Species identification of bacteria of the genus Clostridium isolated from cattle by multiplex real-time PCR

Background. Diseases caused by clostridia are widespread in cattle. In addition to pathogenic clostridia, non-pathogenic species are constantly living in the organism of animals, and to make a correct diagnosis it is necessary to differentiate isolated cultures of these bacteria. A promising direction for solving this problem is the search for highly specific and sensitive methods of genetic analysis. Based on the application of real-time polymerase chain reaction (PCR), which makes it possible to detect and determine the species affiliation of microorganisms in a short period of time.

Purpose.  To develop a multiplex real-time polymerase chain reaction for the detection of toxigenic Clostridium species: Clostridium sporogenes, Clostridium perfringens and Clostridium sordellii in mixed and pure bacterial cultures, to determine its sensitivity and specificity.

Materials and methods. In our studies we used samples of biological material from sick animals, from which clostridia cultures were isolated on artificial nutrient media with their subsequent identification based on the results of culture and biochemical properties. Primers and probes were designed using the PrimerQuest tool, and then the sensitivity and specificity of PCR were determined using reference strains and bacterial isolates.

Results. In the period from 2023 to 2024, 90 samples of biomaterial from cattle collected in livestock farms of the Novosibirsk Region were investigated. As a result of bacteriological studies, 44 isolates of clostridia of nine species of the genus Clostridium were obtained. For the selection of primers and probes, we analysed three pairs of primers and probes, which were used to detect the gerKA gene in C. sporogenes, C. perfringens - plc, C. sordelli - NanS. In addition, a pair of primers was used to detect species of the genus Clostridium by the 16S RNA gene. In order to determine the working concentrations of primers and probes that would provide the necessary sensitivity of the analysis, a series of studies were carried out to optimise the conditions of the analysis. Analysing the results of the studies, we determined the sensitivity of the reaction, which was not less than 102 CFU/ml for pure bacterial cultures of C. sporogenes, C. perfringens and C. sordelli species, and for other species of Clostridia spp. 101 CFU/ml.

Conclusion. The developed multiplex real-time PCR allows short-term diagnosis of bovine clostridiosis and species identification of pathogens. In addition to C. perfringens, C. sporogenes, C. sordellii, other species of Clostridium spp. may be involved in the etiology of bovine clostridiosis. It is necessary to improve diagnostic methods of clostridiosis and expand the range of detectable clostridium species.