Mucins possess potential binding sites for both commensal and pathogenic organisms and may perform a defensive role during establishment of the intestinal barrier. To observe the effects of bacteria on intestinal goblet cell mucin production during posthatch development, differences in the small intestine of conventionally reared (CR) and low bacterial load (LBL) broiler chicks were examined. Jejunal and ileal goblet cells were stained with either periodic acid-Schiff stain or high iron diaminealcian blue pH 2.5 to discriminate among neutral, sulfated, and sialylated acidic mucins. Total goblet cell numbers and morphology of goblet cells containing neutral and acidic mucins did not differ significantly between CR and LBL birds. However, significant differences in acidic mucin composition from primarily sulfated to an increase in sialylated sugars at d 4 posthatch were observed in CR chicks, with greater numbers of jejunal and ileal goblet cells displaying this mucin type (CR, 0.5 +/- 0.1 x 10(3) cells/mm(2); LBL, 0.04 +/- 0.02 x10(3) cells/mm(2)). This change in mucin profile in response to bacterial colonization suggests a potential role as a protective mechanism against pathogenic invasion of the intestinal mucosa during early development.

Solvent‐extracted soybean meal (SESBM) has been reported to cause subacute enteritis in certain fish species. Two 34‐day experiments investigated the effects of SESBM and soy protein concentrate (SPC) on the intestinal mucus layer and development of subacute enteritis in the hindgut of yellowtail kingfish (Seriola lalandi) at 22 and 18 °C. Fish were fed increasing levels of SESBM (Exp. 1: 0 g kg−1, 100 g kg−1, 200 g kg−1, 300 g kg−1) and SPC (Exp. 2: 0 g kg−1, 200 g kg−1, 300 g kg−1, 400 g kg−1). No visual signs of inflammation in the hindgut were observed in either experiment. However, increasing dietary SESBM significantly reduced mucus layer thickness. Neutral and acidic goblet cell mucin composition increased at 18 and 22 °C, respectively. A significant positive linear relationship was evident between goblet cell number and SESBM inclusion at 18 °C. SPC inclusion and water temperature had no significant effect on mucus layer thickness or mucin composition. However, at 18 °C, goblet cell numbers decreased with SPC inclusion. Results suggest the early stages of subacute enteritis may have been manifesting in SESBM fed fish. In the long term, mucus layer alterations associated with feeding SESBM may compromise fish health. Longer‐term studies should investigate the effects of feeding SESBM to yellowtail kingfish, particularly at suboptimal water temperatures.

Clostridial infection of the intestine can result in necrotic enteritis (NE), compromising production and health of poultry. Mucins play a major role in protecting the intestinal epithelium from infection. The relative roles of different mucins in gut pathology following bacterial challenge are unclear. This study was designed to quantify the expression of mucin and mucin-related genes, using intestinal samples from an NE challenge trial where birds were fed diets with or without in-feed antimicrobials. A method for quantifying mucin gene expression was established using a suite of reference genes to normalize expression data. This method was then used to quantify the expression of 11 candidate genes involved in mucin, inflammatory cytokine, or growth factor biosynthesis (IL-18, KGF, TLR4, TFF2, TNF-α, MUC2, MUC4, MUC5ac, MUC5b, MUC13, and MUC16). The only genes that were differentially expressed in the intestine among treatment groups were MUC2, MUC13, and MUC5ac. Expression of MUC2 and MUC13 was depressed by co-challenge with Eimeria spp. and Clostridium perfringens. Antimicrobial treatment prevented an NE-induced decrease in MUC2 expression but did not affect MUC13. The expression of MUC5ac was elevated in birds challenged with Eimeria spp./C. perfringens compared with unchallenged controls and antimicrobial treatment. Changes to MUC gene expression in challenged birds is most likely a consequence of severe necrosis of the jejunal mucosa.

Intestinal health is influenced by a complex set of variables involving the intestinal microbiota, mucosal immunity, digestion and absorption of nutrients, intestinal permeability (IP) and intestinal integrity. An increase in IP increases bacterial or toxin translocation, activates the immune system and affects health. IP in chickens is reviewed in three sections. First, intestinal structure and permeability are discussed briefly. Second, the use of lipopolysaccharide (LPS) as a tool to increase IP is discussed in detail. LPS, a glycolipid found in the outer coat of mostly Gram-negative bacteria, has been reported to increase IP in rats, mice and pigs. Although LPS has been used in chickens for inducing systemic inflammation, information regarding LPS effects on IP is limited. This review proposes that LPS could be used as a means to increase IP in chickens. The final section focuses on potential biomarkers to measure IP, proposing that the sugar-recovery method may be optimal for application in chickens.

Increased intestinal permeability (IP) can lead to compromised health in chickens. As there is limited literature on in vivo biomarkers to assess increased IP in chickens, the objective of this study was to identify a reliable biomarker of IP using DSS ingestion and fasting models. Male Ross chickens (n = 48) were reared until day 14 on the floor pen in an animal care facility, randomized into the following groups: control, DSS and fasting (each with n = 16), and then placed in metabolism cages. DSS was administered in drinking water at 0.75% from days 16 to 21, while controls and fasted groups received water. All birds had free access to feed and water except the birds in the fasting group that were denied feed for 19.5 h on day 20. On day 21, all chickens were given two separate oral gavages comprising fluorescein isothiocyanate dextran (FITC-d, 2.2 mg in 1 ml/bird) at time zero and lactulose, mannitol and rhamnose (LMR) sugars (0.25 g L, 0.05 g M and 0.05 g R in 2 ml/bird) at 60 min. Whole blood was collected from the brachial vein in a syringe 90 min post-LMR sugar gavage. Serum FITC-d and plasma LMR sugar concentrations were measured by spectrophotometry and high-performance ion chromatography respectively. Plasma concentrations of intestinal fatty acid binding protein, diamine oxidase, tight junction protein (TJP), d-lactate and faecal α-antitrypsin inhibitor concentration were also analysed by ELISA. FITC-d increased significantly (p < 0.05) after fasting compared with control. L/M and L/R ratios for fasting and L/M ratio for DSS increased compared with control chickens (p < 0.05). TJP in plasma was significantly increased due to fasting but not DSS treatment, compared with controls. Other tests did not indicate changes in IP (p > 0.05). We concluded that FITC-d and LMR sugar tests can be used in chickens to assess changes in IP.

SummaryFasting of up to 24 hr has been shown to increase intestinal permeability (IP) in chickens.The aim of this study was to determine whether fasting duration of 4.5 and 9 hr increased IP and whether l-glutamine (a non-essential amino acid) supplementation before fasting provided some protection of barrier function as shown in other species.Ross 308 male broilers (n = 96) were fed either a control diet or the same diet supplemented with 1% glutamine from d0 to d38 post-hatch. On d37, the birds were assigned to single-bird metabolism cages and were fasted for either 0, 4.5, 9 or 19.5 hr.This study design was 2 × 4 factorial with two levels of glutamine and four levels of fasting. Birds in the 0-hr fasting group had free access to feed. All birds had ad libitum access to water. To measure IP on day 38, following their respective fasting periods, birds were administered two separate oral gavages of fluorescein isothiocyanate dextran (FITC-d) followed by lactulose, mannitol and rhamnose (LMR) sugars, 60 min apart. Whole blood was collected from the jugular vein 90 min post-LMR sugar gavage. FITC-d and L/M/R ratios were measured by spectrophotometry and highperformance ionic chromatography respectively. Lipopolysaccharide (LPS) endotoxins in plasma of the birds fed the control diet were also measured using chicken-specific LPS antibody ELISA. Serum FITC-d and plasma L/M and L/R ratios for 4.5, 9 and 19.5 hr were significantly (p < .05) higher compared to the non-fasting group. However, IP was not different in the glutamine-supplemented group (p > .05) compared to the control group. LPS concentrations measured by the ELISA were below the detectable range. We conclude that fasting periods of 4.5 and 9 hr increased IP compared to non-fasted birds and dietary glutamine supplementation did not ameliorate changes in IP. K E Y W O R D Sfluorescein isothiocyanate dextran, glutamine, lipopolysaccharides, feed withdrawal and sugar ratio

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.

Short-term fasting for 4.5 and 9 hr has been demonstrated to increase intestinal permeability (IP) in chickens. This study aimed to investigate the effects of 0, 4.5, 9 and 19.5 hr fasting on intestinal gene expression and villus-crypt architecture of enterocytes in jejunal and ileal samples. On day 38, Ross-308 male birds were fasted according to their group and then euthanised. Two separate intestinal sections (each 2 cm long, jejunum and ileum) were collected. One section was utilised for villus height and crypt depth measurements. The second section was snap-frozen in liquid nitrogen for quantitative polymerase chain reaction (qPCR) analysis of tight junction proteins (TJP) including claudin-1, claudin-3, occludin, zonula occludens (ZO-1, ZO-2), junctional adhesion molecules (JAM) and E-cadherin. Additionally genes involved in enterocyte protection including glucagon-like peptide (GLP-2), heat-shock protein (HSP-70), intestinal alkaline phosphatase (IAP), mammalian target of rapamycin (mTOR), toll-like receptors (TLR-4), mucin (MUC-2), cluster differentiation (CD-36) and fatty acid-binding protein (FABP-6) were also analysed. Normally distributed data were analysed using one-way analysis of variance ANOVA. Other data were analysed by non-parametric one-way ANOVA. Villus height and crypt depth were increased (p < .05) only in the ileum after fasting for 4.5 and 9 hr compared with non-fasting group. mRNA expression of claudin-3 was significantly reduced in the ileum of birds fasted for 9 and 19.5 hr, suggesting a role in IP modulation. However, all other TJP genes examined were not statistically different from control. Nevertheless, ileal FABP-6 of all fasted groups was significantly reduced, which could possibly be due to reduced bile acid production during fasting.