toxins Article Occurrence and Toxicogenetic Profiling of Clostridium perfringens in Buffalo and Cattle: An Update from Pakistan Muhammad Umar Zafar Khan 1,2 , Muhammad Humza 3,4,† , Shunli Yang 1,2,† , Mughees Aizaz Alvi 5 , Muhammad Zahid Iqbal 6 , Hafiza Zain-ul-Fatima 7 , Shumaila Khalid 8 , Tahir Munir 8 and Jianping Cai 1,2, *   Citation: Khan, M.U.Z.; Humza, M.; Yang, S.; Alvi, M.A.; Iqbal, M.Z.; Zain-ul-Fatima, H.; Khalid, S.; Munir, T.; Cai, J. Occurrence and Toxicogenetic Profiling of Clostridium perfringens in Buffalo and Cattle: An Update from Pakistan. Toxins 2021, 13, 212. https://doi.org/10.3390/ toxins13030212 Received: 9 February 2021 Accepted: 11 March 2021 Published: 13 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 State Key Laboratory of Veterinary Etological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China; umarzafar8@caas.cn (M.U.Z.K.); yangshunli@caas.cn (S.Y.) 2 Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou 225009,China 3 Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs/ Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2017Y90100131@caas.cn 4 Department of Plant Pathology, University of Agriculture, Faisalabad 38000, Pakistan 5 Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad 38000, Pakistan; mugheesaizazalvi@gmail.com 6 Department of Veterinary Medicine, University of Veterinary and Animal Sciences, Outfall Road, Lahore 54000, Pakistan; zahid.iqbal@uvas.edu.pk 7 Veterinary Research Institute, Zarrar Shaheed Road, Lahore Cantt., Lahore 54810, Pakistan; dr.hafiza.zain@gmail.com 8 Department of Livestock and Dairy Development, Lahore 54000, Pakistan; shumailakhalid.sk@gmail.com (S.K.); dr.tahir99@gmail.com (T.M.) * Correspondence: caijianping@caas.cn † Equally contributed as the first author. Abstract: Clostridium perfringens 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, CPE, and NetB. 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 cpa and etx, 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. C. perfringens exhibits a range of toxinotypes that can be diagnosed via genotyping, which is more reliable than classical toxinotyping. Keywords: Clostridium perfringens; toxins; genotypes; occurrence; optimization Key Contribution: This manuscript states the occurrence of toxinotypes isolated from Clostridium perfringens in bovine areas of Punjab province in Pakistan and it can be applied to the proper prediction of the isolated toxinotypes. The genotyping could be helpful in appropriate diagnosis. Toxins 2021, 13, 212. https://doi.org/10.3390/toxins13030212 https://www.mdpi.com/journal/toxins