Application of molecular methods for the identification of Clostridium sporogenes, Clostridium perfringens and Clostridium sordellii isolated from cattle

The results of research on the development of an effective method of species identification of Clostridium sporogenes, Clostridium perfringens and Clostridium sordellii based on polymerase chain reaction (PCR), as well as the possibility of its practical use for the diagnosis of clostridiosis in cattle are presented. A total of 90 samples of biological material collected from diseased animals in the farms of the Novosibirsk region in 2023 were studied by bacteriological and biochemical methods. PCR was performed with real-time detection. The obtained results were confirmed by sequencing of PCR fragments of 16S rRNA gene. As a result of bacteriological studies, culture-morphological and biochemical properties, 44 isolates of clostridia belonging to nine species were identified. Three primer and probe pairs were selected for the amplification of the gene fragments gerKA C. sporogenes, plc C. perfringens, and NanS C. sordellii. As a result of experimental studies, the working concentrations of primers and probes, providing the necessary sensitivity of the method, were determined and the conditions of PCR were optimized. Specificity of the selected oligonucleotides was tested using the strains of other species clostridia and bacteria of other species. The sensitivity of PCR was at least 102 CFU/ml for pure bacterial cultures and at least 105 CFU/ml for suspensions from biomaterial samples. Compared to direct examination of the biomaterial samples in mixed cultures of bacteria isolated from the same samples on nutrient media, the number of positive results was higher (53 and 37%, respectively). The developed method allows for shorter and more effective diagnostics of bovine clostridiosis. Sensitivity of the developed method is higher when examining the bacterial cultures isolated on nutrient media than when directly examining biomaterial samples. Subsequent more detailed study of molecular genetic characterization of the identified clostridium isolates for the presence of toxin genes will help to determine their potential toxigenicity and role in disease development. 

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