Colonic Digesta Research Articles

Effects of pig colonic digesta and dietary fibres on in vitro microbial fermentation profiles

Abstract The effects of soluble soybean polysaccharides (SSPS) and soluble flaxseed gum (SFG) on the production of short chain fatty acids (SCFA) and other acids as well as bacterial communities during in vitro fermentation by pig colonic digesta were investigated with inulin as the reference fibre. Digesta samples from different ages of pigs (2-month- versus 7-month-old) that exhibited different PCR–DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis) bacterial profiles were initially examined for their influence over Lactobacillus and Bifidobacterium populations when used as an inoculum. The influence of digesta from 7-month-old pigs was more obvious and thus chosen for the remaining experiments. All fibre-grown cultures showed a significant increase in acid production compared to the control cultures that contained no fibres. SFG was less degradable than SSPS and inulin by bacteria from the digesta. Inulin-grown cultures produced mainly lactate and SSPS- and SFG-grown cultures generated largely acetate. While SFG appeared to support the production of propionate, SSPS and inulin shared a similarity in favouring butyrate production. PCR–DGGE and quantitative PCR (QPCR) analyses indicated that types of dietary fibre were able to dictate bacterial communities during the fermentation. Both inulin and SSPS were shown to enrich Bifidobacterium . Unlike inulin, SSPS had no enrichment toward Lactobacillus . SFG was able to enrich Bacteroidetes . While the fermentability and bacterial selection of dietary fibres suggest their potential application in fortifying food products, the impact of digesta from different ages of pigs on fermentation/microbiota patterns highlights the need for consideration of ages in further studies of nutritional values of dietary fibres towards colonic health.

Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin

The aim of this study was to determine whether orally ingested ovine serum IgG partly resists digestion in the growing rat. Fifteen Sprague-Dawley male rats were allocated to one of three diets for a 3-week study: a control diet (CON) and two test diets containing either freeze-dried ovine serum immunoglobulin (FDOI) or inactivated ovine serum immunoglobulin (IOI). Samples of stomach chyme and intestinal digesta from the ad libitum-fed rats were subjected to ELISA and Western blot analysis. Amounts of intact ovine IgG for the FDOI diet were found to be 13.9, 20.0, 34.1, 13.0 and 36.9 μg in the total wet digesta from the stomach chyme, duodenal, jejunal, ileal and colonic digesta respectively. Qualitative detection by Western blot revealed the presence of intact ovine serum IgG with a ~150 kDa MW. This was detected in all of the gut segments (stomach chyme, duodenal, jejunal, ileal and colonic digesta) for growing rats fed the FDOI diet. No ovine IgG was detected in the chyme or digesta from rats fed the CON or the IOI diets. Ovine serum IgG partly resisted digestion in the growing rat fed the FDOI diet and was found throughout the digestive tract. These results provide a basis to explain the reported biological effects of orally administered immunoglobulin.

Influence of fermentable carbohydrates or protein on large intestinal and urinary metabolomic profiles in piglets1

It was recently shown that variations in the ratio of dietary fermentable carbohydrates (fCHO) and fermentable protein (fCP) differentially affect large intestinal microbial ecology and the mucosal response. Here we investigated the use of mass spectrometry to profile changes in metabolite composition in colon and urine associated with variation in dietary fCHO and fCP composition and mucosal physiology. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP and low fCHO, low fCP and high fCHO, high fCP and low fCHO, and high fCP and high fCHO. After 21 to 23 d, all pigs were euthanized and colon digesta and urine metabolite profiles were obtained by mass spectrometry. Analysis of mass spectra by partial least squares approach indicated a clustering of both colonic and urinary profiles for each pig by feeding group. Metabolite identification and annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed increased abundance of metabolites associated with arachidonic acid metabolism in colon of pigs fed a high concentration of fCP irrespective of dietary fCHO. Urinary metabolites did not show as clear patterns. Mass spectrometry can effectively differentiate metabolite profiles in colon contents and urine associated with changes in dietary composition. Whether metabolite profiling is an effective tool to identify specific metabolites (biomarkers) or metabolite profiles associated with gut function and integrity needs further elucidation.

Insoluble nonstarch polysaccharides in diets for weaned piglets

To examine the effect of inclusion insoluble nonstarch polysaccharides (iNSP) in weaned diets on postweaning piglet performance, intestinal activity, and microbial composition 2 experiments were designed. In Exp. 1, 180 piglets were weaned at 28 d of age and divided over 3 treatments: positive control (PC; highly digestible protein), negative control (NC; standard cereal-based diet), and an experimental diet {iNSP; standard + 15% iNSP [wheat straw and oat (Avena sativa) hull]}. Diets were fed during 14 d after weaning. The ADFI, ADG, and G:F were determined. The inclusion of iNSP increased (P < 0.001) ADFI and G:F during days 0 to 14 after weaning and increased the ADG (P = 0.008) during days 0 to 7 after weaning compared with the NC diet. In Exp. 2, 36 piglets were fed the same diets as in Exp. 1. On days 5 and 14 after weaning, 18 piglets were euthanized to determine enzyme activity, intestinal morphology, and microbial population in the ileum and colon and organ weight. The iNSP diet reduced the concentration of Escherichia coli bacteria in the ileum (P = 0.021) and in the colon (P = 0.002) digesta and tended to increase (P = 0.060) the amylase activity. The iNSP diet stimulated the physical adaptation of the gastrointestinal tract because stomach weight as percentage of BW was heavier (P = 0.004) than for the NC diet. In conclusion, the consumption of diets with higher iNSP content in the early weaning period affects the microbial colonization without reducing enzyme activity or animal performance compared with a standard weaned pig diet.

Interactions of Saccharomyces cerevisiae fermentation product and in-feed antibiotic on gastrointestinal and immunological responses in piglets challenged with Escherichia coli K88+1

Interactive effects between in-feed antibiotics (PC; 0.04% chlortetracycline + 0.004% Denagard) and a Saccharomyces cerevisiae fermentation product (XPC) were investigated. Five pens of 3 pigs/pen were randomly allotted to one of six diets consisting of control (NC; no additives), XPC alone (0.2%), PC alone, or PC plus one of three levels of XPC (0.1, 0.2, or 0.4%). On day 7, all pigs were bled for baseline serum and then orally inoculated with a 6-mL dose of 2 x 10(9) cfu/mL of ciprofloxacin-resistant Escherichia coli K88(+) (ETEC). Severity of diarrhea, using fecal score (FS), was assessed on days 8, 9, and 10. On day 10, pigs were bled and 1 pig/pen was killed to obtain gastrointestinal (GIT) samples. Remaining pigs were monitored for another 4 d and then killed to obtain GIT samples. Pigs fed PC and XPC in combination of PC had a lower (P = 0.02) FS than NC. The PC pigs had lower (P = 0.02) serum interleukin-6 and XPC pigs had higher (P < 0.01) serum tumor necrosis factor-α than NC pigs. Ileal mucosal ETEC count was lower on day 10 (P = 0.04) and day 14 (P < 0.01) when additives were fed in combination. Microbiome analysis of day 10 digesta using pyrosequencing showed that pigs fed the PC with XPC had enriched (P = 0.01) Streptococcaceae family than PC fed alone. On day 10, pigs fed PC alone and in combination with XPC at a lower dose had shallower (P = 0.01) crypt depth than those fed higher doses of XPC, XPC alone, or NC. Pigs fed XPC had a thicker colon wall on day 10 (P < 0.01) compared to those fed non-XPC diets. Ileal and colonic digesta NH(3) concentration was lower (P < 0.05) for pigs fed additives-containing diets on days 10 and 14. Feeding PC or XPC reduced diarrhea and the number of ETEC attached to the ileal mucosa; however, responses suggest independent and interactive effects that might be associated with the nature of these additives.

The porcine gut microbial metagenomic library for mining novel cellulases established from growing pigs fed cellulose-supplemented high-fat diets1

The porcine gut microbiome is a novel genomic resource for screening cellulose-degrading enzymes. A plasmid metagenomic expression library was constructed from the hindgut microbiota of 6 Yorkshire growing pigs (25 to 40 kg) fed a high-fat basal diet supplemented with 10% Solka-Floc for 28 d. Fresh cecal and colonic digesta samples were collected, flash-frozen in liquid N, and stored under -80°C. Metagenomic DNA was extracted, mechanically sheared, and cleaned to remove small DNA fragments (<1.0 kb). The resulting DNA fragments were subjected to blunt-end polishing, fractionation, and purification by using commercial kits. The end-modified DNA fragments were ligated to pCR4Blunt-TOPO vector and transformed into competent Escherichia coli TOPO10 cells. Metagenomic plasmid libraries were screened for carboxymethyl cellulolytic activities by using lysogeny broth agar plates. The average insert size of the resulting library was approximately 4.2 kb. Screening for the ability to hydrolyze carboxymethyl cellulose yielded 14 positive colonies, giving an estimated 430 Mb of metagenomic DNA in the approximately 102,000 E. coli clones with an overall hit rate of 0.14%. The 11 assembled insert sequences included 4 function-related gene clusters, and a total of 18 putative carbohydrate active enzyme genes were identified. This included genes encoding 11 cellulases, 4 hemicellulases, 1 polygalacturonas, 1 glycoside hydrolase family 26 mannanase-family 5 cellulase chimeric enzyme gene, and 1 cellobiose phosphorylase. In conclusion, the coupling of functional metagenomic mining with biochemical characterization of fiber-degrading enzymes is a powerful strategy for exploring the enzymological underpinnings of the anaerobic fermentation of dietary fiber in the complex animal gut environment.

Hematological Abnormalities in Aged Telomerase RNA Component Deficient Mice Are Associated with Intestinal Epithelial Degeneration and Bone Marrow Stromal Dysfunction

AbstractAbstract 1244Mice carrying germline deletion of the telomerase RNA component (Terc−/−) have functional alterations in various tissues but can breed and live normally for six generations. We acquired Terc−/− mice at G3 generation under the C57BL/6 (B6) background, backcrossed to normal inbred B6 mice, interbred between Terc+/− offspring to generate Terc−/−, Terc+/− and Terc+/+ mice at later generations, and analyzed cytological changes in hematological tissues using animals at young (3–6 months), middle (12–18 months) and old (20–26 months) ages. Cellular composition was relatively normal in peripheral blood (PB), bone marrow (BM) and spleen in young Terc−/− and Terc+/− mice relative to Terc+/+ littermates. However, middle and old Terc−/− mice had significantly lower levels of red blood cells, hemoglobin and hematocrit, and significantly higher percentage red cell distribution width and neutrophil counts in comparison to old Terc+/+ mice and young B6 controls. Middle and old Terc−/− mice also showed typical signs of lineage bias with significantly higher CD11b cell percentage and significantly lower CD45R cell percentage in BM, PB and spleen. Interestingly, BM cells from middle and old Terc−/− mice contained higher percentage and total number of Lin−Sca1+Kit+CD150+ hematopoetic stem and progenitor cells than those from young Terc−/− and young B6 controls. Transplantation of old Terc−/− BM cells into lethally irradiated young B6 recipients resulted in effective donor cell engraftment with relatively normal levels of blood cell counts and normal CD11b and CD45R cell percentages at four to twelve months after transplantation, indicating that the anemia, neutrocytosis and hematopoietic lineage bias seen in aged Terc−/− mice were not intrinsic to hematopoietic stem or progenitor cells. We therefore hypothesize that the age-related hematological abnormalities in Terc−/− mice were responses secondary to inflammatory stresses, or were results of BM stromal dysfunction, or both.Since hematological abnormalities were easily detectable in middle Terc−/− (12–18 months) mice, we used animals of this age group in our future studies. Middle Terc−/− mice were smaller in size compared to littermates, lethargic, in a hunched posture, and produced a soft stool instead of fecal pellets. At necropsy, the walls of the ileum, cecum and colon were thickened; the ceca were diminished in size, and the cecal and colonic digesta was soft and pasty. Histologic changes in the small intestine, cecum and colon consisted of dysplastic mucosal epithelium with anisocytosis, cytomegaly, and karyomegaly. The small intestinal villi were blunted and fused. Mucosal crypts were lost in the small intestine, cecum and colon along with mucosal ulceration, epithelial attenuation and bacterial colonization of ulcerated areas. There was marked suppurative intestinal inflammation along with severe enteritis, typhlitis and colitis (Figure 1, A & B). In these same animals, BM hematopoiesis was dominated by myeloid precursors with a sharp decline in the presence of erythroid precursors (Figure 1, C & D). In the skin sections, the vast majority of hair follicles were in telogen, a resting phase, and were located close to the epidermis with few sebaceous glands around the hair follicle. Liver, heart, lung and kidney were relatively normal based on microscopic examinations. We analyzed plasma concentrations of selected hematopoietic cytokines and found insignificant declines in GM-CSF and IL-3 and significant increases in G-CSF and IL-6 in middle Terc−/− mice. Flow cytometry analyses revealed a lower percentage of CD3−CD11b−CD45R−CD44+Sca1+ stromal cells in the BM of middle Terc−/− animals. Culturing middle Terc−/− BM cells in vitro in alpha-modified eagle media at 33 degrees for two weeks resulted in the development of stromal feeder layers with normal morphological appearance but reduced functional efficacy to support cobblestone colony formation from freshly-overlaid normal B6 BM cells. We conclude that constitutional Terc deficiency in middle and old Terc−/− mice causes obvious intestinal epithelia degeneration and severe intestinal inflammation that adversely affects normal hematopoiesis to result in neutrocytosis, anemia and lineage bias disfavoring B cells. Dysfunction of BM stromal cells might also contribute to hematological abnormalities in aged Terc-deficient animals. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Casein glycomacropeptide in the diet may reduceEscherichia coliattachment to the intestinal mucosa and increase the intestinal lactobacilli of early weaned piglets after an enterotoxigenicE. coliK88 challenge

Casein glycomacropeptide (CGMP), a glycoprotein originating during cheese manufacture, has shown promising effects by promoting the growth of some beneficial bacteria in vitro, although its activity has not been well explored. The present study was designed to evaluate the effects of CGMP against enterotoxigenic Escherichia coli (ETEC) K88 in vitro (Trial 1) and in vivo (Trial 2). In Trial 1, increasing concentrations of CGMP (0, 0.5, 1.5 or 2.5 mg/ml) were tested regarding its ability to block the attachment of ETEC K88 to ileal mucosa tissues obtained from piglets. Increasing the concentration of CGMP resulted in a gradual decrease in ETEC K88 attachment to the epithelial surface. In Trial 2, seventy-two piglets were distributed in a 2 × 2 factorial combination including or omitting CGMP in the diet (control diet v. CGMP) and challenged or not with ETEC K88 (yes v. no). Inclusion of CGMP increased crude protein, ammonia and isoacid concentrations in colon digesta. CGMP also increased lactobacilli numbers in ileum and colon digesta, and reduced enterobacteria counts in mucosa scrapings and the percentage of villi with E. coli adherence measured by fluorescence in situ hybridisation. The inclusion of CGMP in the diets of challenged animals also prevented the increase of enterobacteria in ileal digesta. We can conclude that CGMP may improve gut health by diminishing the adhesion of ETEC K88 to the intestinal mucosa, by increasing the lactobacilli population in the intestine and by reducing the overgrowth of enterobacteria in the digestive tract of piglets after an ETEC K88 challenge.

Fructan extracts from wheat stem and barley grain stimulate large bowel fermentation in rats

The benefits of inulin-type fructans for bowel health are well established, but less so for other fructan sources. In vitro data suggest that fructans extracted from cereals are readily fermented and produce favorable short-chain fatty acid profiles; however, whether this occurs in vivo is unknown. We hypothesized that in rats, fructans extracted from wheat stem and barley grain would have similar effects on fermentation as oligofructose (OF). Fifty-six male Sprague-Dawley rats were randomly assigned to 1 of 7 dietary treatments that contained either 2% or 5% fructan, provided by a barley grain fructan extract (BGFE), a wheat stem fructan extract, or OF or no added fructan (control). The duration of the feeding study was 14 days. Rats fed diets containing 5% fructan had higher cecal digesta weights; larger acetate, propionate, and total short-chain fatty acid pools; and lower pHs in comparison with the control group. In addition, only the 5% OF and 5% BGFE groups increased cecal butyrate pools, and 5% BGFE was the only group in which colonic digesta pH was lower than that of the control. Diets containing 2% fructan did not affect any of these fermentation end points. Whereas bifidobacteria numbers in cecal digesta of 2% and 5% OF were higher than that in the control group, they were not different from those in rats fed diets containing BGFE and wheat stem fructan extract. Barley grain and wheat stem fructans produced similar large bowel fermentation patterns to OF when fed to rats at 5% of the diet.

Enzyme deactivation treatments did not decrease the beneficial role of oat food in intestinal microbiota and short‐chain fatty acids: an in vivo study

Steaming and roasting treatments are widely used enzyme deactivation methods in the oat food industry in China. Whether or not the enzyme deactivation treatments affect the nutritional function of oat foods is unknown. In the current study, we examined the effects of 4-week ingestion of steamed or roasted oat foods on the intestinal bacteria and short-chain fatty acids of rats. Compared with rats taking no oat foods, rats taking normal oat foods or enzyme-deactivated oat foods showed significantly higher (P < 0.05) counts of Lactobacillus spp. and Bifidobacterium spp. in colon, significantly lower (P < 0.05) counts of Enterococcus spp. and coliforms in colon, and significantly higher (P < 0.05) levels of butyrate and acetate in colonic digesta. In addition, rats taking infrared roasting (IR)-treated oat foods also demonstrated significantly higher (P < 0.05) fecal Lactobacillus spp. counts and significantly lower (P < 0.05) cecal and fecal counts of E. coli, Enterococcus spp. and coliforms than rats taking no oat foods. As for the comparison between the enzyme-undeactivated oat group and the three enzyme-deactivated oat groups, there were no significant differences in most of the parameters (P > 0.05), though a few exceptions did exist. Enzyme deactivation treatments did not decrease the beneficial role of oat food in the intestinal microbes and short-chain fatty acids of rats.

Chain length of inulin affects its degradation and the microbiota in the gastrointestinal tract of weaned piglets after a short-term dietary application

Dietary inulin can affect the composition and metabolic activity of the gastrointestinal microbiota in piglets. To investigate whether the chain length of inulin may influence its stability in the gut and the bacterial community, 18 weaned piglets were housed 2 per cage, with 1 female and 1 castrated male animal each. The piglets received a control diet without or with 4% inulin, defined by an average degree of polymerisation (DP) of 31 (short-chain, I31) or 57 (long-chain, I57), with 6 piglets/diet. After a short feeding period of 6d, fructan concentrations, selected bacterial groups, lactic acid, short-chain fatty acid concentrations, and the pH were determined in the digesta of different segments of the gastrointestinal tract. The results indicated that differences in the microbial degradation of inulin were depending on the DP. Compared to the short-chain inulin, the concentrations of the long-chain inulin were numerically greater in the small intestine and caecum, and greater in the digesta of the ascending colon. Differences were also observed in the bacterial composition of the digesta, showing greater cell numbers of enterococci (P=0.029), bifidobacteria (P=0.029), and Lactobacillus mucosae (P=0.028) in the ileum in group I57 compared to group I31. However, most bacteria tended to be numerically reduced in the ileum in group I31 compared to both control and I57 groups. Minor effects were observed in the ascending colon: L. reuteri and L. amylovorus were decreased in group I57 compared to the control group (P=0.031 and 0.034, respectively), and L. mucosae was decreased in group I31 compared to the control animals (P=0.029). The concentrations of bacterial metabolites were distinctively changed in the large intestine of the piglets fed inulin. The pH was lower in the rectum contents in group I57 compared to the control piglets (P=0.026), but lactic acid and total short-chain fatty acid concentrations were not affected. The molar ratios of propionic acid increased in the caecal contents (P=0.040) and in both, the ascending and descending colonic digesta (P=0.017 and 0.013, respectively) in group I57 compared to the control group, while acetic acid decreased (P<0.001) and n-valeric acid increased (P<0.001 and P=0.011, respectively) in the digesta of the ascending and descending colon in group I57. In conclusion, the microbial degradation of inulin was dependent on its chain length. Long-chain inulin affected the microbial fermentation more pronounced compared to short-chain inulin. The effects were already observed after 6d, a relatively short application period, indicating that inulin may be used specifically during the sensitive post-weaning period for piglets.

Changes in Bowel Microbiota Induced by Feeding Weanlings Resistant Starch Stimulate Transcriptomic and Physiological Responses

The ability to predictably engineer the composition of bowel microbial communities (microbiota) using dietary components is important because of the reported associations of altered microbiota composition with medical conditions. In a synecological study, weanling conventional Sprague-Dawley rats (21 days old) were fed a basal diet (BD) or a diet supplemented with resistant starch (RS) at 5%, 2.5%, or 1.25% for 28 days. Pyrosequencing of 16S rRNA genes and temporal temperature gradient electrophoresis (TTGE) profiles in the colonic digesta showed that rats fed RS had altered microbiota compositions due to blooms of Bacteroidetes and Actinobacteria. The altered microbiota was associated with changes in colonic short-chain fatty acid (SCFA) concentrations, colonic-tissue gene expression (Gsta2 and Ela1), and host physiology (serum metabolite profiles and colonic goblet cell numbers). Comparisons between germ-free and conventional rats showed that transcriptional and serum metabolite differences were mediated by the microbiota and were not the direct result of diet composition. Altered transcriptomic and physiological responses may reflect the young host's attempts to maintain homeostasis as a consequence of exposure to a new collection of bacteria and their associated biochemistry.

Fermentable Fiber Ameliorates Fermentable Protein-Induced Changes in Microbial Ecology, but Not the Mucosal Response, in the Colon of Piglets1–3

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21–23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Equivalent Effects on Fecal Reactive Oxygen Species Generation with Oral Supplementation of Three Iron Compounds: Ferrous Sulfate, Sodium Iron EDTA and Iron Polymaltose

Background: In any context of iron supplementation in the prenatal prophylaxis or therapeutic dosage range, a large amount will remain unabsorbed and pass through the intestinal tract into the colonic digesta possibly causing increased oxidation. Aim: To compare the generation of fecal reactive oxygen species (ROS) in situ after daily consumption of 100 mg of elemental iron in three frequently used forms of iron supplements. Methods: Ten healthy, iron-repleted adult males were investigated before and during supplementation with three oral iron compounds: 100 mg of oral iron were given as ferrous sulfate, Na Fe-EDTA and iron polymaltose for 6 days to each subject in an individually stratified sequence. Stool samples were collected and analyzed for iron content and the in situ generation of fecal ROS. Results: Significant increases in fecal ROS generation were observed during oral iron supplementation. No statistical differences were seen in either residual concentrations of non-heme iron in stool or the level of fecal ROS generation between the three Fe compounds. There was, however, a significant association between the iron concentration in the stool and ROS generation. Conclusion: In spite of the differences in their chemical characteristics, none of the three distinct iron complexes reduced oxidative stress in the intestinal lumen.

Weaned pig responses to Escherichia coli K88 oral challenge when receiving a lysozyme supplement1,2

Lysozyme is a low-molecular-weight protein with antimicrobial properties. An experiment was conducted to investigate the response of piglets receiving a water-soluble lysozyme supplement [Entegard (EG), Neova Technologies Inc., Abbotsford, British Columbia, Canada; 4,000 lysozyme units/mg] after oral challenge with enterotoxigenic Escherichia coli (ETEC). A total of 36 individually housed weanling pigs were randomly allotted to 1 of the 4 treatments, with 9 replicates per treatment. Treatments were a control (CONT, no additive), antibiotic (AB; 2.5 g/kg of feed of antibiotic with chlortetracycline, sulfamethazine, and penicillin), and EG delivered in the drinking water at concentrations of 0.1% (EG1) and 0.2% (EG2). All pigs received a basal diet similar in composition and nutrients, except for pigs receiving the AB diet, which had an added antibiotic. Pigs were acclimated to treatments for a 7-d period to monitor growth performance. On d 8, blood samples were collected from each pig to obtain serum, and each pig was gavaged with 6 mL (2 × 10(9) cfu/mL) of ETEC solution. Pigs were monitored for another 7 d to assess incidences of diarrhea and growth performance, and then all pigs were killed to obtain intestinal tissue and digesta samples. Treatments did not influence growth performance throughout the study. Greater ETEC counts were observed in the ileal mucosal scrapings (P = 0.001) and colonic digesta (P = 0.025) of pigs in the CONT group compared with pigs in the AB and EG1 groups. Pigs receiving AB and EG1 had greater (P < 0.05) small intestinal weights and ileal villus heights than pigs receiving CONT; however, the ileal villus height-to-crypt depth ratio was greater in pigs fed the AB diet (1.69) compared with those fed the CONT diet (1.34), whereas pigs receiving EG1 were intermediate. Pigs in the EG1 group showed greater (P < 0.001) serum tumor necrosis factor α and IL-6 concentrations before ETEC challenge; however, at 7 d postchallenge, pigs receiving EG2 showed the least (P < 0.05) circulating tumor necrosis factor α and IL-6 concentrations. Overall, better intestinal growth and development, as well as decreased ETEC counts on the intestinal mucosa and serum proinflammatory cytokines, suggest that EG can maintain gut health and function in piglets commensurate with antibiotics. However, it is noteworthy that at the largest dose tested, EG seemed to have a dramatic effect on proinflammatory cytokines but had a minimal or no effect on the other response criteria.

Ovine Serum Immunoglobulin Has Immunomodulatory Effects in Growing Rats Gavaged with Salmonella enteritidis1

AbstractIn this study, we aimed to determine whether orally administered ovine serum Ig modulate aspects of immunity and associated gut microflora in growing rats challenged with Salmonella enteritidis. The 4 groups consisted of rats fed a casein-based control diet (BD; ungavaged) and 3 groups of rats gavaged with 1 107 viable Salmonella enteritidis and fed a BD diet, a BD diet containing freeze-dried ovine Ig (FDOI), or a BD diet containing inactivated ovine Ig (IOI). The rats were randomly allocated to 1 of the 4 diets (n = 15) and consumed it for 18 d. They were orally gavaged on d 15. Phagocytic activity of peripheral blood leukocyte and lymphocyte proliferation in the presence of the concanavalin A (ConA) were greater (P < 0.05) in the ungavaged BD- and gavaged FDOI-fed rats than in the gavaged rats fed either the BD or IOI diet. ConA-stimulated Peyer's patch cells and splenocytes from the gavaged rats fed the FDOI diet produced more IFN?, IgA, and IgG than the gavaged rats fed either the BD or IOI diet (P < 0.05). The gavaged FDOI-fed rats had higher ileal and colonic digesta and plasma concentrations of anti-Salmonella secretory sIgA and secretory sIgG (P < 0.05). DNA analysis of a denatured gradient gel electrophoresis profile revealed that 6 of 10 bands had sequence similarity to probiotic strains of bacteria in the ileum and colon of the gavaged FDOI-fed rats. In conclusion, an ovine Ig fraction modulated various indices of immune function and associated gut microflora in growing rats inoculated with Salmonella.

Cultivation-Independent Analysis of the Development of the Lactobacillus spp. Community in the Intestinal Tract of Newborn Piglets

Abstract Molecular diversity and development of the Lactobacillus community in the intestinal tract, as influenced by age and intestinal compartment, were studied in one litter of 12 conventionally raised piglets. Piglets were euthanized at each week (3 animals per time). Digesta and tissue samples from stomach, duodenum, jejunum, ileum, caecum, colon, and rectum were collected and analysed by using 16S ribosomal RNA-based methods. DGGE (denaturing gradient gel electrophoresis) profiles revealed that the Lactobacillus communities throughout the GI tract from duodenum to rectum showed good stability at same age. This indicates that fecal Lactobacillus communities can effectively represent the intestinal community. Two dominant bands were found in tissue samples of the small intestine, suggesting that the lactobacilli can adhere to the small intestinal wall. The Lactobacillus communities in different GI tract compartments developed over time. A successional change of Lactobacillus communities was observed from birth, through creep feeding to one week after weaning, showing a trend from simple to complex and back to simple. Furthermore, a clone library of Lactobacillus spp. 16S rRNA gene sequences were generated from jejunal and colonic chymes. Six dominant DGGE bands generated from jejunal chymes were matched with sequences that show 94-98% similarity to the bands derived from L. reuteri, L. delbrueckii, and L. crispatus. Seven dominant DGGE bands generated from colon chymes were matched with sequences that show 88-99% similarity to those derived from L. reuteri, L. delbrueckii, L. amylovorus/L. sobrius, and L. acidophilus. Amplicons related to L. reuteri were found in all DGGE fingerprints from jejunal digesta of age of weeks 1, 3, and 4. Amplicons related to L. amylovorus/L. sobrius were present in all DGGE fingerprints from colonic digesta of age of week 1, 3, and 4. Amplicons related to L. delbrueckii were found before weaning, L. crispatus after creep feeding before weaning and L. acidophilus after weaning. This indicates that L. reuteri and L. amylovorus/L. sobrius probably belong to the permanent composition, while L. delbruckii, L. acidophilus, and L. crispatus probably belong to the temporal groups of Lactobacillus communities in the GI tract of piglets.

Dietary resistant and butyrylated starches have different effects on the faecal bacterial flora of azoxymethane-treated rats

Epidemiological studies have suggested that dietary fibre lowers the risk of colorectal cancer, which may be due to increased butyrate production from colonic fermentation of a type of fibre, resistant starch (RS). The present study investigated the effects of dietary RS and butyrylated RS on the faecal microbiota of rats treated with azoxymethane. A total of four groups of nine rats were fed diets containing either standard maize starch (low-amylose maize starch (LAMS), low RS), LAMS with 3 % tributyrin (LAMST), cooked 10 % high-amylose maize starch (HAMS, high RS) or cooked 10 % butyrylated HAMS (HAMSB). Faecal samples were examined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Multivariate analysis demonstrated no differences between faecal microbiota before treatment but revealed differences in DGGE patterns between diet groups, with the exception of the two low-RS groups (LAMS and LAMST). Subsequent analysis identified eleven DGGE bands contributing significantly to the differentiation between diets. These phylotypes belonged to Clostridiales (five), Lactobacillus (one) and Bacteroidetes (five) lineages. Rats fed HAMS had increased concentration of propionate in their distal colonic digesta and developed faecal populations containing Ruminococcus bromii-like bacteria. HAMSB increased propionate and butyrate concentrations in distal colonic digesta and was associated with the appearance of two non-butyrate-producing bacteria, Lactobacillus gasseri and Parabacteroides distasonis. In conclusion, supplementation with specific dietary RS leads to changes in faecal microbiota profiles that may be associated with improved bowel health.

Dietary addition of Lactobacillus rhamnosus GG impairs the health of Escherichia coli F4-challenged piglets

Lactobacillus rhamnosus GG (LGG) is a probiotic for humans and is normally not found in pigs; however, it has been shown to protect the human-derived intestinal Caco-2 cells against the damage induced by an important intestinal pathogen, enterotoxigenic Escherichia coli F4 (ETEC). An experiment was conducted to test whether the dietary addition of LGG improves the growth and health of weaned pigs when orally challenged by E. coli F4. Thirty-six pigs were weaned at 21 days and assigned to a standard weaning diet with or without 1010 CFU LGG (ATCC 53103) per day. The pigs, individually penned, were orally challenged with 1.5 ml of a 1010 CFU E. coli F4 suspension on day 7 and slaughtered on day 12 or 14. With the addition of LGG, the average daily gain and the average daily feed intake were reduced after the challenge with ETEC and for the entire trial (P < 0.05). The average faecal score tended to worsen from day 11 to the end of the trial and the concentration of ETEC in the faeces tended to increase (P = 0.07) with the LGG supplementation. The counts of lactic acid bacteria, enterobacteria and yeasts in the colonic digesta were not affected. The pH values in ileal, colonic and caecal digesta, and the small intestine size were also unchanged. Regardless of the site of measurement (duodenum, jejunum or ileum), a trend of decreased villus height was seen with LGG (P = 0.10). Crypt depth and villus to crypt ratio were unchanged by the diet. A gradual increase of total seric IgA was seen after 1 week and after the challenge, in the control (P < 0.05), but not in the treated group. After the challenge, the LGG reduced the total IgA in the blood serum (P < 0.05), v. the control. The total IgA in the saliva and in the jejunum secretion were not affected by the diet. The F4-specific IgA activity was not affected by the diet at all the samplings. Our result shows that, the administration of LGG do not prevent or reduce the detrimental effect of the E. coli F4 infection on the growth performance and health status of weaned piglet.

Dietary supplementation with ovine serum immunoglobulin is associated with an increased gut luminal mucin concentration in the growing rat

The mucus layer covering the gut epithelium is pivotal to host defence and is affected by various dietary components. Part of the reported beneficial effect of dietary immunoglobulins (Igs) on gut health may be due to effects on the gut mucus layer. The aim was to determine whether orally administered ovine serum Ig influence goblet cell count, mucin gene expression and digesta mucin protein content in the gut of the growing rat. Fourteen Sprague–Dawley male growing rats were used in a 21-day study and were fed either a casein-based control diet (CON; no Ig) or a similar diet but containing freeze-dried ovine Ig (FDOI). Daily food intake and growth rate were not affected by the dietary treatments. When compared to the rats consuming CON diet, those consuming the FDOI diet had significantly (P < 0.05) more intact and cavitated goblet cells in the intestinal villi. A similar result was found for crypt goblet cells in the small intestine and colon. Ileal Muc2, Muc3, Muc4 and stomach Muc5Ac mRNA expressions for the FDOI animals were higher (P < 0.05) compared to the the CON animals. Mucin protein content was higher (P < 0.05) in the stomach, ileum and colonic digesta of rats fed the FDOI diet. In conclusion, orally administered FDOI influenced gut mucins in the growing rat as evidenced by increased mucin gene expression and digesta mucin protein concentrations as well as an increased goblet cell count.