Pentavalent Recombinant Vaccine Using Escherichia coli Bacterins Against Major Clostridium Toxins in Ruminants

Abstract Clostridium perfringens and Clostridium botulinum are anaerobic spore-forming bacteria that produce potent toxins and are major causative agents of severe toxicoinfections in ruminants. Although vaccination remains the primary preventive strategy, current commercial vaccines are based on crude toxoid preparations, which present several limitations, including labor-intensive manufacturing processes, high biological risk, and batch-to-batch variability. This study aimed to evaluate the immunogenicity of a pentavalent recombinant bacterin vaccine formulation containing antigens derived from C. perfringens (CPA, CPB, and ETX) and C. botulinum (BoNT/C and BoNT/D) in cattle, sheep, goats, and buffaloes. Individual and chimeric recombinant antigens were expressed in Escherichia coli and incorporated into a vaccine formulation containing three inactivated bacterial components (bacterins) adsorbed onto aluminum hydroxide. A total of 92 ruminants (23 cattle, 23 buffaloes, 23 sheep, and 23 goats) were included in the study and allocated to receive the recombinant vaccine, a commercial vaccine, or saline as a negative control. Neutralizing antibody titers were determined by serum neutralization assays. The recombinant vaccine was well tolerated across all species and induced neutralizing antibody titers of 10 IU/mL against CPB, 5 IU/mL against BoNT/C, and 4 IU/mL against BoNT/D. The response to ETX was species-dependent, with cattle and sheep reaching antibody titers of 4 IU/mL, whereas buffaloes and goats reached 2 IU/mL. Neither the recombinant bacterin nor the commercial vaccine elicited detectable neutralizing antibody responses against CPA, highlighting the need for further strategies to improve the immunogenicity of this antigen. Nevertheless, the overall findings support the potential of the pentavalent recombinant vaccine to confer protective immunogenicity against multiple clostridial toxins. Incorporating recombinant nontoxic antigenic domains into a bacterin-based platform represents a safe, scalable, and antigen-sparing alternative to conventional toxoid vaccines, with promising implications for improving the prevention and control of clostridial diseases in ruminant livestock.