostridium perfringens type B and C cause various diseases in animals, and the use of toxoids is an important prophylactic measure against such diseases. Promising recombinant toxoids have been developed recently. However, both soluble and insoluble proteins expressed in Escherichia coli can interfere with the production and immunogenicity of these antigens. In this context, bioinformatics tools have been used to design new versions of the beta toxin, and levels of expression and solubility were evaluated in different strains of E. coli. The immunogenicity in sheep was assessed using the molecule with the greatest potential that was selected on analyzing these results. In silico analyzes, greater mRNA stability (-169.70 kcal/mol), solubility (-0.755), and better tertiary structure (-0.12) were shown by rCPB-C. None of the strains of E. coli expressed rFH8-CPB, but a high level of expression and solubility was shown by rCPB-C. Higher levels of total and neutralizing anti-CPB antibodies were observed in sheep inoculated with bacterins containing rCPB-C. Thus, this study suggests that due to higher productivity of rCPB-C in E. coli and immunogenicity, it is considered as the most promising molecule for the production of a recombinant vaccine against diseases caused by the beta toxin produced by C. perfringens type B and C.

Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.