Design of methods to monitor and evaluate Clostridium chauvoei under industrial settings for generating a fermentation map

Abstract

Blackleg is a disease that affects cattle caused by the anaerobic and spore-forming bacteria Clostridium chauvoei. It possesses several virulence factors that act in synergy to cause the typical lesions. Flagellin (FliC) is a cell surface antigen that provides protective immunity to vaccinated animals against C. chauvoei infection. It is related to virulence and pathogenicity, thus, demonstrating a relationship between flagellar antigen quantity and final vaccine efficacy for blackleg disease, as demonstrated in several immunoassays. Clostridium chauvoei toxin A (CctA) is a pore-forming toxin secreted to the supernatant. It causes lysis by perforating the cell membrane, and it is known to be the main virulence factor of the pathogen. Other proteins such as sialidase and hyaluronidase are considered the main virulence factors of Clostridium chauvoei. Because blackleg is of economic importance, and as a disease that advances rapidly, vaccination has been available as a prevention method. However, since its development, the vaccine remains as a whole-cell formalin-inactivated culture due to the lack of knowledge of specific conditions to generate enough antigens to induce protection. Additionally, there is a variability of batch-to-batch immunogenic properties and final efficacy, which may be explained by the antigen’s quantity variation throughout the process. Although final batch testing is currently applied in the industry, there are no methods to monitor the fermentation dynamics and consistency of the overall production. Consistency can be achieved by setting parameters that constitute a product profile that satisfies final product requirements. The expression and purification of a recombinant FliC allowed the development of an indirect ELISA to measure the flagellin antigen quantity of Clostridium chauvoei fermentation samples. While the assay is not validated to predict the potency and final efficacy of the vaccine, it can be used as part of a battery of methods to provide a more characterized product profile to monitor trends and dynamics in the production. In addition, a rapid micro-plate bioactivity assay was developed to measure the hemolysis percentage caused by the supernatant of samples of C. chauvoei. The assays represent valuable methods to generate data and monitor the fermentation dynamics of C. chauvoei cultures under industrial conditions to achieve production consistency.