Early-Life Intervention Using Exogenous Fecal Microbiota Alleviates Gut Injury and Reduce Inflammation Caused by Weaning Stress in Piglets

Abstract

Fecal microbiota transplantation (FMT) could shape the structure of intestinal microbiota in animals. This study was conducted to explore the changes that happen in the structure and function of microbiota caused by weaning stress, and whether early-life FMT could alleviate weaning stress through modifying intestinal microbiota in weaned piglets. Diarrheal (D) and healthy (H) weaned piglets were observed, and in the same farm, a total of nine litters newborn piglets were randomly allocated to three groups: sucking normally (S), weaned at 21 d (W), and early-life FMT + weaned at 21 d (FW). The results demonstrated that differences of fecal microbiota existed in group D and H. Early-life FMT significantly decreased diarrhea incidence of weaned piglets. Intestinal morphology and integrity were improved in the FW group. Both ZO-1 and occludin (tight junction proteins) of jejunum were greatly enhanced, while the zonulin expression was significantly down-regulated through early-life FMT. The expression of IL-6 and TNF-α (intestinal mucosal inflammatory cytokines) were down-regulated, while IL-10 (anti-inflammatory cytokines) was up-regulated by early-life FMT. In addition, early-life FMT increased the variety of the intestinal microbial population and the relative amounts of some beneficial bacteria such as Spirochaetes, Akkermansia, and Alistipes. Functional alteration of the intestinal microbiota revealed that lipid biosynthesis and aminoacyl-tRNA biosynthesis were enriched in the FW group. These findings suggested that alteration of the microbiota network caused by weaning stress induced diarrhea, and early-life FMT alleviated weaning stress in piglets, which was characterized by decreased diarrhea incidence, improved intestinal morphology, reduced intestinal inflammation, and modified intestinal bacterial composition and function.

Keywords: diarrhea; fecal microbiota transplantation; intestinal microbiota; piglets; weaning stress.

FIGURE 1

Diversity analysis of fecal microbiota between groups D and H. (A) Alpha diversity is the index of species variety in a single sample, including the observed species, chao1, shannon, and simpson. (B) Principal Coordinates Analysis (PCoA) was used to further demonstrate the differences in species diversity between samples, it can reveal the magnitude of the differences between samples. PCoA analysis results of species diversity between samples, if two samples are close to each other, it means that the species composition of these two samples is similar (D, Diarrhea; H, Health). *difference between the two groups is significant(p < 0.05).

FIGURE 2

Microbiota structure analysis of two groups. (A) According to the results of species annotation, the corresponding histogram of crop species profiling in phyla, family, and genus can be used to intuitively check the species with higher relative abundance and their proportion in different classification levels of each group. (B) LDA Effect Size (LEfSe analysis): LEfSe used linear discriminant analysis (LDA) to estimate the impact of the abundance of each component (species) on the difference effect and to identify the community or species that had a significant impact on the sample division (The LDA threshold is 2). (C) Boxplot result of genus level differential species. (D, Diarrhea; H, Health).

FIGURE 3

Correlation analysis of fecal microbiota. Spearman correlation heat map among species. At all taxonomic levels, the correlation coefficients between species with differences in abundance Top 30 are positively correlated in blue and negatively correlated in red on the right. The darker the color, the stronger the correlation between species.

FIGURE 4

Function analysis of fecal microbiota. PICRUSt (phylogenetic investigation of Communities by reconstruction of unobserved States) studies the phylogenetic investigation of community system evolution through the recessive state. The abscisic is the log value obtained by LDA of KEGG pathway with significant roles in two groups, and different colors indicated that the KEGG pathway was enriched in different groups of samples. (D, Diarrhea; H, Health).

FIGURE 5

ADG (A) and diarrhea incidence (B) of group S, W, and FW. The symbol “*” represented that the W and FWs group were compared with the S group, the symbol “#” represented that the FW group was compared with the W group. Data were shown as mean ± SEM (*p < 0.05, **p < 0.01, and ^#p < 0.05) (S, Sucking; W, Weaned; FW: FMT+Weaned). (1) ADG was calculated as weight gain (final body weight-initial body weight) divided by the number of treatment days. (2) The incidence of diarrhea (%) was calculated as the total number of diarrheal piglets during the period divided by the total number of piglets multiplies duration of the trial.

FIGURE 6

Jejunum sections were used to analyze intestinal morphology. (A) Stained with H&E (bars, 200 μ m). (B) The villus height (mm) and crypt depth (mm) were measured. Data were represented as mean ± SEM for the three different experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001). (C) SEM images (150×). (D) TEM images (8,000×). (S, Sucking; W, Weaned; FW, FMT+Weaned).

FIGURE 7

Intestinal mucosal tight junction proteins expression of three groups. (A,B) Western blot measurements of the expression levels of ZO-1 and occludin in jejunum and colon. (C,D) The distributions of ZO-1 and occludin in jejunum and colon were determined by immunohistochemical staining (150×). (E,F) Western blot measurements of the expression levels of zonulin in jejunum and colon. The symbol “*” represented that the W and FW groups were compared with the S group, the symbol “#” represented that the FW group was compared with the W group. Data are shown as mean ± SEM for the three different experiments (*p < 0.05, **p < 0.01, ^#p < 0.05, and ^##p < 0.01) (S, Sucking; W, Weaned; FW, FMT+Weaned).

FIGURE 8

Intestinal mucosal cytokines level of three groups. The relative mRNA expression of proinflammatory cytokines IL-6 (A), TNF-α (B), and anti-inflammatory IL-10 (C) were determined by qRT-PCR. The symbol “*” represented that the W and FW groups were compared with the S group, the symbol “#” represented that the FW group was compared with the W group. Data were shown as mean ± SEM for the three different experiments (*p < 0.05, **p < 0.01, ***p < 0.001, and ^#p < 0.05) (S, Sucking; W, Weaned; FW: FMT+Weaned).

FIGURE 9

Richness and biodiversity of Intestinal Microbiota. (A) Rarefaction curves determined at the 97% similarity level and Venn diagram of OTUs in the three groups. (B) α-diversity indexes of bacterial community. (C) Scatter plots obtained from PCoA based on the unweighted UniFrac. (D) UPGMA hierarchical clustering analysis based on the unweighted UniFrac (S, Sucking; W, Weaned; FW, FMT+Weaned).

FIGURE 10

Intestinal microbial community structure of three groups. (A) Relative abundance of microbial community in the colon at phylum, family, and genus level. (B) Cladogram and LDA value distribution histogram based on LEfSe analysis showed significant differences of the microbial community in the three groups. (C) Differences of the relative abundance of Bacteroidetes, Prevotellaceae, Alloprevotella, Firmicutes, Peptostreptococcaceae, Stretococcus, Oscillibacter, and Veillonella between the W and FW group (S, Sucking; W, Weaned; FW, FMT+Weaned). *p < 0.05 and **p < 0.01.

FIGURE 11

Network analysis of microbial community. (A) Microbial co-occurrence network analysis in three groups based on the calculation of Spearman’s correlation coefficients. The nodes represented different genus and their size represented the average relative abundance of the genus. The thickness of the connection between the nodes is positively correlated with the absolute value of the correlation coefficient of species interaction. The red lines indicated positive correlations and blue lines indicated negative correlations. (B) Typical coefficients derived from network analysis in three groups. CC, Clustering coefficient; GD, graph density; AD, Average degree (S, Sucking; W, Weaned; FW, FMT+Weaned).

FIGURE 12

The comparison of predicted microbial function between the W and FW groups. STAMP analysis was applied to identify the significant differences in the predictive functions at the third level of KEGG pathways between two groups. The P-values were shown at right (W, Weaned; FW, FMT+Weaned).