Quantitative PCR analysis of intestinal microbiota in suckling piglets of different age groups with diarrhea

Abstract

The article presents the results of quantitative PCR detection of intestinal intestinal microbiota in suckling piglets of different age groups with diarrhea. To determine the etiology of diarrhea in piglets, 4 groups of animals were formed – two experimental and two control aged 3–5 and 17–21 days of life. Each group consisted of 6 piglets. The piglets in the experimental groups showed signs of diarrhea, and the pigs in the control groups were clinically healthy. Rectal smears were taken from piglets and immediately frozen at a temperature of -18 °C – -22 °C and stored in this form until laboratory examination. Isolation of genetic material from rectal smears was performed using a set of reagents for DNA/RNA extraction manufactured by Biosellal (France) on an automatic device “KingFisher Duo Prime” (USA). Performed mechanical lysis of biological material on the device for homogenization “FastPrep-24” (France). Specific regions of rotavirus RNA types A and C (PRV A, PRV C), the causative agent of swine diarrhea (PEDV) and Cl DNA fragments were determined in the purified nucleic acid solution. perfringens, E. coli, Cryptosporidium spp, Eimeria spr, Cystoisospora suis, as well as toxin-producing genes A and B Cl. difficile. Determinations of pathogenic E. coli was performed by toxin genes – STa, STb, LT, STX1, STX2 and EAE, as well as for by coding genes – F4, F5, F18 and F41. The obtained results of the study of the genetic material of microorganisms extracted from rectal smears from clinically healthy pigs and with manifestations of diarrhea characterize the state of the microbiome and the distribution of microorganisms in the intestines of suckling piglets. It was found that the species composition of intestinal microorganisms in clinically healthy suckling piglets is represented by two Cl bacteria. perfringens and E. coli with dominance of the latter, in the population of which there are trace amounts of enterotoxigenic forms. At the same time, it was found that diarrhea in piglets aged 3–5 days of age is induced by a pathogenic effect on the body of rotavirus type C, which is complicated by the effect of toxin synthesizing Cl. difficile, and in pigs 17–21 days of age – co-infection of rotavirus type A and enterotoxigenic forms of E. coli.