Fig 1.
Flow diagram showing experimental paradigm through analyses of fecal microbiome and intestinal transcriptome.
Female piglets (5 weeks old) were fed a weaner diet or a weaner diet mixed with liquid probiotics containing L. plantarum strain JDFM LP11 (1.25×109 CFU/kg of diet) for 4 weeks (n = 3 per group). Rectal feces and ileal tissues were collected at the end of animal trial for analyses of fecal microbiome and intestinal transcriptome to study roles of probiotics in modulation of gut microbiota and immune associated gene expression in weaned piglets.
Fig 2.
Photomicrographs of epithelial layers of the small intestine, cecum and colon in piglets between the control and probiotics groups.
The villus height and crypt depth in intestinal segments were increased by probiotic supplementation.
Fig 3.
Microbial diversity and richness in fecal samples of piglets between the control and probiotics groups.
(A) Observed index; (B) Simpson index; (C) Shannon index; (D) Chao1 index; (E) ACE index. Differences are p<0.05 as measured by T-test. Alpha-diversity analyses indicate probiotic supplementation increased the microbial diversity and richness.
Fig 4.
Principal coordinates analysis (PCoA) plots in fecal samples of piglets between the control and probiotics groups.
(A) Unweighted UniFrac analysis. (B) Weighted UniFrac analysis. Each point represents an individual piglet. The probiotics group was clearly separated from the control group microbiota in both unweighted and weighted UniFrac analyses. These data indicate that probiotics supplementation changes the shape of fecal microbial community in piglets.
Fig 5.
Histogram representing taxonomic composition and relative abundance (>0.1%) at family level in fecal samples between the control and probiotics groups.
A total of 19 different families of bacteria were found in two groups. Prevotellaceae and Ruminococcaceae were the most abundant families.
Fig 6.
Expression pattern comparative analysis between the control and probiotics piglets.
(A) Principal component analysis (PCA) plot between the control and probiotics RNA-seq samples. (B) Heatmap of DEGs distinguishes the control and probiotics piglets. The color scale represents a low expression value in blue and a high expression value in red. (C) Expression of DEGs between control and probiotics piglets.
Table 1.
Gene ontology (GO) analysis of differentially expressed genes (biological process).
Fig 7.
PICRUSt prediction of functional profiling of the microbial communities based on the 16S rRNA gene sequences.
A total of 28 KEGG pathways were significantly changed in the probiotics group compared with the control group. Extended error bar plot indicating differences in functional profiles of the control and probiotics microbiota (at taxonomic Level 3). All unclassified reads were removed and q-value greater than 0.05 is displayed. And then, effect size (difference between proportions) was less than 0.03. Categories are sorted by effect sizes calculated using two-sided Welch’s t-test and multiple test correction is Benjamini-Hochberg FDR. Bar plots on the left side displayed the mean proportion of each KEGG pathway. Dot plots on the right show the differences in mean proportions between the two indicated groups using q-values.
Fig 8.
Comparison of serum IgG between the control and probiotics groups.
(A) is control group and (B) is probiotics group. Blood samples were collected on days 0, 14 and 28 for measurement of serum immunoglobulin G (IgG) during a 4 week trial. The serum IgG was increased with consistency by time in the probiotics group compared to the control group.