Molecular networks affected by neonatal microbial colonization in porcine jejunum, luminally perfused with enterotoxigenic Escherichia coli, F4ac fimbria or Lactobacillus amylovorus.

The development of an early complex gut microbiota may play an important role in the protection against intestinal dysbiosis later in life. The significance of the developed microbiota for gut barrier functionality upon interaction with pathogenic or beneficial bacteria is largely unknown. The transcriptome of differently perfused jejunal loops of 12 caesarian-derived pigs, neonatally associated with microbiota of different complexity, was studied. Piglets received pasteurized sow colostrum at birth (d0), a starter microbiota (Lactobacillus amylovorus (LAM), Clostridium glycolicum, and Parabacteroides) on d1-d3, and a placebo inoculant (simple association, SA) or an inoculant consisting of sow's diluted feces (complex association, CA) on d3-d4. On d 26-37, jejunal loops were perfused for 8 h with either enterotoxigenic Escherichia coli F4 (ETEC), purified F4 fimbriae, LAM or saline control (CTRL). Gene expression of each intestinal loop was analyzed by Affymetrix Porcine Gene 1.1_ST array strips. Gene Set Enrichment Analysis was performed on expression values. Compared to CTRL, 184 and 74; 2 and 139; 2 and 48 gene sets, were up- and down-regulated by ETEC, F4 and LAM, respectively. ETEC up-regulated networks related to inflammatory and immune responses, RNA processing, and mitosis. There was a limited overlap in up-regulated gene sets between ETEC and F4 fimbriae. LAM down-regulated genes related to inflammatory and immune responses, as well as to cellular compound metabolism. In CA pigs, 57 gene sets were up-regulated by CA, while 73 were down-regulated compared to SA. CA up-regulated gene sets related to lymphocyte modulation and to cellular defense in all loop perfusions. In CA pigs, compared to SA pigs, genes for chemokine and cytokine activity and for response to external stimuli were down-regulated in ETEC-perfused loops and up-regulated in CTRL. The results highlight the importance of the nature of neonatal microbial colonization in the response to microbial stimuli later in life.

The effects of starter microbiota and the early life feeding of medium chain triglycerides on the gastric transcriptome profile of 2- or 3-week-old cesarean delivered piglets.

BACKGROUND: The stomach is an underestimated key interface between the ingesta and the digestive system, affecting the digestion and playing an important role in several endocrine functions. The quality of starter microbiota and the early life feeding of medium chain triglycerides may affect porcine gastric maturation. Two trials (T1, T2) were carried out on 12 and 24 cesarean-delivered piglets (birth, d0), divided over two microbiota treatments, but slaughtered and sampled at two or three weeks of age, respectively. All piglets were fed orally: sow serum (T1) or pasteurized sow colostrum (T2) on d0; simple starter microbiota (Lactobacillus amylovorus, Clostridium glycolicum and Parabacteroides spp.) (d1-d3); complex microbiota inoculum (sow diluted feces, CA) or a placebo (simple association, SA) (d3-d4) and milk replacer ad libitum (d0-d4). The The T1 piglets and half of the T2 piglets were then fed a moist diet (CTRL); the remaining half of the T2 piglets were fed the CTRL diet fortified with medium chain triglycerides and 7% coconut oil (MCT). Total mRNA from the oxyntic mucosa was analyzed using Affymetrix©Porcine Gene array strips. Exploratory functional analysis of the resulting values was carried out using Gene Set Enrichment Analysis. RESULTS: Complex microbiota upregulated 11 gene sets in piglets of each age group vs. SA. Of these sets, 6 were upregulated at both ages, including the set of gene markers of oxyntic mucosa. In comparison with the piglets receiving SA, the CA enriched the genes in the sets related to interferon response when the CTRL diet was given while the same sets were impoverished by CA with the MCT diet. CONCLUSIONS: Early colonization with a complex starter microbiota promoted the functional maturation of the oxyntic mucosa in an age-dependent manner. The dietary fatty acid source may have affected the recruitment and the maturation of the immune cells, particularly when the piglets were early associated with a simplified starter microbiota.

The Olfactory Receptor OR51E1 Is Present along the Gastrointestinal Tract of Pigs, Co-Localizes with Enteroendocrine Cells and Is Modulated by Intestinal Microbiota.

UNLABELLED: The relevance of the butyrate-sensing olfactory receptor OR51E1 for gastrointestinal (GIT) functioning has not been considered so far. We investigated in young pigs the distribution of OR51E1 along the GIT, its relation with some endocrine markers, its variation with age and after interventions affecting the gut environment and intestinal microbiota. Immuno-reactive cells for OR51E1 and chromogranin A (CgA) were counted in cardial (CA), fundic (FU), pyloric (PL) duodenal (DU), jejunal (JE), ileal (IL), cecal (CE), colonic (CO) and rectal (RE) mucosae. OR51E1 co-localization with serotonin (5HT) and peptide YY (PYY) were evaluated in PL and CO respectively. FU and PL tissues were also sampled from 84 piglets reared from sows receiving either or not oral antibiotics (amoxicillin) around parturition, and sacrificed at days 14, 21, 28 (weaning) and 42 of age. JE samples were also obtained from 12 caesarean-derived piglets that were orally associated with simple (SA) or complex (CA) microbiota in the postnatal phase, and of which on days 26-37 of age jejunal loops were perfused for 8 h with enterotoxigenic Escherichia coli F4 (ETEC), Lactobacillus amylovorus or saline (CTRL). Tissue densities of OR51E1+ cells were in decreasing order: PL=DU>FU=CA>JE=IL=CE=CO=RE. OR51E1+ cells showed an enteroendocrine nature containing gastrointestinal hormones such as PYY or 5HT. OR51E1 gene expression in PL and FU increased during and after the suckling period (p<0.05). It was marginally reduced in offspring from antibiotic-treated sows (tendency, p=0.073), vs. CONTROL: Jejunal OR51E1 gene expression was reduced in piglets early associated with SA, compared with CA, and in ETEC-perfused loops vs. CTRL (p<0.01). Our results indicate that OR51E1 is related to GIT enteroendocrine activity. Moreover age, pathogen challenge and dietary manipulations influencing the gastrointestinal luminal microenvironment significantly affect the OR51E1 gene expression in GIT tissues presumably in association with the release of microbial metabolites.

Differential gene expression in the oxyntic and pyloric mucosa of the young pig.

The stomach is often considered a single compartment, although morphological differences among specific areas are well known. Oxyntic mucosa (OXY) and pyloric mucosa (PYL, in other species called antral mucosa) are primarily equipped for acid secretion and gastrin production, respectively, while it is not yet clear how the remainder of genes expressed differs in these areas. Here, the differential gene expression between OXY and PYL mucosa was assessed in seven starter pigs. Total RNA expression was analyzed by whole genome Affymetrix Porcine Gene 1.1_ST array strips. Exploratory functional analysis of gene expression values was done by Gene Set Enrichment Analysis, comparing OXY and PYL. Normalized enrichment scores (NESs) were calculated for each gene (statistical significance defined when False Discovery Rate % <25 and P-values of NES<0.05). Expression values were selected for a set of 44 genes and the effect of point of gastric sample was tested by analysis of variance with the procedure for repeated measures. In OXY, HYDROGEN ION TRANSMEMBRANE TRANSPORTER ACTIVITY gene set was the most enriched set compared to PYL, including the two genes for H+/K+-ATPase. Pathways related to mitochondrial activity and feeding behavior were also enriched (primarily cholecystokinin receptors and ghrelin). Aquaporin 4 was the top-ranking gene. In PYL, two gene sets were enriched compared with OXY: LYMPHOCYTE ACTIVATION and LIPID RAFT, a gene set involved in cholesterol-rich microdomains of the plasma membrane. The single most differentially expressed genes were gastrin and secretoglobin 1A, member 1, presumably located in the epithelial line, to inactivate inflammatory mediators. Several genes related to mucosal integrity, immune response, detoxification and epithelium renewal were also enriched in PYL (P<0.05). The data indicate that there is significant differential gene expression between OXY of the young pig and PYL and further functional studies are needed to confirm their physiological importance.

Age-related expression of the polymeric immunoglobulin receptor (pIgR) in the gastric mucosa of young pigs.

To date few studies have addressed the development and function of the porcine gastric mucosal immune system and this is a major limitation to understanding the immunopathogenesis of infections occurring in young pigs. The polymeric immunoglobulin receptor (pIgR) mediates the transport of secretory immunoglobulins until luminal surface of the gut mucosa and the aim of this study was to investigate the time course of pIgR expression and to determine its localization in three functionally different porcine gastric sites during the suckling period and after weaning. An additional goal was to investigate the time course expression of toll-like receptors (TLRs) in relation to pIgR expression. Gastric samples were collected from the cardiac-to-oxyntic transition (Cd), the oxyntic (Ox), and the pyloric (Py) regions in 84 pigs, slaughtered before weaning (14, 21 and 28 days of age; 23, 23 and 19 pigs, respectively) and 14 days post-weaning (42 days of age, 23 pigs). PIgR was expressed in the mucosa of all the three gastric sites, and its transcript levels were modulated during suckling and after weaning, with regional differences. PIgR expression increased linearly during suckling (P=0.019) and also increased post-weaning (P=0.001) in Cd, it increased post-weaning in Py (P=0.049) and increased linearly during suckling in Ox (P=0.036). TLRs expression was also modulated during development: in Cd, TLR2 increased linearly during suckling (P=0.003); in Ox, TLR2 decreased after weaning (P=0.038) while TLR4 increased linearly during suckling(P=0.008). The expression of TLR2, 3 and 4 in Ox was positively correlated with pIgR expression (P<0.001). Importantly, both pIgR protein and mRNA were localized, by immunohistochemistry and in situ hybridization, respectively, in the gastric glands of the lamina propria. These results indicate that pIgR is actively synthesized in the gastric mucosa and suggest that pIgR could play a crucial role in gastric mucosal immune defense of growing pigs.

Effect of susceptibility to enterotoxigenic Escherichia coli F4 and of dietary tryptophan on gut microbiota diversity observed in healthy young pigs.

Healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4 (ETEC) require more tryptophan (Trp) to maximize their performance. This may be related to an effect on intestinal microbiota. We studied the intestinal bacterial diversity of healthy pigs with different susceptibility to ETEC and fed different Trp levels. Thirty-six littermate weaned pigs were selected to obtain a set potentially formed of 50% ETEC-susceptible and 50% non-susceptible pigs, based on a Mucin 4 gene polymorphism. Pigs were fed a diet with 0.17 (TrpL) or 0.22 (TrpH) standardized ileal digestible Trp:Lys ratio for 21 days. Slaughtered pigs were classified into non-susceptible, mildly susceptible, and susceptible, by testing ETEC adhesion to intestinal villi. Bacterial diversity in jejunum content was assessed by the 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting analysis and expressed by the Shannon index. Susceptible pigs had a reduced bacterial diversity, particularly with TrpL diet (p=0.003). The ETEC adhesion class affected the quantification of enterobacteria DNA (p=0.027). One DGGE band, which referred to Clostridium bartlettii, was not evidenced in all the susceptible pigs; less DNA from this microbe was quantified by RT-PCR in the jejunum from TrpH susceptible pigs (p=0.025) compared to TrpL. The gene expression for ß-galactoside α-2,3-sialyltransferase 1 was higher in jejunal tissue of ETEC-susceptible pigs (p=0.019). In studies on pig gut microbiota, the presence of intestinal receptors for ETEC should be considered because of their contribution to a reduced bacterial diversity. This effect could be partially reversed by dietary Trp addition.