Early-Life Intervention Using Fecal Microbiota Combined with Probiotics Promotes Gut Microbiota Maturation, Regulates Immune System Development, and Alleviates Weaning Stress in Piglets

Abstract

Previous studies have suggested that immune system development and weaning stress are closely related to the maturation of gut microbiota. The early-life period is a "window of opportunity" for microbial colonization, which potentially has a critical impact on the development of the immune system. Fecal microbiota transplantation (FMT) and probiotics are often used to regulate gut microbial colonization. This study aims to test whether early intervention with FMT using fecal microbiota from gestation sows combined with Clostridium butyricum and Saccharomyces boulardii (FMT-CS) administration could promote the maturation of gut microbiota and development of immune system in piglets. Piglets were assigned to control (n = 84) and FMT-CS treatment (n = 106), which were treated with placebo and bacterial suspension during the first three days after birth, respectively. By 16S rRNA gene sequencing, we found that FMT-CS increased the α-diversity and reduced the unweighted UniFrac distances of the OTU community. Besides, FMT-CS increased the relative abundance of beneficial bacteria, while decreasing that of opportunistic pathogens. FMT-CS also enhanced the relative abundance of genes related to cofactors and vitamin, energy, and amino acid metabolisms during the early-life period. ELISA analysis revealed that FMT-CS gave rise to the plasma concentrations of IL-23, IL-17, and IL-22, as well as the plasma levels of anti-M.hyo and anti-PCV2 antibodies. Furthermore, the FMT-CS-treated piglets showed decreases in inflammation levels and oxidative stress injury, and improvement of intestinal barrier function after weaning as well. Taken together, our results suggest that early-life intervention with FMT-CS could promote the development of innate and adaptive immune system and vaccine efficacy, and subsequently alleviate weaning stress through promoting the maturation of gut microbiota in piglets.

Keywords: early-life intervention; fecal microbiota transplantation; gut microbiota; immune system; piglet; weaning stress.

Figure 1

α- and β-diversity of fecal microbiota in piglets after early intervention with FMT-CS. (A) Chao1 estimator, ACE estimator, Simpson index, and Shannon diversity index between control group and treatment group. (B) NMDS analysis of the fecal microbiota structure between the control groups and treatment groups. (C) β-diversity based on the unweighted UniFrac distances of the OTU community.

Figure 2

Microbiota composition determined by 16S rRNA gene sequencing of fecal samples. The relative abundance of the phyla (A) and genera (B) of fecal bacteria present in suckling piglets of both the control and treatment groups.

Figure 3

BugBase analysis based on the 16S rRNA gene sequencing dataset. The outcome was grouped according the modules (x-axis). Blue dots represent the control group and red dots represent the treatment group. The relative abundance is presented on the y-axis. Pairwise Mann–Whitney–Wilcoxon tests (p₁) and FDR-corrected pairwise tests (p₂) were performed for data analysis.

Figure 4

Differential enrichment of fecal microbiota. LefSe analysis of fecal microbiota at the age of 7 d (A), 27 d (B), 35 d (C), and D 56 (D) after early intervention with FMT-CS. The cladogram shows the microbial species with significant differences between groups in LEfSe analysis. Red indicates the treatment group and green indicates the control group.

Figure 5

Hierarchical clustering and heatmap of the differential genes at the phylum level (A) and genus level (B) between groups at different time based on the results of the metastats analysis.

Figure 6

Differences in metabolic functional genes in piglets of the control and treatment groups at the age of 7 d, 27 d, 35 d, and 56 d. Blue dots and red dots represent the control and treatment groups, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Effect of early-life intervention with FMT-CS on the development of type 3 innate lymphoid cells. (A) Cytokines (IL-1β, IL-10, and IL-23) associated with regulation of ILC3, and (B) cytokines (IL-17, IL-22, and IFN-γ) secreted by ILC3s in the plasma of piglets at the age of 7 d. (C–E) Plasma concentrations of IL-17, IL-22, and IFN-γ at indicated time points after birth. Values are expressed as mean ± SEM. ^# 0.05 < p < 0.1, * p < 0.05, ** p < 0.01.

Figure 8

Effects of early-life intervention with FMT-CS on vaccine antibody responses. Commercial ELISA kits were utilized to test anti-M.hyo antibody (A), anti-PCV2 antibody (B), anti-CSFV antibody (C), and anti-PRV antibody (D). (S-N)/(P-N) ratio: S, sample OD630; N, negative control OD630; P, positive control OD630. (S-N)/(P-N) _(M.hyo) ≥ 0.4, positive; (S-N)/(P-N) _(M.hyo) < 0.4, negative. (S-N)/(P-N) _(PCV2) ≥ 0.4, positive; (S-N)/(P-N) _(PCV2) < 0.4, negative. Blocking rate = (negative control OD450–sample OD450)/negative control OD450. Blocking rate _(CSFV) ≥ 40%, positive; blocking rate _(CSFV) < 40%, negative. S/N ratio = sample OD450/negative control OD450. S/N _{(PRV gB)} ≤ 0.6, positive; S/N _{(PRV gB)} > 0.6, negative. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.