Blackleg, also known as clostridial myositis caused by Clostridium chauvoei, is an acute and often fatal disease primarily affecting cattle and buffalo posing a threat to livestock health and productivity worldwide. Despite routine vaccination against Blackleg in Pakistan, recurrent outbreaks continue to occur, highlighting the need for improved understanding of circulating strains and their toxigenic potential. In the present study, tissue samples were collected from 15 clinically suspected blackleg cases, from which five C. chauvoei isolates were confirmed based on morphological, biochemical, and molecular characterization. Initial identification was performed using culture-based methods under anaerobic conditions, followed by Gram and spore staining along with biochemical profiling. Molecular confirmation was achieved through amplification of the 16S rRNA gene, yielding ~1500bp product. Toxinotyping was conducted using gene-specific PCR assays targeting genes including a specie specific gene (CCF516) yielding an amplicon of 516bp while cctA, and CCTO2AL genes, yielded amplicons of 1120bp, and 1400bp, respectively, confirming the alpha toxin associated genes. Optimization of alpha toxin production was evaluated under different physical conditions, including variations in pH, temperature, and incubation time. Toxin activity was quantified using hemolytic assays with sheep red blood cells, revealing maximum activity at 37°C and neutral pH after 24 hours, while increased temperature and prolonged incubation resulted in a marked decline in toxin activity. These findings highlight the variability in toxin production among local C. chauvoei isolates, underscoring the need to characterize circulating strains to enhance diagnostics and inform better control strategies for Blackleg disease.

To Cite This Article: Tariq N, Ghafoor A, Yaqub T and Ijaz M, 2026. Characterization and alpha toxinotyping of Clostridium chauvoei from cattle and buffaloes in punjab, pakistan. Pak Vet J. http://dx.doi.org/10.29261/pakvetj/2026.131