Identification of the taxonomic affiliation of Cryptosporidium spp. in piglets in the conditions of the north-west of the Russian Federation using molecular genetic methods

Abstract

The purpose of the research is to determine taxa of the genus Cryptosporidium species in pigs using molecular genetic methods in the north-west of the Russian Federation.Materials and methods. Fecal samples were taken from pigs of different age groups on farms of different types of incorporation that differ in climatic and geographical zones and animal keeping technologies in the Vologda Region. Microscopic research methods identified “positive” samples in which Cryptosporidium species were present; they were sorted out and deep-frozen. Then the samples were examined using the equipment of the resource center «Genomic Technologies, Proteomics and Cell Biology» of ARRIAM. The Cryptosporidium species in the fecal samples from farm animals were identified using high-throughput sequencing of 18S rRNA gene amplicon libraries obtained by a nested polymerase chain reaction (PCR) assay.Results and discussion. A primer system was designed for the nested PCR to amplify a potentially species-specific 393 bp fragment of the 18S rRNA gene. The sequence of the ILL_R2_ Zheng primer was modified with included degenerated positions to make the primer more versatile. As a result of sequencing of the libraries of 18S rRNA gene fragments obtained with the selected primers and subsequent taxonomic analysis of the nucleotide sequences, it was shown that all the studied samples included representatives of only one species, Cryptosporidium scrofarum.