The role of gut microbiota in the formation of immune system in newborn piglets

The intestinal microbiome is one of the important components of the human and animal body. It serves as a significant target for environmental factors and as a selective agent shaping the adaptive evolution of the mammalian diet, phenotypic plasticity, and gut morphology. Studying the microbial “quorum”, its influence on the development of the immune system, and the activation of innate immunity in piglets during the early neonatal period is a relevant task. This study is the first to establish relationships between the gut microbiota and the development of the immune system in piglets during the early neonatal period, namely a lower content of Enterococcus cecorum, Clostridium sensu stricto 2 disporicum , Atopobium fossor , and Faecalicoccus pleomorphus bacteria, reduced expression of the immune response genes IFNa and TGFβ, and reduced nuclear expression of Ki-67 in the small intestine of animals with low body weight, indicating insufficient immunological reactivity of innate immunity. The aim of the work was to study the role of the gut microbiota in the development of the immune system of newborn piglets. The research was conducted on Landrace piglets of the neonatal period, obtained from sows of the 3rd-4th farrowing at a large industrial pig farm in the Voronezh Province in 2023-2024. When forming the groups and making the diagnosis of hypotrophy (growth retardation), the main focus was on the animals' weight. The average weight in the group of hypotrophic piglets (n = 11) was 0.724±0.07 kg, and in the group of normotrophic piglets (n = 11) it was 0.927±0.04 kg. After group formation and before colostrum intake, the animals were euthanized, and biological material (intestinal contents, ileum) was collected from piglets of each group. High-throughput sequencing of 22 samples of intestinal contents was performed. Immunohistochemical studies of intestinal samples were also conducted, assessing the nuclear expression of markers CD3, Ki-67, and PAX-5. The expression of immune response genes in the intestine (IL-1β, IFNα, TGFβ, β-actin gene) was evaluated using PCR with universal primers. The sequencing data of the gut microbiota from normotrophic and hypotrophic piglets identified 72 bacterial species. In normotrophic piglets, the species composition of the meconium microbiome was more diverse compared to hypotrophic piglets, which will subsequently directly affect feed utilization efficiency, metabolism, and immune status. In a differential assessment of microbiota abundance in the studied samples, Enterococcus cecorum , Clostridium sensu stricto 2 disporicum , Atopobium fossor , and Faecalicoccus pleomorphus predominated in normotrophic piglets. According to the results of immunohistochemical studies of the intestine, higher nuclear expression of Ki-67 and membrane expression of CD3 (by 14.8 % and 32 %, respectively) were found in normotrophic piglets compared to hypotrophic ones, indicating more active cell proliferation and organ development. The expression of the transforming growth factor-beta gene (TGFβ) in hypotrophic piglets was 3.0 times lower than in normotrophic ones, which may indicate an inability of hypotrophic piglets to adequately regulate the growth and differentiation of various cell types. The expression of the interferon-alpha gene (IFNα) in hypotrophic piglets was 2.2 times lower compared to normotrophic ones, which may further affect resistance to viral agents, as well as the regulation of the adaptive immune response and the development of immune cells. Conversely, the expression of the interleukin-1 beta gene (IL-1β) was 48.0 % higher in the group of hypotrophic piglets compared to the group of normotrophic piglets, which, in our opinion, may indicate increased synthesis of acute-phase proteins initiating inflammatory processes in hypotrophic piglets. Thus, piglets with congenital hypotrophy were characterized by weak species diversity of the gut microbiota, resulting in low immunological reactivity of innate immunity.