Sequencing of the 16S rRNA gene V3-V4 region to determine the composition and relationships of the microbiota during inflammation of the udder and reproductive tract in cows (Bos taurus)

Inflammatory diseases of the mammary gland and reproductive tract of cattle cause the greatest economic damage to dairy industry. 16S rRNA gene sequencing has significantly expanded the knowledge of microbiomes and uncultured in vitro bacteria that were not previously known to exist. This paper aims to expand the understanding of the composition of the microbiota of the reproductive tract and mammary gland of cattle with the identification of non-culturable microorganisms. New etiologically significant pathogens were identified for the first time. The role of microbiota of these animal loci in the development of inflammatory diseases was established. Our goal was to determine the common bacterial etiology of the inflammatory process in the mammary gland and reproductive tract of cattle. 16S rRNA gene variable regions sequencing was carried out in order to compare the composition of microbiota in mammary gland and the reproductive tract of cattle with inflammation. Four experimental groups were formed from each farm in five districts of the Sverdlovsk Province, animals without signs of inflammation of the mammary gland and reproductive tract (NP, group 1); animals with inflammation of the mammary gland, but without inflammation of the reproductive tract (M, group 2); animals with inflammation of the reproductive tract, but without inflammation of the mammary gland (E, group 3); animals with inflammation of the mammary gland and reproductive tract (EM, group 4). Samples of biological material (mammary gland secretion, cervical swabs) were obtained from each cow of all experimental groups; 16S rRNA gene sequencing was used for further studies. Sequence analysis of the 16S rRNA variable regions showed that the vast majority of identified ОТU belong to the Bacteria domain, the rest belong to the Euryarchaeota type of the Archaea domain (0.38 % in mammary gland secretion samples, 0.44 % in cervical swabs samples). In mammary gland secretions, we reveled 19 bacterial types, including 43 classes, 85 orders, 165 families, and 484 genera. Half of the bacterial ОТU are the Firmicutes phylum (680 ОТU, 51.7 %), while Actinobacteria and Bacteroidetes are the second and third largest phyla (14.5 % and 11.3 %, respectively). Members of 22 bacterial phyla were found in cervical swabs, including 50 classes, 93 orders, 172 families and 365 genera. The predominant bacterial phylum is the Firmicutes phylum (876 ОТU, 55.3 %), the second and third largest phyla are the Bacteroidetes (13.3 %) and the Actinobacteria (11.6 %), respectively. By the 16S rRNA gene sequencing, we revealed bacteria of the class Clostridia and the genus Facklamia not detectable by cultural methods. This confirms clinical significance of the 16S rRNA gene sequencing method for clarifying etiological agents in the case of unculturable or difficult-to-culture bacteria. We revealed the interrelation between the microbiota of the mammary gland and the reproductive tract during inflammatory processes. The data contribute to a deeper understanding of the role of bacterial microbiota in the etiology and pathogenesis of inflammatory diseases of the mammary gland and the reproductive tract of animals.