Jan van der Meulen

Intestinal barrier function and absorption in pigs after weaning: a review

Under commercial conditions, weaning of piglets is associated with social, environmental and dietary stress. Consequently, small-intestinal barrier and absorptive functions deteriorate within a short time after weaning. Most studies that have assessed small-intestinal permeability in pigs after weaning used either Ussing chambers or orally administered marker probes. Paracellular barrier function and active absorption decrease when pigs are weaned at 3 weeks of age or earlier. However, when weaned at 4 weeks of age or later, the barrier function is less affected, and active absorption is not affected or is increased. Weaning stress is a critical factor in relation to the compromised paracellular barrier function after weaning. Adequate feed intake levels after weaning prevent the loss of the intestinal barrier function. Transcellular transport of macromolecules and passive transcellular absorption decrease after weaning. This may reflect a natural intestinal maturation process that is enhanced by the weaning process and prevents the pig from an antigen overload. It seems that passive and active absorption after weaning adapt accurately to the new environment when pigs are weaned after 3 weeks of age. However, when weaned at 3 weeks of age or earlier, the decrease in active absorption indicates that pigs are unable to sufficiently adapt to the new environment. To improve weaning strategies, future studies should distinguish whether the effect of feed intake on barrier function can be directed to a lack of a specific nutrient, i.e. energy or protein.

Mannose-specific interaction of Lactobacillus plantarum with 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.

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.

Transcription networks responsible for early regulation of Salmonella -induced inflammation in the jejunum of pigs

BackgroundThe aim of this study was to identify transcription factors/regulators that play a crucial role in steering the (innate) immune response shortly (within a few hours) after the first contact of the intestinal mucosa with an inflammatory mediator, and to test whether the processes regulated by these factors/regulators can be modulated by chemical substances of natural origin.MethodsWe experimentally induced inflammation by perfusion of surgically applied jejunal loops with Salmonella enterica subspecies enterica serovar Typhimurium DT104 in three pigs. Segments of mock and Salmonella treated loops were dissected after 2, 4 and 8 hours of perfusion. IL8 and IL1-beta mRNA expression levels were measured in mucosal scrapings of all segments. Furthermore, intra-animal microarray comparisons (isogenic) between Salmonella and mock treated segments after 8 hours, and inter-animal comparisons between similar Salmonella-treated loops of each pig at 2 and 4 hours, were performed.ResultsIL-1beta and IL8 mRNA levels, and intra-animal microarray comparisons at 8 hours between Salmonella and mock treated segments showed that the response-time and type of response to Salmonella was different in all three pigs. This plasticity allowed us to extract a comprehensive set of differentially expressed genes from inter-animal comparisons at 2 and 4 hours. Pathway analysis indicated that many of these genes play a role in induction and/or tempering the inflammatory response in the intestine. Among them a set of transcription factors/regulators known to be involved in regulation of inflammation, but also factors/regulators for which involvement was not expected. Nine out of twenty compounds of natural origin, which according to literature had the potential to modulate the activity of these factors/regulators, were able to stimulate or inhibit a Salmonella-induced mRNA response of inflammatory-reporter genes IL8 and/or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in cultured intestinal porcine epithelial cells.ConclusionsWe describe a set of transcription factors/regulators possibly involved in regulation of “very early” immune mechanism which determines the inflammatory status of the intestine later on. In addition, we show that these mechanisms may be modulated by chemical substances of natural origin.

Transcriptomics of enterotoxigenic Escherichia coli infection. Individual variation in intestinal gene expression correlates with intestinal function

Acute secretory diarrhea is a major cause of morbidity and mortality in young animals and humans. Deaths result from excessive fluid and electrolyte losses. The disease is caused by non-invasive bacteria such as Vibrio cholerae and Escherichia coli which produce enterotoxins, however, much less is known about the role of individual host responses. Here we report the response of intact porcine small intestinal mucosa to infection with enterotoxigenic E. coli (ETEC). Jejunal segments in four piglets were infused with or without ETEC, and perfused for 8h, and net absorption measured. Microarray analysis at 8h post-infection showed significant differential regulation of on average fifteen transcripts in mucosa, with considerable individual variation. Differential net absorption varied between animals, and correlated negatively with the number of up regulated genes, and with one individual gene (THO complex 4). This shows that quantitative differences in gene regulation can be functionally linked to the physiological response in these four animals.

A high molecular weight soluble fraction of tempeh protects against fluid losses in Escherichia coli -infected piglet small intestine

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhoea in children and piglets. Infection of ETEC results in fluid secretion and electrolyte losses in the small intestine. In this study the effects of tempeh, a traditional fungal fermented soybean product, on fluid losses induced by ETEC infection in piglets was investigated. Pairs of ETEC-infected and non-infected small intestinal segments of piglets were perfused simultaneously for 8 h with pre-digested tempeh, its supernatant and saline as an internal control. In saline perfused segments, ETEC infection reduced net fluid absorption by more than 500 microl/cm2, whereas this reduction was significantly less for pre-digested tempeh and its supernatant (75 and 282 microl/cm2, respectively). The supernatant of pre-digested tempeh was also compared with its permeate and retentate fractions. These fractions were created by ultra-filtration and contained respectively low and high molecular weight (>5 kDa) compounds. Again ETEC infection caused a significant reduction of net fluid absorption when perfused with saline (386 microl/cm2) and also with the permeate fraction (300 microl/cm2), but much less with the supernatant and the retentate fraction (125 and 140 microl/cm2, respectively). The reduction in net fluid absorption upon ETEC infection when perfused with supernatant of either undigested or pre-digested tempeh was not different. Therefore from this study it can be concluded that a high molecular weight soluble fraction of tempeh is able to protect against fluid losses induced by ETEC, suggesting that this could play a potential role in controlling ETEC-induced diarrhoea.

Bioactivity of tempe by inhibiting adhesion of ETEC to intestinal cells, as influenced by fermentation substrates and starter pure cultures

Soya bean tempe is known for its bioactivity in reducing the severity of diarrhoea in piglets. This bioactivity is caused by an inhibition of the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells. In this paper, we assessed the bioactive effect of soya tempe on a range of ETEC target strains, as well as the effect of a range of cereal and leguminous substrates and starter pure cultures. Soya bean tempe extracts strongly inhibited the adhesion of ETEC strains tested. All tempe made from other leguminous seeds were as bioactive as soya bean tempe, whereas tempe made from cereals showed no bioactivity. Using soya beans as substrate, fermentation with several fungi (Mucor, Rhizopus spp. and yeasts) as well as Bacillus spp. resulted in bioactive tempe, whereas fermentation with lactobacilli showed no bioactivity. The active component is released or formed during the fermentation and is not present in microbial biomass and only partly in unfermented substrates. The bioactivity being not specific for a single ETEC strain, makes the bioactive tempe relevant for applications in animal husbandry.

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

Transfer of pyrrolizidine alkaloids from various herbs to eggs and meat in laying hens

ABSTRACT To investigate the potential transfer of pyrrolizidine alkaloids (PAs), laying hens were fed for 14 days with diets containing 0.5% of dried common ragwort, common groundsel, narrow-leaved ragwort or viper’s bugloss, or 0.1% of common heliotrope. This resulted in total PA levels in feed of respectively 5.5, 11.1, 53.1, 5.9 and 21.7 mg kg–1, with varying composition. PAs were transferred to eggs, in particular yolk, with steady-state levels of respectively 12, 21, 216, 2 and 36 µg kg–1. Overall transfer rates for the sum of PAs were estimated between 0.02% and 0.23%, depending on the type of PAs in the feed. In animals slaughtered shortly after the last exposure, levels in meat were slightly lower than those in eggs, levels in livers somewhat higher. When switched to clean feed, levels in eggs gradually decreased, but after 14 days were still above detection limits in the hens exposed to higher PA levels. Similar was the case for meat and especially kidneys and livers. It is concluded that the intake of PA containing herbs by laying hens may result in levels in eggs and meat that could be of concern for consumers, and as such should be avoided.

Case studies on genetically modified organisms (GMOs): Potential risk scenarios and associated health indicators

Within the frame of the EU-funded MARLON project, background data were reviewed to explore the possibility of measuring health indicators during post-market monitoring for potential effects of feeds, particularly genetically modified (GM) feeds, on livestock animal health, if applicable. Four case studies (CSs) of potential health effects on livestock were framed and the current knowledge of a possible effect of GM feed was reviewed. Concerning allergenicity (CS-1), there are no case-reports of allergic reactions or immunotoxic effects resulting from GM feed consumption as compared with non-GM feed. The likelihood of horizontal gene transfer (HGT; CS-2) of GMO-related DNA to different species is not different from that for other DNA and is unlikely to raise health concerns. Concerning mycotoxins (CS-3), insect-resistant GM maize may reduce fumonisins contamination as a health benefit, yet other Fusarium toxins and aflatoxins show inconclusive results. For nutritionally altered crops (CS-4), the genetic modifications applied lead to compositional changes which require special considerations of their nutritional impacts. No health indicators were thus identified except for possible beneficial impacts of reduced mycotoxins and nutritional enhancement. More generally, veterinary health data should ideally be linked with animal exposure information so as to be able to establish cause-effect relationships.