Results of the research of intestinal microbial profiles of Equus ferus caballus by NGS sequencing

The symbiotic microbiome of the gastrointestinal tract of animals plays a vital role in the digestion and assimilation of feed nutrients, the development of immunity, disease resistance, and the breakdown of toxins. Significant amounts of starch are introduced into the diet of horses specialized for riding, in some cases (for example, before participating in exhibitions). This can lead to serious dysbiotic disorders of the microbiome. Disorders of the microbial community of the intestine can adversely affect animal health that become the cause of metabolic disorders, such as acidosis, a decrease in the digestibility of diet components, primarily fiber, hoof diseases, etc. The digestive system of Equus ferus caballus has a number of unique features compared to other mammals. In this work, for the first time in Russia, the diversity of the equine intestinal microbiome composition was demonstrated using the 16S metagenomics method. The study aimed to evaluate the microbiomes of the contents of the rectum of horses of different ages, physiological status, diets, sexes and breeds using NGS sequencing. The experiment was carried out in the summer 2017 in the Malanichev Farm (Grishkino settlement, Leningrad Province, Tosnensky District,) with horses ( Equus ferus caballus ) specialized for riding and hippodrome trials. Samples of 10-50 g (in triplicate) were taken from the rectum of three stallions of the Hanoverian breed (3 years old), a mare (6 years old) and a stallion (7 years old) of the Trakehner breed. Five days before sampling, the mare was foaled. The diets of stallions and mares were different. The stallions’ diet included grass (20 kg), hay (9 kg), carrots (1 kg), oats (3 kg), table salt (29 g). The mares’ diet consisted of grass (26 kg), carrots (1 kg), rolled oats (2.5 kg), table salt (27 g). Total DNA from the samples was extracted using Genomic DNA Purification Kit (Fermentas Inc., Lithuania). Amplification for subsequent NGS sequencing was carried out on a Verity DNA amplifier (Life Technologies, Inc., USA) using eubacterial primers (IDT) 343F 343F (5'-CTCCTACGGRRSGCAGCAG-3') and 806R (5'-GGACTACNVGGGTWTCTAAT-3') flanking the V1V3 region of the 16S rRNA gene. Metagenomic sequencing was performed on a MiSeq instrument (Illumina, Inc., USA). The taxonomic affiliation of microorganisms to genus was determined using the RDP Classifier program (https://rdp.cme.msu.edu/classifier/classifier.jsp). In five different studied individuals of E. ferus caballus , fairly similar microbiomes of intestinal profiles were revealed, regardless of the type of nutrition, physiological status, age, gender, and breed. High values of the Shannon and Simpson diversity indices testified to the species richness and biodiversity of the intestinal contents of horses. In the rectum, 25 phyla of microorganisms were found. The dominant phyla were Firmicutes (ranged from 32±1.9 to 40±3.8 %) and Bacteroidetes (from 34±2.1 to 40±4.7 %). It is important to emphasize that we revealed in the microflora a significant number of microorganisms associated with feed digestion, especially those decomposing cellulose. So, the content of bacteria synthesizing cellulases reached significant values, up to 23.8±1.30 % for Bacteroidales , up to 14.7±2.80 % for Lachnospiraceae , up to 10.2±3.30 % for Ruminococcaceae , and up to 6.6±0.6 % for Clostridiaceae . A number of microorganisms were identified that can be associated with various diseases, e.g. horse with colic, acidosis, laminitis, etc. For example, in all samples of the rectum contents, we detected undesirable members of the order Lactobacillales , such as Streptococcus equinus and Str . bovis, which are associated with the occurrence of acidosis and laminitis in horses. The genus Treponema bacteria was revealed (from 2.2±0.22 to 6.5±0.40 %) which are associated with the occurrence of periodontitis in horses. The enterobacteria of the genera Enterobacter , Serratia , and Escherichia were detected, among which gastroenteritis pathogens can be often found. Further study of the intestinal microbiota profiles may contribute to the improvement of diagnosis and treatment of equine diseases.