| Summary: | <i>Clostridium perfringens</i> is a Gram-positive bacterium that possess seven toxinotypes (A, B, C, D, E, F, and G) that are responsible for the production of six major toxins, i.e., α, β, ε, ι, <i>CPE</i>, and <i>NetB</i>. The aim of this study is to find out the occurrence of toxinotypes in buffalo and cattle of Punjab province in Pakistan and their corresponding toxin-encoding genes from the isolated toxinotypes. To accomplish this aim, six districts in Punjab province were selected (i.e., Lahore, Sahiwal, Cheecha Watni, Bhakkar, Dera Ghazi Khan, and Bahawalpur) and a total of 240 buffalo and 240 cattle were selected for the collection of samples. From isolation and molecular analysis (16S rRNA), it was observed that out of seven toxinotypes (A-G), two toxinotypes (A and D) were found at most, whereas other toxinotypes, i.e. B, C, E, F, and G, were not found. The most frequently occurring toxinotype was type A (buffalo: 149/240; cattle: 157/240) whereas type D (buffalo: 8/240 cattle: 7/240) was found to occur the least. Genes encoding toxinotypes A and D were <i>cpa </i>and <i>etx</i>, respectively, whereas genes encoding other toxinotypes were not observed. The occurrence of isolated toxinotypes was studied using response surface methodology, which suggested a considerable occurrence of the isolated toxinotypes (A and D) in both buffalo and cattle. Association between type A and type D was found to be significant among the isolated toxinotypes in both buffalo and cattle (P < 0.05). Correlation was also found to be positive and significant between type A and type D. <i>C. perfringens</i> exhibits a range of toxinotypes that can be diagnosed via genotyping, which is more reliable than classical toxinotyping.
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