Adam J. Moeser

The Fecal Virome of Pigs on a High-Density Farm

ABSTRACT Swine are an important source of proteins worldwide but are subject to frequent viral outbreaks and numerous infections capable of infecting humans. Modern farming conditions may also increase viral transmission and potential zoonotic spread. We describe here the metagenomics-derived virome in the feces of 24 healthy and 12 diarrheic piglets on a high-density farm. An average of 4.2 different mammalian viruses were shed by healthy piglets, reflecting a high level of asymptomatic infections. Diarrheic pigs shed an average of 5.4 different mammalian viruses. Ninety-nine percent of the viral sequences were related to the RNA virus families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae, while 1% were related to the small DNA virus families Circoviridae, and Parvoviridae. Porcine RNA viruses identified, in order of decreasing number of sequence reads, consisted of kobuviruses, astroviruses, enteroviruses, sapoviruses, sapeloviruses, coronaviruses, bocaviruses, and teschoviruses. The near-full genomes of multiple novel species of porcine astroviruses and bocaviruses were generated and phylogenetically analyzed. Multiple small circular DNA genomes encoding replicase proteins plus two highly divergent members of the Picornavirales order were also characterized. The possible origin of these viral genomes from pig-infecting protozoans and nematodes, based on closest sequence similarities, is discussed. In summary, an unbiased survey of viruses in the feces of intensely farmed animals revealed frequent coinfections with a highly diverse set of viruses providing favorable conditions for viral recombination. Viral surveys of animals can readily document the circulation of known and new viruses, facilitating the detection of emerging viruses and prospective evaluation of their pathogenic and zoonotic potentials.

Early weaning stress impairs development of mucosal barrier function in the porcine intestine

Early life stress is a predisposing factor for the development of chronic intestinal disorders in adult life. Here, we show that stress associated with early weaning in pigs leads to impaired mucosal barrier function. Early weaning (15- to 21-day weaning age) resulted in sustained impairment in intestinal barrier function, as indicated by reductions in jejunal transepithelial electrical resistance and elevations in mucosal-to-serosal flux of paracellular probes [(3)H]mannitol and [(14)C]inulin measured at 5 and 9 wk of age, compared with that shown in late-weaned pigs (23- to 28-day weaning age). Elevated baseline short-circuit current was observed in jejunum from early-weaned pigs and was shown to be mediated via enhanced Cl(-) secretion. Jejunal barrier dysfunction in early-weaned pigs coincided with increased lamina propria immune cell density particularly mucosal mast cells. The mast cell stabilizer drug sodium cromoglycolate ameliorated barrier dysfunction and hypersecretion in early-weaned pigs, demonstrating an important role of mast cells. Furthermore, activation of mast cells ex vivo with c48/80 and corticotrophin-releasing factor (CRF) in pig jejunum mounted in Ussing chambers induced barrier dysfunction and elevations in short-circuit current that were inhibited with mast cell protease inhibitors. Experiments in which selective CRF receptor antagonists were administered to early-weaned pigs revealed that CRF receptor 1 (CRFr1) activation mediates barrier dysfunction and hypersecretion, whereas CRFr2 activation may be responsible for novel protective properties in the porcine intestine in response to early life stress.

CRF induces intestinal epithelial barrier injury via the release of mast cell proteases and TNF-α.

Background and Aims Psychological stress is a predisposing factor in the onset and exacerbation of important gastrointestinal diseases including irritable bowel syndrome (IBS) and the inflammatory bowel diseases (IBD). The pathophysiology of stress-induced intestinal disturbances is known to be mediated by corticotropin releasing factor (CRF) but the precise signaling pathways remain poorly understood. Utilizing a porcine ex vivo intestinal model, the aim of this study was to investigate the mechanisms by which CRF mediates intestinal epithelial barrier disturbances. Methodology Ileum was harvested from 6–8 week-old pigs, mounted on Ussing Chambers, and exposed to CRF in the presence or absence of various pharmacologic inhibitors of CRF-mediated signaling pathways. Mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) and transepithelial electrical resistance (TER) were recorded as indices of intestinal epithelial barrier function. Results Exposure of porcine ileum to 0.05–0.5 µM CRF increased (p<0.05) paracellular flux compared with vehicle controls. CRF treatment had no deleterious effects on ileal TER. The effects of CRF on FD4 flux were inhibited with pre-treatment of tissue with the non-selective CRF1/2 receptor antagonist Astressin B and the mast cell stabilizer sodium cromolyn (10−4 M). Furthermore, anti-TNF-α neutralizing antibody (p<0.01), protease inhibitors (p<0.01) and the neural blocker tetrodotoxin (TTX) inhibited CRF-mediated intestinal barrier dysfunction. Conclusion These data demonstrate that CRF triggers increases in intestinal paracellular permeability via mast cell dependent release of TNF-α and proteases. Furthermore, CRF-mast cell signaling pathways and increases in intestinal permeability require critical input from the enteric nervous system. Therefore, blocking the deleterious effects of CRF may address the enteric signaling of mast cell degranulation, TNFα release, and protease secretion, hallmarks of IBS and IBD.

Early Weaning Stress in Pigs Impairs Innate Mucosal Immune Responses to Enterotoxigenic E. coli Challenge and Exacerbates Intestinal Injury and Clinical Disease

Background and Aims The clinical onset and severity of intestinal disorders in humans and animals can be profoundly impacted by early life stress. Here we investigated the impact of early weaning stress in pigs on intestinal physiology, clinical disease, and immune response to subsequent challenge with enterotoxigenic F18 E. coli (ETEC). Methodology Pigs weaned from their dam at 16 d, 18 d, and 20 d of age were given a direct oral challenge of F18 ETEC at 26 d of age. Pigs were monitored from days 0 to 4 post-infection for clinical signs of disease. On Day 4 post-ETEC challenge, ileal barrier function, histopathologic and inflammatory cytokine analysis were performed on ileal mucosa. Results Early weaned pigs (16 d and 18 d weaning age) exhibited a more rapid onset and severity of diarrhea and reductions in weight gain in response to ETEC challenge compared with late weaned pigs (20 d weaning age). ETEC challenge induced intestinal barrier injury in early weaned pigs, indicated by reductions in ileal transepithelial electrical resistance (TER) and elevated FD4 flux rates, in early weaned pig ileum but not in late weaned pigs. ETEC-induced marked elevations in IL-6 and IL-8, neutrophil recruitment, and mast cell activation in late-weaned pigs; these responses were attenuated in early weaned pigs. TNF levels elevated in ETEC challenged ileal mucosa from early weaned pigs but not in other weaning age groups. Conclusions These data demonstrate the early weaning stress can profoundly alter subsequent immune and physiology responses and clinical outcomes to subsequent infectious pathogen challenge. Given the link between early life stress and gastrointestinal diseases of animals and humans, a more fundamental understanding of the mechanisms by which early life stress impacts subsequent pathophysiologic intestinal responses has implications for the prevention and management of important GI disorders in humans and animals.

Spray-Dried Porcine Plasma Influences Intestinal Barrier Function, Inflammation, and Diarrhea in Weaned Pigs

The objective of this study was to evaluate the effects of dietary inclusion levels of spray-dried porcine plasma (SDPP) on postweaning (PW) intestinal barrier function, mucosal inflammation, and clinical indices of gut health in pigs. Ex vivo Ussing chamber studies were conducted to measure Ileal and colonic barrier function in terms of transepithelial electrical resistance and paracellular flux of (3)H-mannitol and (14)C-inulin. Intestinal inflammation was assessed by histological analysis and mucosal levels of proinflammatory cytokines. Dietary inclusion of 2.5 and 5% SDPP reduced colonic paracellular permeability of (14)C-inulin compared with controls (0% SDPP) on d 7 PW. Both 2.5 and 5% dietary SDPP reduced ileal (3)H-mannitol and (14)C-inulin permeability on d 14 PW. The 5% SDPP diet reduced colonic short-circuit current, an index of net electrogenic ion transport, and fecal scores when measured on d 7 and 14 PW compared with the control and 2.5% SDPP groups (P < 0.05). Histological analysis revealed fewer lamina propria cells in ileum and colon from pigs fed diets containing 2.5 and 5% SDPP on d 7 and 14 PW. Levels of the proinflammatory cytokine TNFα were reduced in the colon but not ileum from pigs fed the 5% SDPP on d 7 and 14 PW compared with controls (P < 0.05). IFNγ levels were lower than in controls in both of the SDPP-fed groups in the ileum and colon on d 7 but not on d 14 PW. Overall, this study demonstrated that dietary inclusion of SDPP had beneficial effects on intestinal barrier function, inflammation, and diarrhea in weaned pigs.

Carbon monoxide and heme oxygenase-1 prevent intestinal inflammation in mice by promoting bacterial clearance.

BACKGROUND & AIMS Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. METHODS Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium. RESULTS In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. CONCLUSIONS Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.

Iron Transporters Are Differentially Regulated by Dietary Iron, and Modifications Are Associated with Changes in Manganese Metabolism in Young Pigs

To investigate the effects of dietary iron (Fe) on manganese (Mn) metabolism, 24 weaned pigs (21 d old) were blocked by litter and weight and randomly assigned to the following treatments: 1) no supplemental Fe [low Fe (L-Fe)]; 2) 100 mg supplemental Fe/kg [adequate Fe (A-Fe)]; and 3) 500 mg supplemental Fe/kg [high Fe (H-Fe)]. The basal diet was analyzed to contain 20 mg Fe/kg. Tissues were harvested after 32 d of feeding. Daily gain (least square means +/- SEM) was greater in A-Fe pigs (328.3 +/- 29.9 g/d) than in L-Fe pigs (224.0 +/- 11.2 g/d). Hemoglobin concentrations on d 32 were lower in L-Fe pigs (62 +/- 3.5 g/L) than in A-Fe pigs (128 +/- 5.6 g/L) and did not differ between pigs fed A-Fe and H-Fe (133 +/- 12.0 g/L). Liver Fe increased with increasing dietary Fe. Relative hepatic hepcidin expression was greater in pigs fed A-Fe and H-Fe than in those fed L-Fe. Relative expressions of duodenal divalent metal transporter 1 (DMT1) and solute carrier family 39 member 14 (ZIP14) were increased in L-Fe pigs compared with H-Fe pigs. Liver copper (Cu) was higher in L-Fe (0.56 +/- 0.04 mmol/kg) and H-Fe (0.58 +/- 0.04 mmol/kg) pigs than in A-Fe pigs (0.40 +/- 0.04 mmol/kg). Liver Mn was lower in H-Fe pigs (0.15 +/- 0.01 mmol/kg) than in A-Fe (0.23 +/- 0.02 mmol/kg) or L-Fe pigs (0.20 +/- 0.02 mmol/kg). Duodenal Mn concentrations were greater in L-Fe pigs than in A-Fe or H-Fe pigs. Fe deficiency in pigs increased gene expression of duodenal metal transporters (DMT1 and ZIP14) and supplementation with H-Fe reduced expression of DMT1 and ZIP14, which may have decreased absorption of Mn.

Animal models of ischemia-reperfusion-induced intestinal injury: progress and promise for translational research

Research in the field of ischemia-reperfusion injury continues to be plagued by the inability to translate research findings to clinically useful therapies. This may in part relate to the complexity of disease processes that result in intestinal ischemia but may also result from inappropriate research model selection. Research animal models have been integral to the study of ischemia-reperfusion-induced intestinal injury. However, the clinical conditions that compromise intestinal blood flow in clinical patients ranges widely from primary intestinal disease to processes secondary to distant organ failure and generalized systemic disease. Thus models that closely resemble human pathology in clinical conditions as disparate as volvulus, shock, and necrotizing enterocolitis are likely to give the greatest opportunity to understand mechanisms of ischemia that may ultimately translate to patient care. Furthermore, conditions that result in varying levels of ischemia may be further complicated by the reperfusion of blood to tissues that, in some cases, further exacerbates injury. This review assesses animal models of ischemia-reperfusion injury as well as the knowledge that has been derived from each to aid selection of appropriate research models. In addition, a discussion of the future of intestinal ischemia-reperfusion research is provided to place some context on the areas likely to provide the greatest benefit from continued research of ischemia-reperfusion injury.

High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves

A 56-d experiment was designed to examine the effect of high dietary Fe on metal transporters involved in Fe and Mn metabolism. Fourteen weaned Holstein calves were stratified by weight and randomly assigned to 1 of 2 treatments: 1) no supplemental Fe (normal Fe) or 2) 750mg of supplemental Fe/kg of dry matter (high Fe). Jugular blood was collected on d 0, 35, and 56. At the end of the trial, 6 calves per treatment were humanely killed and duodenal scrapings, liver, and heart were collected for analysis. Additionally, proximal duodenum was mounted on Ussing chambers to assess intestinal barrier integrity. Calves receiving high dietary Fe displayed decreased transepithelial resistance and increased apical-to-basolateral flux of radiolabeled mannitol, suggesting that high Fe created increased intestinal permeability. Feeding calves a diet high in Fe decreased average daily gain, dry matter intake, and feed efficiency. Hemoglobin and serum Fe concentrations did not differ due to dietary treatment. High dietary Fe increased concentrations of Fe in the liver, but did not affect heart or duodenal Fe concentrations. Duodenal Mn concentrations were lowered by feeding a high Fe diet, but liver and heart Mn concentrations were not affected. As determined by real-time reverse transcription PCR, relative hepatic expression of the gene that encodes the Fe regulatory hormone hepcidin was 5-fold greater in calves fed high dietary Fe. Hepcidin is released in response to increased Fe status and binds to the Fe export protein ferroportin causing ferroportin to be degraded, thereby reducing dietary Fe absorption. Confirmation of this result was achieved through Western blotting of duodenal protein, which revealed that ferroportin was decreased in calves fed high dietary Fe. Duodenal protein expression of divalent metal transporter 1 (DMT1), a Fe import protein that can also transport Mn, tended to be reduced by high dietary Fe. Transcript levels of several genes involved in Fe metabolism in liver and duodenum were unchanged by treatment. In summary, feeding calves a diet high in Fe induced a signal cascade (hepcidin) designed to reduce absorption of Fe (via reduced protein expression of ferroportin and DMT1) in a manner similar to that reported in rodents. Additionally, reduced levels of DMT1 protein appeared to decrease duodenal Mn, suggesting that Mn may also be a substrate for DMT1 in cattle.

Early life stress triggers persistent colonic barrier dysfunction and exacerbates colitis in adult IL-10-/- mice.

Background:It has become increasingly evident that disease flares in the human inflammatory bowel diseases are influenced by life stress. It is known that life stress can trigger disturbances in intestinal barrier function and activate proinflammatory signaling pathways, which are important contributors to intestinal inflammation and clinical disease; however, the exact mechanisms of stress-induced inflammatory bowel disease exacerbations remain to be elucidated. Here, we presented a model of early life stress–induced exacerbation of colitis in interleukin (IL)-10−/− mice. Methods:C57Bl/6 wild-type and IL-10−/− mice were exposed to neonatal maternal separation (NMS) stress on postnatal days 1 to 18 and reared under normal conditions until 10 to 12 weeks of age. At this time, histopathology, colitis scores, intestinal barrier function, proinflammatory cytokine expression, and mast cell activity were evaluated. Results:NMS increased the severity of colitis IL-10−/− mice indicated by greater colitis scores and colonic proinflammatory cytokine concentrations. NMS and IL-10−/− increased colonic permeability; however, NMS alone did not induce colitis. Increased mast cell activation and colonic tryptase release were observed in IL-10−/− mice exposed to NMS, indicating mast cell activation. Conclusions:This study demonstrates that colitis in IL-10−/− mice can be exacerbated by NMS stress. The precise mechanisms of enhanced colitis severity in NMS IL10−/− mice are unclear but persistent defects in intestinal barrier function likely play a contributing role. NMS serves as a novel model to investigate the mechanisms by which early life stress influences the development and course of inflammatory bowel disease in adulthood.