Small Intestinal Segment Perfusion Research Articles

Lactoferrin Decreases Enterotoxigenic Escherichia coli-Induced Fluid Secretion and Bacterial Adhesion in the Porcine Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) infections are one of the most prevalent causes of post-weaning diarrhea in piglets, resulting in morbidity, mortality and elevated use of antibiotics. The emergence and further spread of antimicrobial resistance together with the growing demand for high quality animal protein requires the identification of novel alternatives for antimicrobials. A promising alternative is lactoferrin, as we previously showed that it can both inhibit the growth and degrade bacterial virulence factors of porcine ETEC strains in vitro. Aiming to confirm these findings in vivo, we performed a small intestinal segment perfusion experiment in piglets. Here, we showed that lactoferrin could not only decrease ETEC-induced fluid secretion, but also their ability to colonize the small intestinal epithelium. Furthermore, while ETEC infection induced pro-inflammatory cytokine mRNA expression in this experiment, lactoferrin was not able to counteract these responses. In addition, a bacterial motility assay showed that lactoferrin can reduce the motility of ETEC. Our findings further support the use of lactoferrin as an alternative for antimicrobials and also show its potential for the prevention of ETEC infections in pigs.

PSVII-16 Galactosylated chitosan-oligosaccharides have anti-adhesive effect against enterotoxigenic Escherichia coli in piglets

Abstract Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets. In vitro, high molecular weight β-galactosylated chitosan-oligosaccharides (Gal-COS) had strong anti-adhesive activity against ETEC-expressing K88 fimbriae (ETEC K88) binding to porcine erythrocytes. This study assessed the effects of Gal-COS differing in structure on anti-adhesive properties against ETEC in a small intestinal segment perfusion (SISP) model in 8 piglets (BW 10 kg; 5-wk old). With 10 jejunal segments in each pig, 5 segments were infected with ETEC K88, and the other 5 segments were infused with saline (non-ETEC). Every 2 paired segments (ETEC or non-ETEC) from the same pig were treated for 8 h with 64 ml of 10 g L-1 of one of the following test products: 1) α-Gal-COS; 2) β-Gal-COS; 3) exopolysaccharides produced by Lactobacillus reuteri; and 4) raffinose in a double 4 × 4 Latin square with a saline control. Infection by ETEC K88 was verified by quantitative PCR. Net fluid loss was calculated as difference of fluid loss between ETEC segment and its paired non-ETEC segment. Data were analyzed using the mixed model with segment and test product as fixed effects, and pig as random effect. Number of eubacterial rRNA genes was 10-fold greater (P < 0.001) in ETEC segments than non-ETEC segments, indicating that ETEC K88 accounted for > 90% of bacterial gene counts. Test product did not affect (P > 0.10) the number of ETEC bacteria in the outflow fluid. Furthermore, net fluid loss caused by ETEC tended (P = 0.08) to be decreased by β-Gal-COS compared to all other treatments. In conclusion, the in vivo SISP model confirmed that Gal-COS had anti-diarrheal effects, indicating that β-Gal-COS is a potential feed additive to reduce the ETEC-induced diarrhea in piglets.

Exopolysaccharides synthesized by Lactobacillus reuteri protect against enterotoxigenic Escherichia coli in piglets.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter(-1) of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.

Effects of faba bean and faba bean hulls on expression of selected genes in the small intestine of piglets

In a small intestinal segment perfusion (SISP) study in pigs, effects were studied of intestinal perfusion of ground faba beans (Vicia faba), faba bean hulls, or saline on intestinal net fluid absorption in intestinal segments either challenged or not with an enterotoxigenic Escherichia coli (ETEC). After an 8-h perfusion, piglets were euthanized and small intestinal mucosa samples were collected for analysis of expression level of a selected set of genes (APOC3, TIMP1, AQP8, MMP1, MUC13, and PAP) using real-time quantitative PCR (qPCR). Perfusion with ground faba beans and faba bean hulls and challenge with ETEC affected (P < 0.05) expression of several of genes in the intestinal mucosa. Expression of APOC3, TIMP1, AQP8, MMP1, and PAP was correlated with net fluid absorption in the small intestine of pigs.

Optimization of a small intestinal segment perfusion model for heat-stable enterotoxin A induced secretion in pigs

Enterotoxigenic Escherichia coli (ETEC) are a major cause of infectious diarrhea both in human and pigs. After ingestion of contaminated food or water, ETEC bacteria colonize the small intestine where they produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, which induce watery diarrhea. We investigated the possibility of eliciting STa-induced secretion in jejunal segments of anesthetized pigs using a small-intestinal segment perfusion (SISP) model. Five consecutive mid-jejunal segments of anaesthetized piglets were perfused for 6h with different concentrations of STa in a physiologic salt solution. Changes in intestinal net fluid absorption were measured. From the results we could conclude that the STa response was dose-dependent and that continuous perfusion with 50nM of STa or more was required to reduce net absorption. This concentration was sufficient to reduce net absorption compared to control segments in 12 out of 14 piglets. STa-induced responses however showed relative high variation between different jejunal segments of one pig, similar to the inter-segment variation seen in control animals where segments were perfused with physiologic salt solution. These results indicate that more optimization is required before this model could be used to test compounds that could interfere with the STa-induced fluid secretion.

Role of Heat-Stable Enterotoxins in the Induction of Early Immune Responses in Piglets after Infection with Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat - labile (LT) enterotoxins are cause of post – weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac+, LT+ STa+ STb+ ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

In vitro inhibition of ETEC K88 adhesion by pea hulls and of LT enterotoxin binding by faba bean hulls.

Enterotoxigenic Escherichia coli (ETEC) expressing K88 (F4) adhesins are associated with post-weaning diarrhoea in piglets. Different grain fractions from pea (Pisum sativum) and faba bean (Vicia faba) were tested in vitro for their capacity to counteract aetiological factors, which contribute to the development of diarrhoea. In detail, adhesion of E.coli O149:K91:K88ac (ETEC K88ac) to grain legume products, intended to impair the colonization of the host, was studied as well as interference with receptor binding of the pathogen's heat-labile enterotoxin LT, intended to reduce toxin-inflicted gut cell damage. When comparing different pea and faba bean products tested for their binding capacity of ETEC K88ac, especially pea hulls, but also whole pea meal, starch-enriched and protein-enriched pea meal, and digestion-resistant pea hull and meal fractions showed a higher binding of ETEC K88ac than faba bean products. In contrast to the ETEC K88ac adhesion results, bean hulls proved more effective than pea hulls in preventing GM1 receptor binding of LT. Previous small intestinal segment perfusion experiments we performed with ETEC K88ac-challenged piglets indicated that both pea and bean hulls have the potential for successful application in diarrhoea prophylaxis and treatment, which is in agreement with and refined by our detection of their different modes of functioning.

Intestinal Barrier Physiology

Abstract Enterotoxigenic Escherichia coli (ETEC) are a major cause of infectious diarrhea both in human and pigs. After ingestion of contaminated food or water, ETEC bacteria colonize the small intestine where they produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, which induce watery diarrhea. We investigated the possibility of eliciting STa-induced secretion in jejunal segments of anesthetized pigs using a small-intestinal segment perfusion (SISP) model. Five consecutive mid-jejunal segments of anaesthetized piglets were perfused for 6 h with different concentrations of STa in a physiologic salt solution. Changes in intestinal net fluid absorption were measured. From the results we could conclude that the STa response was dose-dependent and that continuous perfusion with 50 nM of STa or more was required to reduce net absorption. This concentration was sufficient to reduce net absorption compared to control segments in 12 out of 14 piglets. STa-induced responses however showed relative high variation between different jejunal segments of one pig, similar to the inter-segment variation seen in control animals where segments were perfused with physiologic salt solution. These results indicate that more optimization is required before this model could be used to test compounds that could interfere with the STa-induced fluid secretion.

Small intestinal segment perfusion test in piglets: future applications in studying probiotics-gut crosstalk in infectious diarrhoea?

Worldwide infectious diarrhoea, mainly caused by rotavirus and enterotoxigenic Escherichia coli (ETEC), accounts for a large part of deaths in children. ETEC is also the main cause of traveller's diarrhoea. Probiotics are promising for prevention and treatment of diarrhoea, but there is insufficient evidence to support the use of any specific probiotic or probiotics in general. Because of the sensitivity of suckling and weaned piglets for ETEC, piglets are a good model for infectious diarrhoea in infants and traveller's diarrhoea. Just as in human the efficacy of probiotics in diminishing diarrhoea and improving growth in suckling and weaned piglets is not uniform. A piglet model of infectious diarrhoea provides access to intestinal compartments that are not easily accessible in infants. In an in situ piglet model of secretory diarrhoea, the functional physiological response to ETEC and the concomitant host genome response to ETEC and probiotics may be tested. This will provide new insights in the complex crosstalk between ETEC, probiotics and the gut in the future.

Effect of pea and faba bean fractions on net fluid absorption in ETEC-infected small intestinal segments of weaned piglets

After weaning piglets frequently have diarrhoea associated with an enterotoxigenic Escherichia coli (ETEC) infection. Alternative plant protein sources such as peas, faba beans and lupins may contribute in preventing gastrointestinal problems. In the small intestinal segment perfusion model, the anti-diarrhoea effect of pea and faba bean fractions on net absorption of fluid was investigated. Segments with a cranial inflow tube and a caudal outflow tube were fitted in the small intestine of 5-week old anaesthetized piglets. The odd numbered segments were infused with ETEC and the even segments with PBS, whereupon the segments were perfused over 8 h. Pea meal, pea hulls, protein-enriched pea meal and starch-enriched pea meal were perfused in 4 piglets in 4 pairs of segments (an uninfected and an adjacent ETEC-infected) with saline as a control in another pair of segments. Faba bean meal, faba bean hulls, protein-enriched faba bean meal and starch-enriched faba bean meal were perfused in another 4 piglets. Net fluid absorption was calculated from the difference between the volumes of inflow and outflow divided by the surface area. ETEC induced loss of net intestinal fluid absorption was shown to be decreased in the segments perfused with pea hulls and faba bean hulls, while there was no effect of the other pea or faba bean products. Although hulls of pea and faba bean did not completely eliminate the decreased fluid absorption after ETEC challenge, these results indicate that hulls of pea and faba bean may promote net fluid absorption in piglets during post weaning diarrhoea.

Mannose-specific interaction ofLactobacillus plantarumwith porcine jejunal epithelium

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.

Expression of β-defensins pBD-1 and pBD-2 along the small intestinal tract of the pig: Lack of upregulation in vivo upon Salmonella typhimurium infection

Defensins are antimicrobial peptides that play an important role in the innate immune response in the intestine. Up to date, only one β-defensin (pBD-1), has been described in pig, which was found to be expressed at low levels in the intestine. We set-up a quantitative PCR method to detect the gene expression of pBD-1 and a newly discovered porcine β-defensin, pBD-2. Expression of pBD-1 mRNA increased from the proximal to the distal part of the intestine whereas pBD-2 expression decreased. The main gene expression sites for pBD-2 were kidney and liver, whereas pBD-1 was mainly expressed in tongue. The porcine small intestinal segment perfusion (SISP) technique was used to investigate effects of Salmonella typhimurium DT104 on intestinal morphology and pBD-1 and pBD-2 mRNA levels in vivo. The early responses were studied 2, 4 and 8 h post-infection in four separate jejunal and ileal segments. Immunohistochemistry showed invasion of the mucosa by Salmonella and changes in intestinal morphology. However, no concomitant changes in expression of either pBD-1 or pBD-2 were observed. We conclude that at least two defensins are differentially expressed in the intestine of pigs, and that expression of both defensins is not altered by S. typhimurium under these conditions.

Effect of osmolality on net fluid absorption in non-infected and ETEC-infected piglet small intestinal segments

In the small intestinal segment perfusion model the effect of osmolality on net fluid absorption in enterotoxigenic Escherichia coli (ETEC)-infected and non-infected small intestinal segments of piglets was investigated. In ETEC-infected segments net fluid absorption was reduced. Lowering the osmolality from 375 to 150 mOsmol/l by reducing sodium chloride concentrations increased net fluid absorption. There was a linear relationship between osmolality and net fluid absorption for both non-infected and ETEC-infected segments. Below 150 mOsmol/l the inverse relationship between osmolality and net fluid absorption disappeared. Substitution of sodium chloride with mannitol decreased net fluid absorption since mannitol cannot be actively absorbed. In ETEC-infected segments perfused with oral rehydration solution net fluid absorption significantly increased compared to a sodium chloride solution of similar osmolality, probably because of the high glucose concentration. Supplying hypotonic sodium chloride fluids to piglets can be beneficial in replenishing water and electrolyte losses in case of infectious diarrhoea.

Development of a porcine small intestinal cDNA micro-array: characterization and functional analysis of the response to enterotoxigenic E. coli

The intestine is a complex and dynamic ecosystem, in which nutrients, exogenous compounds and micro-flora interact, and its condition is influenced by the complex interaction between these factors and host genetic elements. Furthermore, interactions of immune cells with the other components of the intestinal mucosa are essential in the defense against pathogens. The outcomes of these complex interactions determine resistance to infectious diseases. The development of genomic tools and techniques allows for analysis of multiple and complex host responses. We have constructed a porcine small intestinal micro-array, based on cDNA from jejunal mucosal scrapings. Material from two developmental distinct stages (4- and 12-week-old pigs) was used in order to assure a reasonably broad representation of mucosal transcripts. The micro-array consists of 3468 cDNAs spotted in quadruplicate. Comparison of the 4-week-old versus 12-week-old pigs revealed a differential expression in at least 300 spots. Furthermore, we report the early gene expression response of pig small intestine jejunal mucosa to infection with enterotoxigenic E. coli (ETEC) using the small intestinal segment perfusion (SISP) technique. A response pattern was found in which a marker for innate defense dominated, demonstrating the strength of this applied technology. Further analysis of these response patterns will contribute to a better understanding of enteric health and disease in pigs. The great similarity between pig and human suggest results from these continuing studies should be applicable for both agricultural and human biomedical purposes.