Microbial diversity studies of the porcine gastrointestinal ecosystem during weaning transition

At the time of weaning, major quantitative and qualitative changes occur in the composition of the intestinal microbiota of piglets, influenced by diet, environmental factors, and the host. Within a short period of time, the intestinal microbiota must ultimately develop from a simple, unstable community into a complex and stable one. Here we present data on the development of the intestinal microbiota based on 16S rRNA gene sequence diversity. In addition to a PCR-based analysis of the 16S rRNA gene by cloning and denaturing gradient gel electrophoresis (DGGE), data on fluorescent in situ hybridisation (FISH) are presented to quantify the total bacterial communities, major Lactobacillus populations and specific Lactobacillus species. The results reported here indicate that the addition of non-digestible, fermentable carbohydrates (= prebiotics) leads to an enrichment of lactobacilli in the small intestine, and increased stability and diversity of the bacterial community in the colon. The data support the hypothesis that changes of the diet can modulate the composition of the microbiota in the intestine. These findings may have potentially major implications for the development of dietary strategies aiming to improve animal health during the weaning process

Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro

Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (F-cytosol/F-nucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells.Peer reviewe

Effects of threshold choice on the results of gene expression profiling, using microarray analysis, in a model feeding experiment with rat

Global gene expression studies using microarray technology are widely employed to identify biological processes which are influenced by a treatment e.g. a specific diet. Affected processes are characterized by a significant enrichment of differentially expressed genes (functional annotation analysis). However, different choices of statistical thresholds to select candidates for differential expression will alter the resulting candidates list. This study was conducted to investigate the effect of applying a False Discovery Rate (FDR) correction and different fold change thresholds in statistical analysis of microarray data on diet-affected biological processes based on a significantly increased proportion of differentially expressed genes. In a model feeding experiment with rats fed genetically modified food additives, animals received a supplement of either lyophilized inactivated recombinant VP60 baculovirus (rBV-VP60) or lyophilized inactivated wild type baculovirus (wtBV). Comparative expression profiling was done in spleen, liver and small intestine mucosa. We demonstrated the extent to which threshold choice can affect the biological processes identified as significantly regulated and thus the conclusion drawn from the microarray data. In our study, the combined application of a moderate fold change threshold (FC≥1.5) and a stringent FDR threshold (q≤0.05) exhibited high reliability of biological processes identified as differentially regulated. The application of a stringent FDR threshold of q≤0.05 seems to be an essential prerequisite to reduce considerably the number of false positives. Microarray results of selected differentially expressed molecules were validated successfully by using real-time RT-PCR

Effects of threshold choice on the results of gene expression profiling, using microarray analysis, in a model feeding experiment with rat

Global gene expression studies using microarray technology are widely employed to identify biological processes which are influenced by a treatment e.g. a specific diet. Affected processes are characterized by a significant enrichment of differentially expressed genes (functional annotation analysis). However, different choices of statistical thresholds to select candidates for differential expression will alter the resulting candidates list. This study was conducted to investigate the effect of applying a False Discovery Rate (FDR) correction and different fold change thresholds in statistical analysis of microarray data on diet-affected biological processes based on a significantly increased proportion of differentially expressed genes. In a model feeding experiment with rats fed genetically modified food additives, animals received a supplement of either lyophilized inactivated recombinant VP60 baculovirus (rBV-VP60) or lyophilized inactivated wild type baculovirus (wtBV). Comparative expression profiling was done in spleen, liver and small intestine mucosa. We demonstrated the extent to which threshold choice can affect the biological processes identified as significantly regulated and thus the conclusion drawn from the microarray data. In our study, the combined application of a moderate fold change threshold (FC≥1.5) and a stringent FDR threshold (q≤0.05) exhibited high reliability of biological processes identified as differentially regulated. The application of a stringent FDR threshold of q≤0.05 seems to be an essential prerequisite to reduce considerably the number of false positives. Microarray results of selected differentially expressed molecules were validated successfully by using real-time RT-PCR

Effect of weaning on intestinal lactate concentration and lactobacilli load of piglets.

<p><b>(A)</b> Lactate concentration <b>(B)</b> <i>Lactobacillus</i> load and <b>(C)</b> pH in chyme of suckling and weaned piglets along the intestinal axis was measured. Statistical analysis was performed with the procedure “MIXED” (SAS, version 8) and comparison between pre- and postweaning using the t-test (p<0.01).</p

Attachment and structural effects of lactobacilli on IPEC-1 cells.

<p><b>(A)</b> Three dimensional reconstruction of lactobacilli attached to IPEC-1 cells. <i>Lactobacillus amlylovorus</i> strain GRL1110 (arrows) was applied for 6 h on confluent IPEC-1 cells. The bacterial suspension was then carefully removed; cells and attached bacteria were fixed and stained with DAPI (nuclei, blue) and CM-Dil (plasma membrane, red). Confocal sections were reconstructed to obtain a 3D view. Note the thin, barely visible cytosolic layer above nucleus (*). (<b>B-H</b>) Actin structure of IPEC-1 cells treated with lactate and <i>Lactobacillus amylovorus</i> supernatants. IPEC-1 cells were cultured on glass, incubated as indicated and stained for F-actin (Phalloidin-PromoFluor 546) and nuclei (DAPI). (<b>B</b>) control buffer pH7, (<b>C</b>) Na-DL-lactate (25 mM, pH 7), (<b>D</b>) control buffer pH 4, (<b>E</b>) lactic acid pH 4. (<b>F-H</b>) supernatants of <i>Lactobacillus amylovorus</i> strains. (<b>F</b>) GRL1110, (<b>G</b>) GRL1112, (<b>H</b>) GRL1115. Predominantly basal F-actin fibre structure was reduced by lactic acid and bacterial supernatants (pH 4), (<b>I</b>) F-actin staining of freeze-section of porcine jejunal epithel. Note positive F-actin signal indicating the apical terminal web (arrowhead), the low F-actin signal at the lateral side and the faint signal in the basal region of the enterocytes (arrow). Bar: 5 μm.</p

Quantification of cytosol / nucleus NADH ratio in IPEC-1 cells after 1 h treatment with glucose and lactate without further challenge.

<p>Cellular autofluorescence of IPEC-1 cells was monitored at ex: 340 nm / em: 510 nm. <b>(A)</b> Change in spatial distribution of autofluorescence in response to Anti A (100 μM). <b>(B)</b> Cytosolic autofluorescence in response to complex III inhibitor Anti A (100 μM) and uncoupler FCCP (1 μM). <b>(C)</b> IPEC-1 cells were treated as indicated for 1 h. Glucose (gluc) concentration was 1 g/L if not otherwise indicated. Cells were treated with lactate (lac) 25 mM or increased gluc 4.5 g/L. Autofluorescence measured after 1 h and ratio of cytosolic (F_cytosol) and nuclei (F_nucleus) was calculated (ex: 340 nm / em: 510 nm) from individual cells without further stimulation. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p

Visualisation of adherence of bacteria on IPEC-1 cells.

<p>Three different strains of <i>Lactobacillus amylovorus</i> (GRL1110 (<b>B</b>); GRL1112 (<b>C</b>); GRL1115 (<b>D</b>)) were applied on confluent IPEC-1 cells at the cell:bacteria ratio 1:1000 for 6 h in cell culture medium. Bacterial suspensions were removed, cells and attached bacteria were fixed and stained with DAPI. Bacterial DNA indicates a different degree of adherence of bacteria to epithelial cell surface (<b>B-D</b>) in comparison to non-treated (<b>A</b>) cells. Representative micrographs of three independent experiments are shown. Bar: 5 μm.</p

Effect of glucose and lactate on cellular ATP content in IPEC-1 cells after 1 h treatment.

<p>IPEC-1 cells were grown to confluence (7 d) and treated for 1 h with HBSS-based solutions. Supernatant was removed and ATP content was measured by a luminescence based assay. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p