Salmosan, a ß-Galactomannan-Rich Product, Protects Epithelial Barrier Function in Caco-2 Cells Infected by Salmonella enterica Serovar Enteritidis.

BACKGROUND: One promising strategy for reducing human salmonellosis induced by Salmonella Enteritidis is to supplement animal diets with natural feed additives such as mannan oligosaccharides (MOSs). OBJECTIVE: We sought to investigate the potential role of Salmosan (S-ßGM), an MOS product extremely rich in ß-galactomannan, in preventing epithelial barrier function disruption induced by S. Enteritidis colonization in an in vitro model of intestinal Caco-2 cells in culture. METHODS: Differentiated Caco-2 cells were incubated for 3 h with S. Enteritidis at a multiplicity of infection of 10 in the absence or presence of 500 µg S-ßGM/mL. Paracellular permeability (PP) was assessed by transepithelial electrical resistance (TER), d-mannitol, and fluorescein isothiocyanate-dextran (FD-4) flux. Tight junction proteins and cytoskeletal actin were also localized by confocal microscopy. Reactive oxygen species (ROS) and lipid peroxidation products were evaluated. Scanning and transmission electron microscopy were used to visualize S. Enteritidis adhesion to, and invasion of, the Caco-2 cell cultures. RESULTS: Compared with controls, TER was significantly reduced by 30%, and d-mannitol and FD-4 flux were significantly increased by 374% and 54% in S. Enteritidis-infected cultures, respectively. The presence of S-ßGM in infected cultures induced total recoveries of TER and FD-4 flux to values that did not differ from the control and a partial recovery of d-mannitol flux. These effects were confirmed by immunolocalization of actin, zonula occludens protein 1, and occludin. Similar results were obtained for Salmonella Dublin. The protection of S-ßGM on PP in infected cultures may be associated with a total recovery of ROS production to values that did not differ from the control. Moreover, S-ßGM has the capacity to agglutinate bacteria, leading to a significant reduction of 32% in intracellular S Enteritidis. CONCLUSION: The results demonstrate that S-ßGM contributes to protecting epithelial barrier function in a Caco-2 cell model disrupted by S. Enteritidis.

Effect of Saccharomyces cerevisiae var. Boulardii and ß-galactomannan oligosaccharide on porcine intestinal epithelial and dendritic cells challenged in vitro with Escherichia coli F4 (K88).

Probiotic and prebiotics, often called "immune-enhancing" feed additives, are believed to deal with pathogens, preventing the need of an immune response and reducing tissue damage. In this study, we investigated if a recently developed ß-galactomannan (ßGM) had a similar protective role compared to Saccharomyces cerevisiae var. Boulardii (Scb), a proven probiotic, in the context of enterotoxigenic Escherichia coli (ETEC) infection. ETEC causes inflammation, diarrhea and intestinal damage in piglets, resulting in large economic loses worldwide. We observed that Scb and ßGM products inhibited in vitro adhesion of ETEC on cell surface of porcine intestinal IPI-2I cells. Our data showed that Scb and ßGM decreased the mRNA ETEC-induced gene expression of pro-inflammatory cytokines TNF-α, IL-6, GM-CSF and chemokines CCL2, CCL20 and CXCL8 on intestinal IPI-2I. Furthermore, we investigated the putative immunomodulatory role of Scb and ßGM on porcine monocyte-derived dendritic cells (DCs) per se and under infection conditions. We observed a slight up-regulation of mRNA for TNF-α and CCR7 receptor after co-incubation of DC with Scb and ßGM. However, no differences were found in DC activation upon ETEC infection and Scb or ßGM co-culture. Therefore, our results indicate that, similar to probiotic Scb, prebiotic ßGM may protect intestinal epithelial cells against intestinal pathogens. Finally, although these products may modulate DC activation, their effect under ETEC challenge conditions remains to be elucidated.

Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens.

The current study was conducted to examine the effects of deoxynivalenol (DON) at different levels (5 and 15 mg/kg feed) on the metabolism, immune response and welfare parameters of male broiler chickens (Ross 308) at 42 days old. Forty-five 1 day-old broiler chickens were randomly distributed into three different dietary treatments: (1) control, (2) DON-contaminated diet with 5 mg DON/kg of feed (guidance level), and (3) DON-contaminated diet with 15 mg DON/kg of feed. Five replicated cages with three birds each were used for each treatment in a randomized complete block design. The results showed that DON was detected in excreta of birds fed contaminated diets compared with controls. The metabolite DON-3 sulphate (DON-3S) was detected in plasma and excreta in both treated groups, as well as in the liver (but only at 15 mg/kg feed). The increase in the level of DON decreased the hemoglobin concentration (p < 0.001), whereas the erythrocyte counts were only decreased at 15 mg DON/kg feed. No effect of DON on the responses to common vaccines was observed. In plasma, interleukin 8 levels in both contaminated groups were significantly higher than in the control group. The expression of interleukin 6, interleukin 1ß and interferon-γ increased in jejunum tissues of broilers fed 5 mg/kg of DON compared with controls. The stress index (heterophil to lymphocyte ratio) was not affected by DON-contaminated diets compared with controls. The plasma corticosterone level was significantly lower in both DON groups compared with controls. In conclusion, DON-3S could be used as a specific biomarker of DON in different biological matrices, while the immune response in broiler chickens is stimulated by the presence of DON at the guidance level, but no adverse effect was observed on physiological stress parameters.

Biomarkers of Deoxynivalenol Toxicity in Chickens with Special Emphasis on Metabolic and Welfare Parameters.

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium species, is the most widespread mycotoxin in poultry feed worldwide. Long term-exposure from low to moderate DON concentrations can produce alteration in growth performance and impairment of the health status of birds. To evaluate the efficacy of mycotoxin-detoxifying agent alleviating the toxic effects of DON, the most relevant biomarkers of toxicity of DON in chickens should be firstly determined. The specific biomarker of exposure of DON in chickens is DON-3 sulphate found in different biological matrices (plasma and excreta). Regarding the nonspecific biomarkers called also biomarkers of effect, the most relevant ones are the impairment of the productive parameters, the intestinal morphology (reduction of villus height) and the enlargement of the gizzard. Moreover, the biomarkers of effect related to physiology (decrease of blood proteins, triglycerides, hemoglobin, erythrocytes, and lymphocytes and the increase of alanine transaminase (ALT)), immunity (response to common vaccines and release of some proinflammatory cytokines) and welfare status of the birds (such as the increase of Thiobarbituric acid reactive substances (TBARS) and the stress index), has been reported. This review highlights the available information regarding both types of biomarkers of DON toxicity in chickens.

Effects of Deoxynivalenol-Contaminated Diets on Productive, Morphological, and Physiological Indicators in Broiler Chickens.

The present study with 1-day-old male broilers (Ross 308) was conducted to evaluate the effects of deoxynivalenol (DON) at different levels (5 and 15 mg/kg feed) on growth performance, relative weight of organs, morphology of the small intestine, serum biochemistry, and welfare parameters of broiler chickens. Forty-five broiler chicks were randomly divided into three different experimental groups with five replicates each: (1) control group received a non-contaminated diet, (2) contaminated diet with 5 mg DON/kg of feed, and (3) contaminated diet with 15 mg DON/kg of feed for 42 days. Results showed that feed artificially contaminated with DON at guidance level (5 mg/kg diet) did not affect growth performance parameters. However, 15 mg/kg reduced body weight gain and altered feed efficiency. DON at two assayed levels significantly increased the absolute and relative weight of thymus and the relative weight of gizzard and decreased the absolute and the relative weight of the colon. Compared to controls, both doses affected small intestine morphometry parameters. In terms of biochemical indicators, DON at 5 mg/kg reduced the creatine kinase level and at 15 mg/kg DON reduced the cholesterol level. Furthermore, DON at 15 mg/kg induced more fear in broilers compared to broilers fed the guidance level. It was concluded that even the guidance level of DON did not affect the chickens' performance. However, its toxic effect occurred in some organs and biochemical parameters.

Oligosaccharide structure determines prebiotic role of ß-galactomannan against Salmonella enterica ser. Typhimurium in vitro.

Prebiotics and probiotics are considered natural alternatives to dietary antibiotics in animal production. Plant extracts and yeast cell walls are mannose-rich products that can be used as substrate for adhesion of Gram-negative bacteria. We assessed whether the structure of these saccharides is relevant to develop their role as prebiotics and therefore, their suitability to be used as alternatives to antibiotics to prevent intestinal infections in pigs. The prebiotic functionality of ß-galactomannan (ßGM), mannanoligosaccharide from yeast Saccharomyces cerevisiae (Mannan SC) and monosaccharide D-Mannose were studied in porcine intestinal epithelial cells (IPI-2I) challenged with Salmonella enterica ser. Typhimurium. Results showed that in vitro challenge with Salmonella induces the secretion of proinflammatory cytokine IL6 and chemokine CXCL8 compared with control without infection. Both ßGM and Mannan SC, attenuate Salmonella-induced secretion of IL6 and CXCL8. Interestingly, cells treated with D-mannose showed similar levels of proinflammatory IL6 and CXCL8 compared with the control of infection. These data suggest that prebiotic role of ßGM is related to its oligosaccharide structure.

ß-Galactomannan and Saccharomyces cerevisiae var. boulardii modulate the immune response against Salmonella enterica serovar Typhimurium in porcine intestinal epithelial and dendritic cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed ß-galactomannan (ßGM) prebiotic compared to the proven probiotic Saccharomyces cerevisiae var. boulardii on porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) cocultured in vitro with Salmonella. We observed that both S. cerevisiae var. boulardii and ßGM inhibited the association of Salmonella with IECs in vitro. Our data indicated that ßGM has a higher ability than S. cerevisiae var. boulardii to inhibit Salmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, ßGM and S. cerevisiae var. boulardii induced some effects on DCs that were not observed on IECs: ßGM and S. cerevisiae var. boulardii showed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of ßGM or S. cerevisiae var. boulardii on DCs cocultured with Salmonella showed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control with Salmonella. In conclusion, the addition of ßGM inhibits Salmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.