Saccharomyces cerevisiae boulardii accelerates intestinal microbiota maturation and is correlated with increased secretory IgA production in neonatal dairy calves

Neonatal calves have a limited capacity to initiate immune responses due to a relatively immature adaptive immune system, which renders them susceptible to many on-farm diseases. At birth, the mucosal surfaces of the intestine are rapidly colonized by microbes in a process that promotes mucosal immunity and primes the development of the adaptive immune system. In a companion study, our group demonstrated that supplementation of a live yeast probiotic, Saccharomyces cerevisiae boulardii (SCB) CNCM I-1079, to calves from birth to 1 week of age stimulates secretory IgA (sIgA) production in the intestine. The objective of the study was to evaluate how SCB supplementation impacts the intestinal microbiota of one-week-old male calves, and how changes in the bacterial community in the intestine relate to the increase in secretory IgA. A total of 20 calves were randomly allocated to one of two treatments at birth: Control (CON, n = 10) fed at 5 g/d of carrier with no live yeast; and SCB (n = 10) fed at 5 g of live SCB per day (10 × 109 CFU/d). Our study revealed that supplementing calves with SCB from birth to 1 week of age had its most marked effects in the ileum, increasing species richness and phylogenetic diversity in addition to expediting the transition to a more interconnected bacterial community. Furthermore, LEfSe analysis revealed that there were several differentially abundant taxa between treatments and that SCB increased the relative abundance the family Eubacteriaceae, Corynebacteriaceae, Eggerthellaceae, Bacillaceae, and Ruminococcaceae. Furthermore, network analysis suggests that SCB promoted a more stable bacterial community and appears to reduce colonization with Shigella. Lastly, we observed that the probiotic-driven increase in microbial diversity was highly correlated with the enhanced secretory IgA capacity of the ileum, suggesting that the calf’s gut mucosal immune system relies on the development of a stable and highly diverse microbial community to provide the necessary cues to train and promote its proper function. In summary, this data shows that supplementation of SCB promoted establishment of a diverse and interconnected microbiota, prevented colonization of Escherichia Shigella and indicates a possible role in stimulating humoral mucosal immunity

Potential Regulatory Role of MicroRNAs in the Development of Bovine Gastrointestinal Tract during Early Life

<div><p>This study aimed to investigate the potential regulatory role of miRNAs in the development of gastrointestinal tract (GIT) during the early life of dairy calves. Rumen and small intestinal (mid-jejunum and ileum) tissue samples were collected from newborn (30 min after birth; n = 3), 7-day-old (n = 6), 21-day-old (n = 6), and 42-day-old (n = 6) dairy calves. The miRNA profiling was performed using Illumina RNA-sequencing and the temporal and regional differentially expressed miRNAs were further validated using qRT-PCR. Analysis of 16S rRNA gene copy numbers was used to quantify total bacteria, <i>Bifidobacterium</i> and <i>Lactobacillus</i> species. The expression of miR-143 was abundant in all three gut regions, at all time points and it targets genes involved primarily in the proliferation of connective tissue cells and muscle cells, suggesting a role in regulating rapid tissue development during the early life of calves. The expression of miR-146, miR-191, miR-33, miR-7, miR-99/100, miR-486, miR-145, miR-196 and miR-211 displayed significant temporal differences (FDR <0.05), while miR-192/215, miR-194, miR-196, miR-205 and miR-31 revealed significant regional differences (FDR <0.05). The expression levels of miR-15/16, miR-29 and miR-196 were positively correlated with the copy numbers of 16S rRNA gene of <i>Bifidobacterium</i> or <i>Lactobacillus</i> species or both (<i>P</i><0.05). Functional analysis using Ingenuity Pathway Analysis identified the above mentioned differentially expressed miRNAs as potential regulators of gut tissue cell proliferation and differentiation. The bacterial density-associated miRNAs were identified as modulators of the development of lymphoid tissues (miR-196), maturation of dendritic cells (miR-29) and development of immune cells (miR-15/16). The present study revealed temporal and regional changes in miRNA expression and a correlation between miRNA expression and microbial population in the GIT during the early life, which provides further evidence for another mechanism by which host-microbial interactions play a role in regulating gut development.</p></div

Expression levels of the most significant regionally DE miRNAs detected by miRNA sequencing in rumen (RU, open bar), mid-jejunum (MJ, solid black bar) and ileum (IL, solid grey bar).

<p>“*” represents miRNA differentially expressed between two tissues (FDR <0.05, >1.5 fold). No bar is shown when miRNA expression was not detected.</p

Expression of temporal DE miRNAs detected by qRT-PCR and miRNA-seq.

<p>MiRNA expressions from qRT-PCR are shown by line graphs on the top and values are shown on the right Y-axis as relative expression (ΔCt). Lower ΔCt values represent higher miRNA expression levels and <i>vice versa</i>. MiRNA expressions from miRNA-seq are shown by bar graphs on the bottom and values are shown on the left Y-axis as log₂ (normalised reads number). A, B, C, D - indicate significant expression difference between pairs at <i>P</i><0.05 (D7 <i>vs</i> D0, D21 <i>vs</i> D7, D42 <i>vs</i> D21) detected via qRT-PCR. a, b, c, d - indicate significant expression difference between pairs at FDR <0.05 (D7 <i>vs</i> D0, D21 <i>vs</i> D7, D42 <i>vs</i> D21) detected by miRNA-seq. Data are presented as Mean±Standard deviation.</p

Numbers of miRNAs with significant correlations between their expression and the copy number of microbial 16S rRNA gene.

<p>The correlation analysis was performed between normalized miRNAs expression and log copy number of total bacteria, <i>Lactobacillus</i> and <i>Bifidobacterium</i> spp. in each location (RU: rumen; MJ: mid-jejunum; IL: ileum) and at each time point (D0: blue bar; D7: black bar; D21: red bar; D42: yellow bar). The horizontal axis shows the number of miRNAs correlated with microbial density. “# of negatively correlated miRNAs” means the number of miRNAs with a negative correlation (P<0.05, r<−0.8) with microbial density. “# of positively correlated miRNAs” means the number of miRNAs with a positive correlation (<i>P</i><0.05, r>0.8) with microbial density.</p

Conservation analysis of known miRNAs detected in calf GIT.

<p>Proportion of different miRNAs conservation categories in total identified known miRNAs (Total), in rumen (RU), in mid-jejunum (MJ), and in ileum (IL). Highly conserved (blue bar): conserved across most vertebrates; Conserved (black bar): conserved across most mammals, but usually not beyond placental mammals; Poorly conserved: miRNAs that do not belong to the above two groups, including bovine specific (red bar, belong to poorly conserved group, and seed region sequence only reported in cattle) and other poorly conserved miRNAs (yellow bar).</p

Detected temporally DE miRNAs based on comparisons D7 <i>vs.</i> D0 (red), D21 <i>vs.</i> D7 (green), and D42 <i>vs.</i> D21 (black).

<p>The X and Y axes show log2 (fold change) and log2 (normalised reads number) of each DE miRNA, respectively. (A). DE miRNAs detected in rumen (RU) tissue. (B). DE miRNAs identified in tissue collected from mid-jejunum (MJ). (C). DE miRNAs detected in tissue collected from from ileum (IL). “↑”: the number of up-regulated miRNAs; “↓”: the number of down-regulated miRNA.</p

Detected regionally DE miRNA based on comparisons between different tissues on D0 (red), D7 (green), D21 (black), and D42 (purple).

<p>(A). Detected DE miRNAs when comparing rumen (RU) <i>vs.</i> mid-jejunum (MJ). (B). Detected DE miRNAs when comparing rumen (RU) <i>vs.</i> ileum (IL). (C). Detected DE miRNAs when comparing mid-jejunum (MJ) <i>vs.</i> ileum (IL). “↑”: the number of up-regulated miRNAs; “↓”: the number of down-regulated miRNA.</p

Expression of regional DE miRNAs detected by qRT-PCR and miRNA-seq.

<p>MiRNA expressions from qRT-PCR are shown by line graphs on the top and values are shown on the right Y-axis as relative expression (ΔCt). Lower ΔCt values represent higher miRNA expression levels and <i>vice versa</i>. MiRNA expressions from miRNA-seq are shown by bar graphs on the bottom and values are shown on the left Y-axis as log₂ (normalised reads number). A, B, C - indicate the significant difference in the relative expression of miRNAs detected via qRT-PCR at <i>P</i><0.05; a, b, c - indicate significant difference in the expression of miRNAs detected from miRNA-seq at FDR <0.05. Data are presented as Mean±Standard deviation. RU, MJ and IL represent rumen, mid-jejunum, and ileum, respectively.</p