Defining and validating gut health criteria in young pigs, based on digestive physiology, microbiology and mucosal immunology investigations for testing alternative strategies to in-feed antibiotics

An in vitro cumulative gas production technique was used to assess microbial activity of a complex community, in relation to fermentation of selected energy sources. This was carried out in combination with an in vivo study to examine the effects of two different diets for weaning piglets, so that microbial activities of faeces could be compared from the same animals on the two different diets in the in vivo experiment. The two diets were: CHO diet (containing added fermentable carbohydrates including sugar beet pulp (SBP) and wheat starch (WST)), and Control diet without any added fermentable CHOs. Neither diet contained antibiotics nor extra added copper. Twenty-four piglets were selected from 12 litters (2 per litter), weaned at 4 weeks of age (neither creep feeding nor any antibiotic treatment before and during the study), and introduced to one of the two diets. After nine days on the diet, faecal samples were collected from selected animals, and tested for their activity in terms of gas production kinetics, and end products such as volatile fatty acids (VFA), ammonia, and dry matter (DM) disappearance of two test substrates SBP and WST. From samples taken before and after in vitro fermentation, the bacterial diversity was also analyzed, using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. There were differences both in kinetics and end-products of the substrates. In terms of gas production, significant differences were detected between inocula, though mainly in terms of fermentation kinetics of the two substrates, rather than total gas. For the CHO inoculum, SBP was fermented faster, than for the Control diet animal faeces, while this effect was reversed for WST. Significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the microbial community enrichment on SBP as compared to WST at the end of fermentation. The difference between the kinetics of SBP and WST fermentation by faecal microbiota from the CHO diet fed piglets, suggests better adaptation of those piglets to SBP fermentation than to WST fermentation. The WST fermentation differences were more unexpected, though it was realized that a significant amount of starch is known to be fermentable by the small intestinal microbiota. This may have meant that the faecal microbiota was therefore less adapted to fermentation of starch. It was concluded that the microbial community composition and activity in the GIT may be changed in response to diet, and that this change can be detected in vitro. Secondly, the response of ileal and colonic microbiota of weaning piglets, to dietary addition of 4 fermentable carbohydrates (inulin, lactulose, wheat starch and sugar beet pulp) was analyzed for an animal feeding trial. The same two diets, either enriched in or lacking these fermentable carbohydrates, were fed to piglets for 4 (n=48), and 10 days (n=48), and the lumen associated microbiota was compared using DGGE analysis of amplified 16S rRNA genes. The results revealed that the addition of fermentable CHOs (= prebiotics) led to a significant enrichment of lactobacilli in the ileum. Furthermore, the bacterial diversity in the ileum and colon of each piglet was measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 +/- 5.5) compared to the ileum (9.7 +/- 4.2) was observed in all samples. In addition, at 10 days after weaning, a significantly higher bacterial diversity was detected in the colon of CHO-fed piglets, compared with the control. Selected samples from the ileum and colon lumen were also investigated using fluorescent in situ hybridization (FISH), and cloning and sequencing of the 16S rRNA. This revealed that Lactobacillus amylovorus- like and L. reuteri- like phylotypes were the most prevalent in the ileum and colon lumen samples of the piglets fed the CHO diet. Newly developed DNA probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. These results indicated that the addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ileum and colon of weaning piglets, and increased the bacterial community diversity in the colon. The data therefore supported the hypothesis, that changes of the diet can modulate the composition of the microbiota in the intestine. More specifically, they show that the addition of a careful selection of fermentable carbohydrates can lead to positive changes in the bacterial population in the gut. Furthermore, the last study suggested a significant role of L. amylovorus-like phylotype in the metabolism of fermentable carbohydrates.

Flaxseed (Linum usitatissimum) and oats (Avena sativa) gain a renewed scientific interest in the context of searching for antibiotic alternatives in diets for piglets after weaning. Both plants possess mucilage-rich compounds, which may serve as a bio-film lining along the surface (mucus layer) of epithelial cells, and thus protecting against pathogenic colonisation, particularly after weaning. The physiological benefits of flaxseed oil are also attributed to the high content of á-linolenic acid, which is essential for mucosal growth and immunity. Flaxseed protein may also stimulate insulin secretion, whereas lignans and other phenolics are known to have strong anti-oxidative and protein-binding properties that may suggest some preventive effect of flaxseed protein in conjunction with phenolics against bacterial overgrowth. Beta glucan from oats is a soluble, viscous fibre found in the bran layer of the grain, which can stimulate the function or secretion of various immunoglobulins and immune system cells, and up-regulated immune mechanisms can enhance resistance to some pathogens. Therefore, the aim of this study was to test the potency of flaxseed and oats in a cereal-based, antibiotic-free diet on the gut health, mucosal integrity and performance of weaned piglets before and after exposure to a controlled infection with enterotoxigenic E. coli K88. In total 48 piglets of approximately 8 kg BW were used during 19 days post-weaning to test if adding 10% flaxseed and 15% oats (as polyfunctional/anti-diarrhoetic ingredients rich in mucilage for protecting the surface of epithelial cells) in diets without antibiotics can positively affect the performance, health and integrity of the digestive tract in weaned piglets exposed to a controlled pathogenic infection with ETEC K88 on day 6-postweaning. They were allotted to 4 dietary treatments: 1) Basal, no E.coli infection (B); 2) Basal+E.coli infection (B+); 3) Basal+10% flaxseed +E.coli infection (BF+); 4) Basal + 15% oats + E.coli infection (BO+). Their responses to the dietary treatments were evaluated in terms of clinical health status, performance, faecal consistency index (scale 0-3), activity index (scale 0-4), breath index (scale 0-4), condition index (scale 0-4), faecal E.coli shedding, mucus content in faeces (days 7-9), blood samples (days 11 and 18), weight/length of duodenum/jejunum/ileum, jejunal/ileal digesta pH and contents of DM and NH3, ileal VFA, morphology, K88 receptor status. Piglets receiving 10% flaxseed and 15% oats consumed more feed (P<0.01) and grew faster (P<0.01) during the first 5 pre-challenge days, as compared to those fed a control diet (Figure 4). Faecal shedding of E.coli K88 by the piglets highly corresponded to the ETEC K88 infection (Figure 5). Thus, this implies that our E.coli infection model was optimally targeted and effective. However, the differences among the dietary treatments were less evident. Conclusions: Irrespective of the diet, piglets challenged with ETEC K88 grew slower (439-457 g/day) than non-challenged piglets (533 g/day), although no major clinical health problems were encountered. Daily gains of E.coli infected piglets fed 10% flaxseed and 15% oats during 18 days of this study were numerically greater (by 52 and 29 g/day, respectively) as compared to the control. Faecal consistency was similar among the treatments, irrespective of the post-challenge period. Of numerous blood indices recorded on the day 5 post-weaning, only WBC, basophiles and IgG titres were found to differ at P<0.05. The gut wall in piglets fed flaxseed was thinner (P<0.01) as compared to the remaining treatments. Also, the number of goblet cells was lower (P<0.05) due to adding flaxseed, whereas the remaining morphological indices (villous length, crypt depth, and their ratios) differed numerically (P>0.05) only. Significant effects of the infection were also found for the jejunal length (P=0.04) and DM contents in the jejunal digesta, whereas the dietary treatments exerted minor (P>0.05) effects on the gut length, weight or digesta composition.

The ban is expected to decrease the productivity of the pig and poultry sectors of the EU. The software package MISS (world trade simplified simulation model) has been used to simulate the international market effects of the ban on fifteen commodities which are produced or used in agriculture in four big countries: EU-15, USA, new EU-members, rest of the world. EU-15 pig farmers will bear most of the cost of the ban with the loss of international market shares, despite the decrease in EU feed prices that derives from the current reforms of the Common Agricultural Policy (CAP). Other farmers than pig producers will benefit from higher world prices of agricultural products than without the ban. Theoretically, the world consumers will face a welfare loss but it might not be statistically significant because they also will adjust their food bundle according to relative price changes (with a little more of beef meat and vegetal oils), and EU consumers' benefit from the CAP reforms. Eventually a loss of social welfare is associated to higher manure per pig in the EU. Whether this social cost will be born by pig producers, taxpayers or directly by the EU consumers as a negative externality will depend on the environmental rules and on their enforcement modalities.

A feeding trial was performed, in which the effect of 4 fermentable carbohydrates (inulin, lactulose, wheat starch and sugar beet pulp) on the intestinal microbiota of weaning piglets was analyzed. Cultivation and molecular 16S ribosomal RNA targeted approaches revealed that the addition of fermentable CHOs (= prebiotics) led to a significant enrichment of lactobacilli in the ileum, namely Lactobacillus reuteri- and amylovorus-related strains. In an attempt to obtain porcine isolates related to Lactobacillus amylovorus, we screened isolates from piglet intestine grown on Lactobacillus-specific MRS agar for hybridization to a phylotype-specific fluorescent 16S rRNA-targeted DNA probe. Six isolates were obtained and further characterized by phenotypic and molecular taxonomic methods. The isolates were Gram-positive, catalase-negative, facultatively anaerobic rods. They had similar phenotypic characteristics and displayed genomic DNA relatedness values of >78%, indicating that they belong to a single species. Comparative 16S rRNA gene sequence analysis demonstrated that the novel isolates were members of Lactobacillus rRNA group I. Based on 16S rRNA sequence identity, L. kitasatonis (99%), L. crispatus (98%), and L. amylovorus (97%) were the nearest relatives of the new isolates, but their DNA relatedness was found to be lower than 49%. One of the isolates, strain OTU171_001 was further characterized using physiological and biochemical tests. Together, the results allowed genotypic and phenotypic differentiation of strain OTU171_001 from those related species that showed 16S rRNA gene sequence similarity values greater than 97%. Strain OTU171_001 T merits species status, and the name Lactobacillus sobrius sp. nov. has been proposed with the type strain OTU171_001T.

The effect of low and high dietary protein level and fermentable carbohydrates content on performance and intestinal characteristics of newly weaned piglets was studied in a 2x2 factorial arrangement. Growth performance data were significantly affected by dietary treatments. In week 1-2 post weaning daily gain and gain/feed were reduced by an increase in fermentable carbohydrate content in the low protein diet. In the high protein diet however, growth performance was slightly (ns) improved by an increase in fermentable carbohydrates. The increase in dietary protein reduced growth performance in the low but not in the high fermentable carbohydrate diet. These results suggest an increase in either crude protein or fermentable carbohydrates can negatively influence growth performance of the piglets. However, the combination of an increase in both nutrients may compensate for these negative effects on growth performance. In week 3-4 post weaning both an increase in crude protein and/or an increase in fermentable fibre reduced feed intake and daily gain. The highest performance was realised on diets with a low crude protein and low fermentable carbohydrates content. Characteristics of the digestive tract were determined on day 7 post weaning. The increase in fermentable carbohydrate content in the diet significantly increased the lactobacilli counts in the small intestine but not in the colon. In addition, fermentable carbohydrates reduced the content of total coliform bacteria in the jejunum and to a lesser extent in the colon, but only for the high protein diets. The ammonia content in the jejunum and to a lesser extent in the colon was increased by the increase in dietary crude protein. The increase in fermentable carbohydrates reduced the ammonia content in both segments of the digestive tract and increased the lactic acid content in the jejunum but not in the ileum. In addition, the increase in fermentable carbohydrates increased the total VFA content in the colon, but not in the jejunum. The villus length and crypt depth were not significantly affected, although the crypt depth was slightly increased on the high fermentable carbohydrate diets. The results of growth performance and digestive tract characteristics indicate that in newly weaned piglets the highest growth performance can probably be realised using diets with a low content of (fermentable) crude protein and fermentable carbohydrates. These diets limit microbial growth and fermentation in the digestive tract and as a result, dietary nutrients can be used by the piglet with lowest losses in the digestive tract. However, the reduced microbial population may compromise the colonisation resistance of the piglet, making the animal more prone to pathogenic bacteria. An increase in dietary crude protein may increase the risk of multiplication of those bacteria. The present experiment shows that inclusion of fermentable carbohydrates stimulates growth of lactobacilli and production of lactic acid in the digestive tract and reduced the number of coliform bacteria. As such, the inclusion of fermentable carbohydrates will likely improve the colonisation resistance of the piglets.

The Partner from Wageningen University (additional contact person: Hauke Smidt, Hesselink van Suchtelenweg 4, 6703 CT, WAGENINGEN, NETHERLANDS, tel +31-3-17483102, hauke.smidt@wur.nl http://www.wur.nl) isolated a Lactobacillus amylovorus -like strain 001 (LabAmy), from pigs fed different fibre sources that showed a good balanced micro-flora. We tested this strain as a probiotic in the diet of weaned piglets orally challenged with E. coli K88. 48 piglets, weaned at 21 days of age, were fed control diet or control diet plus 10 x 10 CFU Lactobacillus amylovorus/day (provided by Partner 3). Pigs were orally challenged with 10 x 10 CFU E. coli K88ac O148 (F4) on day 7, and sacrificed on day 13 or 14. Pigs supplemented with LabAmy grew faster (+ 74%, P<0.05), while did not show different feed intake, before and after challenge, compared with control pigs. Surprisingly, an increased number of days with diarrhoea were observed with the supplementation. Faecal excretion of total E.coli and E. coli K88 was not affected. The length and the weight of small intestine, the pH in stomach, duodenum, jejunum, cecum and colon, the morphology of small intestine, and K88-specific IgA in blood, saliva and intestinal secretion did not change with the supplementation. The Lactobacillus amylovorus -like strain is a valid candidate for supplementation of weaning pig diets, in case of not optimal sanitary conditions in the farm. The same results were not obtained with Bifidobacterium animalis MB 05 and Lactobacillus rhamnosus GG. Furthermore Lactobacillus rhamnosus GG, strain not recovered usually in pigs, impaired growth performance.

The objective of our investigation was the evaluation of ileal digesta - qualitatively and quantitatively - in piglets before and after weaning, with special reference to protein and amino acid digestion. To implement this aim we conducted two main experimental complexes: a long-term slaughter trial for the qualitative aspects and two balance trials for the quantitative part. During the slaughter trial animals were sacrificed at different stages before and after weaning: -6, -4, -2 days pre-weaning, weaning day (28 d of age), +1, +2, +5, +8 and +15 days post-weaning. Dietary regimes comprised sow milk as sole feed (no creep feed) before and after weaning four different starters, two reference diets based on soybean meal and whey and two home-produced diets based on cereals and legume seeds. Reference diets differed in supplementation of in-feed antibiotic avilamycin (+AB, -AB) and home-produced diets in crude fibre content, being either 3 or 8% (LF, HF). In the slaughter trial we applied a wide range of analysis, determining pH, DM, LA, VFA, NH3, biogenic amines and D-alanine and investigating the luminal microflora with classical and molecular techniques at ileal level. Although we observed changes in some parameters during weaning transition - decrease in ammonia and VFA, increase in LA - these scarcely proofed to be statistical significant. Also classical and molecular microbiology revealed a remarkable stability in the universal ileal microflora of piglets receiving sow milk or starter diets. Observed changes were more age-related than influenced by dietary treatment. Only the Lactobacillus spp. group displayed a differential response: here we could establish a diet-dependent change in the Lactobacillus spp. population. The home-produced diets induced a more diverse population than the reference diets, being more comparable to the Lactobacillus spp. population of suckling piglets. Lactic acid producing bacteria are regarded as health beneficial, especially in the GIT of young animals such as weaning piglets. Therefore the promotion of this group by the home-produced diets can be considered as a major benefit for intestinal health. However, more studies are required to receive statistical relevant evidence. Furthermore we detected a negative correlation between the Lactobacillus spp. group and yeast (Pearson Correlation Coefficient r = -0.24, p = 0.02) and a highly positive correlation to amines (putrescine = 0.41, p < 0.0001; histamine r = 0.36, p = 0.0005; cadaverine r = 0.35, r = 0.0006; spermidine r = 0.23, p = 0.03; spermine r = 0.28, p = 0.007). The inverse relationship with yeast was reported, but evidence for biogenic amines in the porcine gut is rare in literature. There are some in vitro studies reporting production of biogenic amines in this bacterial species, but the main contribution to this field comes from food technology rather than from animal physiology research. These results suggest a crucial role of Lactobacillus spp. in the terminal ileum of weaning piglets. Further investigation is required to elucidate those interesting interrelationships in the GIT of farm animals, especially in swine.

Intestinal health, feed intake and growth of weaned piglets fed antibiotic-free diets without and with a precursor for proliferation and differentiation of epithelial cells Butyric acid as a natural metabolite of intestinal microbial activity and/or of exogenous (dietary) origin for piglets may serve as a fuel to stimulate gut metabolic activity (differentiation/ reversion of cells from a neoplastic to a non-neoplastic phenotype and the proliferation index at the bottom of the crypt, thus exerting a trophic effect on absorption of sodium. This trophic effect determines the efficiency of ionic absorption in the gut and could have clinical implications in the genesis of post-weaning diarrhoea (PWD) in piglets. Butyrate can also contribute to energy balance, feed intake stimulation and, thereby, to enhancing piglets' performance. The objective of our study was to evaluate the effect of supplemental calcium butyrate (0.1%) in a practical, antibiotic-free diet for piglets after weaning on their growth, feed conversion ratio, and morphological indices in the terminal duodenum and anterior jejunum (villous height, crypt depth, and villus to crypt ratios). The effects of supplemental calcium butyrate (0.1%) in a practical, antibiotic-free diet for 28-day old piglets (n=16) on their growth, feed conversion ratio, and morphological indices in the terminal duodenum and anterior jejunum (villous height, crypt depth, and villous to crypt ratios) were studied during 7 days post-weaning. Irrespective of the sampling site, no statistically significant differences in the intestinal villous height and crypt depth of weaned piglets fed without and with 0.1% Ca-butyrate were found. This implies that the role of supplemental butyrate as a precursor for proliferation and differentiation of epithelial cells in the small intestine of piglets fed antibiotic-free diets could not be confirmed in this study. However, due to the fact that the piglets consumed very little or nothing during the first 48-72 h after weaning, it is highly probable that the potency of dietary butyrate as a substrate for proliferation and differentiation of epithelial cells was underestimated. To prove a statistically meaningful effect of this compound, its sufficient consumption directly after weaning should be ensured. Supplemental Ca-butyrate (0.1%) in a standard Dutch diet for 28-day old piglets did not influence (P>0.5) on their performance (feed intake and ADG) during 7 days post-weaning. Besides, this dose of Ca-butyrate exerted no effect (P>0.05) on the gut structure, as evaluated in terms of the villous height and crypt depth in the duodenum and jejunum. However, it is highly probable that the potency of dietary butyrate as a substrate for proliferation and differentiation of epithelial cells in this study was underestimated due to the fact that piglets consumed no or very little feed during first 48-72 hours after weaning.

Bacterial therapy for man and farmhouse animals is largely based on the concept that specific strains selected from the commensal gut microbiota are involved in resistance to intestinal diseases. The underlying mechanisms, however, are poorly understood. Here the possible protective effects of two phylogenetically closely related Lactobacillus species, against intestinal damages promoted by enterotoxigenic Escherichia coli (ETEC) strain K88 were examined in vitro. Using fully differentiated swine intestinal cell line IPEC-1, antibacterial activity was measured by assessing the number of the adhering ETEC cells after an inoculation with porcine isolate Lactobacillus amylovorus-like or with a non-porcine strain of L. amylovorus DSMZ. The results revealed that ETEC adhesion was reduced by L. amylovorus-like, but not L. amylovorus DSMZ. Upon L. amylovorus-like treatment, the distribution of tight junctional and cytoskeletal proteins were preserved from ETEC induced damages. To investigate whether these effects were mediated by cytokines and were associated with prevention of inflammation, and whether this was counteracted by the two L. amylovorus strains, the expression of pro- and anti-inflammatory cytokines was analysed. ETEC-induced up regulation of interleukin (IL)-1b and IL-8 was significantly diminished when the cells were treated with L. amylovorus-like, but not with L. amylovorus DSMZ. Together, the data suggests that L. amylovorus-like exerts a protective effect against ETEC adhesion and invasivity and against the membrane barrier disruption promoted by ETEC, possibly via blocking IL-1b and IL-8 expression in IPEC-1 cell line.

Intestinal mucin, a family of glycoproteins secreted by goblet cells, is the main constituent of the mucus protecting the gastrointestinal tract. For optimal mucosal protection, both quantitative and qualitative characteristics of mucin are essential. To evaluate how viscosity influences ileal apparent digestibility and mucin biology, a highly viscous non-fermentable soluble polysaccharide, carboxymethylcellulose (CMC), was fed to weaned piglets during 15 days. The ileal crude mucin concentration was determined by ethanol precipitation and changes in goblet cell subtypes were analyzed by histochemistry of ileal and colonic tissues. As expected, CMC increased significantly the viscosity of ileal digesta and the moisture of feces (P < 0.001). The crude mucin concentration and output at the ileum were significantly higher (P < 0.05) with CMC than with control diet. Increasing intestinal content viscosity with CMC had no significant effects on the ileal apparent digestibility of dry matter, organic matter, nitrogen and minerals. The number of total ileal goblet cell per villi also was significantly higher (+30%, P < 0.05) with the CMC diet as compared to the control. This increase was essentially accounted for by increased numbers of acidic and acidic sulfated mucin-containing cells (+30%, P < 0.05). Trends (P = 0.06) towards decreased numbers of neutral and acidic mucin-containing cells in ileal crypts were also noted. In conclusion, increasing intestinal content viscosity with CMC in weaned piglets increased the ileal mucin output and numbers and maturation of goblet cells in ileal villi without effects on diet apparent digestibility.

The effect of early weaning at 21 days and immune challenge on immune development and function was studied. Lactating sows (large white x landrace) were selected from a commercial pig farm and at 19 days of age there piglets were randomly allocated into two groups. Group 1 remained on the sow throughout the remainder of the study, whilst those in group 2 were weaned and transferred to the Langford weaner pig facility at 21 days of age. Piglets (n=6 per group) from each group were killed at 19, 21, 23, 27 and 33 days of age. Tissue samples were collected from the small intestine, spleen and mesenteric lymph node for cell isolation and immunohistology. Cell and tissue samples were analysed. In the present study showed that weaning at 21 days of age resulted in little or no changes in the proportion of T cell populations in the small intestinal lamina propria. Polyclonal T cell activation of isolated lamina propria did result in significantly reduced live CD4+CD8+ DP lymphocytes compared with un-weaned controls at 2 days post-weaning (23 days of age) It is surprising that in the present study weaning (introduction of new dietary antigens, potential increased exposure to pathogens) had little effect on the small intestinal lamina propria cell populations as measured by T cell phenotype, ability to produce IL-2 & IL-4 following polyclonal activation and activation induced cell death. This contrasts with our earlier studies in which we have reported that weaning is associated with a transient reduction in the ability of intraepithelial lymphocytes to respond to mitogens, and splenic T cells to secrete IL-2. We have also shown that weaning triggers an accumulation of CD2+ cells into the intestinal lamina propria and this led us to postulate that this may be due to the re-localisation of systemic T cells to the gut (for review see Stokes et al 2001). At that time we reasoned that this might lead to the local secretion of Th1 type cytokines and that this could provide a mechanism for the observed changes in gut morphology normally observed at weaning. No changes in gut morphology (crypt, villus height etc) were observed in the present study. To date we have been unable to provide an explanation for these profound differences in response to weaning. The results provided in the presenting study highlighting immunological differences between pigs reared on "closed" organic farms and those under more conventional conditions highlight the sensitivity of the mucosal immune system to a variety of factors such as disease (eg Porcine circovirus 2), environment and genotype. Further studies are required to unravel these critical effects.

The practice of adding organic acid to diets for weaning pigs is quite established. An improvement of growth after weaning has been averagely demonstrated during feeding trials. However data showing that organic acids can positively counteract the presence of post-weaning diarrhoea and of enterotoxigenic E.coli k88 (ETEC) are conflicting. It cannot be excluded that differences in the response to organic acids depend on individual susceptibility to ETEC. Furthermore gastric acidification reduces gastric release and consequently hydrochloric acid secretion, but its long term effects are not enough studied. 60 pigs, weaned at 21 days, were divided into three groups, balanced for number, litter and live weight and fed: standard balanced diet (C), C + 1.2% of free calcium formate (F), C + 1.2% of fat-protected calcium formate (P). In F and P diets, monosodium phosphate and calcium formate were partial substituted for dicalcium phosphate and calcium sulphate of diet C. Pigs were orally challenged with 1.5 ml of a 1010 CFU Escherichia coli K88 O148 (F4) suspension on day 2, and sacrificed on day 7 or 8. The supplementation with free calcium formate improved growth (P<0.01) and feed intake (P<0.05) (Fig.1), while only a trend of increase of growth was observed for fat-protected formate (P<0.10). Both forms of formate addition reduced diarrhoea score, days in diarrhoea, total E. coli faecal excretion (P<0.05), and increased the average of villous heights in duodenum, jejunum and ileum (P<0.05). IgA anti E.coli K88 were reduced by formate treatments (P<0.05), in saliva but not in blood and in jejunum secretion (Data not in figure). In saliva, the total IgA activity tended to be reduced by acidifiers (P=0.07). The expression of TNFalfa gene in stomach and jejunum wall was not affected by the diet. The pH of stomach was not affected, while free formate reduced average pH in ileum, colon and cecum, compared to the other dietary treatments. Pigs fed 12 g/kg calcium formate for 7 or 8 days had a relevant reduction of parietal cells (acid secreting in the stomach), and of expression of H+/K+-ATPase gene (functional to acid secretion) compared to control ones (P<05). This reduction was not observed with fat-protected formate supplementation (same dose). It is confirmed that formic acid (actually calcium formate) has a growth promoting action for the young pig. This action is explained by a reduction of total E.coli persistence. Our data concerning the use of fat-protected formate are not conclusive. Some positive effect was seen on health, but not on growth. The degree of protection was not tested, but data could lead to suspect that the protection was not homogeneous; the reduction of IgA in saliva observed also with the addition of fat-protected formate could be explained by the presence of some free formate in the product too. The use of this fat-protection is justified only to overcome the negative effect of free formate on gastric morphology.

There is increasing evidence that dietary supplementation with prebiotics can be effective in the treatment of intestinal inflammation. As weaning time is characterized by a rapid inflammation, this study investigated the effect of a diet supplemented with four fermentable carbohydrates (lactulose, inulin, sugar beet pulp and wheat starch) on the expression of pro-inflammatory cytokines in newly weaned piglets. Cytokines (IL-1b, IL-6, IL-8, IL-12p40, IL-18 and TNF-a) were analysed by an RT-PCR technique on day 1, 4 and 10 in the ileum and colon of piglets fed the test diet (CHO) or control diet (CON). In addition to the diet, the effect of enforced fasting on cytokine expression was also evaluated. No effect of fasting was observed on pro-inflammatory cytokine expression. Our results showed that the CHO diet induced an up-regulation of IL-6 mRNA expression in the colon of piglets 4 days post-weaning. An increase in IL-1b mRNA expression was also observed at day 4 post-weaning in piglets fed with both the CHO diet and the CON diet. Correlation between pro-inflammatory cytokines and end-product of fermentation demonstrated that the regulation of cytokines is linked with some of the fermentation end products such as Branched Chain Proportion (BCP).

Different organic acids and their salts, such as formic acid, Ca-formate and Na-butyrate, were screened and tested for their ability to protect intestinal cells from ETEC infection. The TEER of Caco-2 cells was unaffected by addition of 1-5mM Ca-formate, whereas it increased with 10-30mM of this organic acid. On the other hand, higher concentration of Ca-Formate (50mM) induced a TEER decrease. When Caco-2 cells were infected with enterotoxigenic Escherichia coli (ETEC) strain K88, the addtion of Ca-Formate was not able to protect the cells against TEER decrease induced by the pathogen. Formic acid (up to 30mM) did not affect the TEER of Caco-2 cells and was able to protect cells against ETEC-induced membrane permeability increase. Na-butyrate (up to 15mM) did not affect membrane permeability of Caco-2 cells and did not induce a TEER increase in Caco-2 cells during differentiation. On the contrary, treatment of IPEC-1 cells with Na-butyrate (2mM) during differentiation induced a significant TEER increase and an establishment of tight junction structure, indicating that this organic acid promotes differentiation of this cell line. When Caco-2 and IPEC-1 cells were infected ETEC Na-butyrate treatment was not able to protect the cells against ETEC-induced membrane damage.

Balance trials were conducted with artificially reared piglets (milk replacer) to assess the pre-weaning quantitative aspects and post-weaning with weaners fed the four starters already applied in the slaughter trial. The 15N tracer technique was used to estimate the endogenous nitrogen loss and the D-alanine method to assess the bacterial nitrogen contribution. The both balance trials aimed to investigate the nitrogen digestibility, endogenous and bacterial nitrogen at ileal in unweaned and weaned piglets fed different diets. The 15N tracer technique was applied to estimate endogenous nitrogen losses and calculate real ileal digestibility of nitrogen. Bacterial nitrogen was assessed by means of D-alanine as bacterial marker. Our results demonstrate that in unweaned piglets nitrogen digestibility was highest and endogenous and bacterial N flow (g/100g DMI resp. CPI) significantly lower compared to weaned pigs. Looking at total ileal N endogenous nitrogen contributed to the greatest and exogenous (dietary) nitrogen to the least part. This underlines the high nitrogen utilisation of milk replacer at the terminal ileum. Similar results were established for the low-fibre starter LF: real nitrogen digestibility was equal to that of milk replacer (98.5 vs. 98.2 %) and endogenous nitrogen was the main contributor to total ileal N. The remaining starters +AB, -AB and HF showed markedly lower RIDN (90.2, 88.3, 94.0 %). In the reference diets endogenous N was lower and exogenous N contribution greater than in home-produced starters and milk replacer. Bacterial nitrogen proofed to be less affected by weaning itself but influenced by dietary regime. Un-weaned animals and those fed +AB and AB exhibited equal bacterial nitrogen contribution (11.6, 11.4, 11.8 % of total N), whereas in piglets receiving LF and HF bacterial contribution was markedly lower (9.1, 7.9 % of total N). This might be attributed to differential microbial population, though these differences were not present in the results of the classical plate counting. One reason could be that the major part of the gastrointestinal microflora belongs to yet uncultured microorganisms, thus not to be proofed by classic microbiological techniques. However, D-alanine as bacterial marker can be assumed to have well covered the yet uncultured bacteria. D-alanine was used as a novel approach in swine. At present it is used successfully in ruminants, but not in monogastric animals. However, indicate its suitability as a bacterial marker in monogastric animals as well. Our results support this notion. Results obtained from both the balance trials imply that the magnitude of the main components of total ileal nitrogen - endogenous and bacterial N - are more dependent on the applied dietary regime, whether being milk replacer or starter diets, than on the actual weaning process. Comprising the experiments of this study - slaughter and balance trials - we could demonstrate that the small intestinal environment is subjected to a short-termed adaptation period during the actual weaning process. During this period the ileum adjusts to a physiological equilibrium, which goes along with minor changes in various parameters such as ammonia, VFA and LA. Postweaning our results showed no advantage of dietary supplementation with the in-feed antibiotic avilamycin compared with the other three starters. In fact we could demonstrate considerable beneficial characteristics of the home-produced low-fibre diet LF: higher nitrogen digestibility (apparent and real ileal N digestibility) as evidenced in the balance trial and a promotion of the Lactobacillus spp. population in the terminal ileum as showed in the slaughter trial. Both features might proof advantageous in the practical application of this diet: higher digestibility has the potential to improve ADG in long-term administration (exceeding 15 days postweaning), which is of great importance for commercial swine production in a financial aspect. Furthermore, enhancing the diversity of Lactobacillus spp. in the terminal ileum indicates that this cereal-based diet has probably prebiotic properties and therefore could be of good use in commercial swine production, particularly with regards to the EU-wide ban of antimicrobial feed additives in weaning piglets.

The economic effects are derived from a survey of experiments. Both on-farm and research station experiments testing different feed-additives are used. Without any in-feed substitutes, AGP ban would increase the fixed costs (buildings, permanent labour force) by about 5%, twice more than the increase in variable feed-costs (2.4%). In-feed alternative growth promoters, acidifiers for piglets and enzymes for fattening pigs in particular, are relatively profitable, especially for fixed production factors. The use of in-feed alternatives reduces the cost impact of the ban to about 2% for both fixed costs and feed costs. Based on experimental data these results do not take into account extra-costs due to additional manure (+3% with AGP substitutes) and additional therapeutic use of antibiotics. Recent data, of the years following the EU partial AGP ban of 1999, show that the therapeutic use of antibiotics is the main short-term substitute of AGPs in real farms. In the long run, animal farm advisors insist on farm workers' training and good farming practices like all-in all-out system, hygiene and vaccines. Although later weaning was also used as a response to the Swedish unilateral AGP ban of 1986, its effectiveness and its economic efficiency remains debated among experts. Better practices take time to be implemented and proved efficient. As an example, the sanitary conditions and the therapeutic use of antibiotics in Swedish pig farms have come back to 1986 levels since 1996 only.

Copious research data from trials in vitro and on experimental animals are available showing that the gut immune system could be down- or up- regulated in order to improve the overall health of the host. In some cases it is claimed that humoral immunity was increased, with special emphasis on the stimulation of specific response against certain pathogens. However the development of specific immune response towards commensal bacteria and probiotics is less documented. Indeed a prolonged secretion of immune globulins is a cost for the host and could also reduce the chance of survival of the probiotic in the gut. Changing levels of Lactobacillus-species specific IgA were observed in mice mono-associated with 2 lactobacilli showing similar in vitro adhesion patterns (L. johnsonii NCC 533 or L. paracasei NCC 2461). Nevertheless the variations of Lactobacillus-species specific IgA are in general not studied in normally fed producing animals. Data refer to blood and saliva samples collected at different times during two different trails in witch L.rhamnosus GG (Lb-R) (Trial A) and strain 001T of the new species Lactobacillus amylovorus-like strain 001 (Lb-S) (Trial B), were respectively supplemented to pigs. The first microbe is a probiotic approved for human use and is considered to be not commensal of pig, while the second was isolated in piglets fed fermentable diets (collaborative work). In both trials, pigs, weaned at 21 days of age, were fed control diet or control diet plus 10x10 CFU probiotic/day. Pigs were orally challenged with 10x10 CFU E. coli K88ac O148 (F4) on day 7, and sacrificed after another week.We wanted to assess at different times after weaning the IgA activity against a pig-specific and a pig-not specific strain. Secondly we wanted to verify if part of this IgA strain-specific activity was partially related to cross-reactivity between two different Lactobacillus-species. Seric IgA anti-L.rhamnosus GG were present at all the samplings, even before the dietary supplementation with L.rhamnosus GG and in the control group. This contrast with the fact that L.rhamnosus GG is not considered a strain typical of the pig. Indeed, cecum samples obtained from control subjects were negative for the presence of DNA from L.rhamnosus GG (at the contrary, most of the probiotic-fed pigs were positive). A similar observation was done in the second trial, where seric IgA anti L.amylovorus-like strain 001 were also detected in control pigs that were negative in ileum for the presence of this particular strain. All these observations can be explained by the presence of a cross reactivity between different bacteria. If the values of probiotic specific IgA are expressed on total IgA content and as a difference between different times of samplings, we can see that the strain-specific IgA relative content in the Lactobacillus group increased between start and 1st week; this was not observed after and for the control group. This observation shows that there was an additional short-term specific immune response in the probiotic group, in agreement with observations on germ-free piglets fed with non-pathogenic E.coli. Cross reactivity index evaluated by pre-absorption test for different samples (blood or saliva) and for different specific IgAs was high. There is a consistent debate in the literature about the presence of antibodies that apparently react with many different microbes. One possible explanation is the similarity of some amino acid and sugar motifs in the structure of the S-layer that protects Gram+ cells; a second hypothesis is that part of these IgA's are "polyreactive" or "natural". Then it is not yet clear the functional significance of the presence of IgA against commensals: it could constrain these bacteria inside the gut lumen, but it could also improve their chance of adhesion to mucous and their persistence in the gut. In any case, when probiotic are supplied to weaning pigs, the possible action of already present secretory IgA should be considered. This could also explain unfavourable results of the probiotic strategy.

Diets for weaning piglets can be manipulated for their protein and fibre composition in order to providing substrates to the commensal microflora and decrease pathogenic bacteria and bacterial metabolites detrimental to the gut. Therefore, the influence of diets differing in the protein source and fibre on the gut was investigated in piglets weaned at 28 days of age. Four diets were formulated: a control with high digestible protein and low fibre, a diet with low digestible protein and low fibre content, and two diets with low digestible protein and a high content of either insoluble or soluble fibre, provided with wheat bran and sugar beet pulp, respectively. These diets were fed for two weeks to piglets according to a balanced incomplete block design. At slaughter, the studied variables included gastrointestinal segmental full and empty weight, ileal flow of digesta, nutrient and mucin, digesta concentration of volatile fatty acids and ammonia, intestinal morphometry and goblet cell histochemistry and finally pancreatic enzyme activities. The high fibre diets usually increased the weight of gastro-intestinal segment tissues and digesta. The amount of fresh digesta in the ileum was higher with both fibre-rich diets. However, no significant differences between diets were found for ileal dry matter and nitrogen output. The ileal mucin output was higher with the three low digestible-protein diets than with the control, the increase with the insoluble fibre diet being less than with the low fibre or soluble fibre-rich diet. Fermentation already took place in the ileum, the flow of acetic acid being higher with the soluble fibre diet as compared to the other diets. The ileal flow of valeric and isovaleric acids was also higher with the soluble fibre diet. The fresh contents in the caecum had a pH lower with the soluble fibre diet. Interestingly, both types of fibres were shown to reduce the concentration of ammonia in the caecum. Conversely, lactic acid concentration in the caecum was higher with the soluble fibre diet, intermediate with the insoluble fibre diet and the lowest with the control and low digestible protein diet. Formic acid concentration in the caecum was reduced with the three diets containing the low digestible protein source. The fibre-rich diets, especially the soluble fibre one, decreased the counts of Clostridium perfringens in the proximal colon. The ileal mucosa weight and jejunal crypt area were higher with the high soluble fibre diet. Maltase, sucrase and aminopeptidase N jejunal specific activities were lower with the high fibre diets, and ileal aminopeptidase N activity tended to be lower with the high soluble fibre diet. No impact of diet on goblet cell histochemistry in colonic crypts was noted. Pancreas weight, trypsin and amylase activities were higher with the low digestible protein diets. It is concluded that dietary fibre can modulate gut tissue and digesta mass and composition, influence intestinal epithelial cell proliferation, decrease digestive enzyme activities and stimulate ileal mucin output in the weaned piglet.

The impact of diet containing fermentable carbohydrates on the development of the intestinal immune system was studied in several experiments. Four to six week old piglets express in the period after their weaning mRNA for the pro-inflammatory cytokines IL-2, IFN-g and IL-12p40 and the anti-inflammatory cytokines IL-4, IL-5 and IL-10 in all parts of the gastrointestinal tract. In the small and large intestine cells containing IL-2-, IL-12p40-, IFN-g-, IL-4- and IL-5-mRNA transcripts were observed in the lamina propria close to the crypts. In experiments under highly controlled hygienic conditions differences in cytokine expression were observed between pigs that had received high-fibre diet and those fed conventional food. In high-fibre diet pigs a marked increase of transcripts for inflammatory and non-inflammatory cytokines were visible in the first days after weaning. This peak had disappeared around day 8 after weaning.

At the time of weaning, major quantitative and qualitative changes occur in the composition of the intestinal microbiota of piglets, influenced by diet, environmental factors, and the host itself. Within a short period of time, the intestinal microbiota must develop from being essentially sterile, to a microbial community of varying complexity. The microflora remains fairly stable in terms of species after this initial colonisation, and for as long as the piglets receive sow milk. However, the sudden introduction of solid food causes major qualitative and quantitative alterations in the microflora which must then change from being a comparatively simple (and potentially unstable) community into a complex and stable one. For example, strict anaerobes such as Bacteroides become established in the large intestine, and this corresponds with a decline in the number of facultative anaerobe organisms. Also, invasion by pathogens is very much dependent on the condition of the animal, including the composition and activity of the GI tract microbiota. The results obtained indicate that the addition of non-digestible, fermentable carbohydrates (= prebiotics) leads to an enrichment of lactobacilli in the small intestine, and increased stability and diversity of the bacterial community in the colon. The data support the hypothesis that changes of the diet can modulate the composition of the microbiota in the intestine. These findings may have important implications for the development of dietary strategies aiming to improve animal health during the weaning process, and should therefore be of high interest to pig farmers and feed manufacturers.

The intestinal immune system has to provide mechanisms for induction of immune defence against pathogens and for the development of oral tolerance versus nutritional antigens. Two major compartments are present to provide these reactions: the normal mucosa and the organised lymphoid tissue e.g. isolated follicles and Peyer's patches. The freqency of Peyer's patches increases along the small intestine, in piglets in the terminal ileum a long continuous Peyer's patch is present. The regulation of the immune reactions is not known yet, especially the role of the intestinal mucosa and that of the Peyer's patches. We therefore analysed the cytokine expression in the intestine. As methods RT-PCR and non-radioactive in-situ hybridisation for cytokine mRNA of IL2, IL4, Il10, IL12, IL18, gammaIFN, TNFalpha, TGFbeta were used. In jejunum, jejunal Peyer's patches, ileum and colon message of all inflammatory and non-inflammatory cytokines was present. After weaning the frequency of cells with transcripts was highest in the colon, fewer transcripts were detected in the ileum, in the jejunum few cells expressing cytokine mRNA were detected. Both, inflammatory and non-inflammatory cytokine mRNA was present in cells close to each other, as shown by the morphological analysis by in situ hybridisation.

Changes in diet composition such as the one that occurs at weaning alter gastric emptying and gastro-intestinal motility. The underlying factors for theses changes may include adaptation of duodenal vagal receptors. The aim of this study was to evaluate the characteristics of multimodal duodenal receptors before and immediately after artificial weaning in 20 anaesthetized 40 kg pigs. Twenty duodenal vagal afferents were recorded from the cervical vagus using the single fibres method. 10 pigs were fed with a milk-based diet (MD) for one month while the diet of the 10 other pigs was changed for plant-based diet (PD) the day preceding the recording session. The behaviour of the receptors was tested before and after challenges with duodenal intralipid and close intra-arterial injection of CCK, 5-HT or Capsaicin with and without isobaric duodenal distensions at 20, 40 and 60 mmHg. All receptors were slowly adapting C type fiber with a receptor field located 6-7 cm distal to the pylorus. Comparisons between groups were achieved by normalizing the firing pattern to that recorded during 20, 40 and 60 mmHg distensions in the unchallenged condition. Intralipid induced an overall reduction in basal discharge irrespective of the diet. In contrast, the rate of discharge during distension (20, 40 and 60 mmHg) combined with duodenal intralipid was significantly larger for MD compared with PD (104 +/- 18.2 vs 49 +/- 15.8 spikes.5s-1 for 20mmHg distension, p<0.05). Similarly, the rate of discharge observed during distensions performed with CCK (90 +/- 14.1 vs 51 +/- 16.3 spikes.5s-1 for 20mmHg distension, p<0.05) and with 5-HT (168 +/- 23.2 vs 43 +/- 14.9 spikes.5s-1 for 20mmHg distension, p<0.05) were greater for MD compared with PD while CCK and 5-HT without distension were equally stimulating for MD and PD. No significant difference was found between groups during Capsaicin infusion irrespective of the stimulating pressure. In conclusion, a plant-based diet, when compared to a milk-based diet, results in an overall decrease in mechanical sensitivity of duodenal neurones during chemical challenge, but not in basal conditions.

An experiment was conducted to examine differences in in-vitro fermentability of 4 carbohydrate-rich feed ingredients, and two weaning piglet diets with and without these ingredients, using both the ileal contents and the faeces of un-weaned piglets as inocula. Cumulative gas production was measured in time, using faecal inocula mixed from 9 crossbred piglets (no creep feed or antibiotics) at 3 weeks of age. Inulin, lactulose, unmolassed sugar beet pulp, wheat starch and the two diets, were used as substrates and fermented in vitro for 72h. Gas production was measured as an indicator of fermentation kinetics. End products including VFA, and ammonia, and organic matter loss, were also measured. To study microbiota changes, PCR/DGGE analyses were done in the initial inoculum and after fermentation. This was repeated one week later, using ileal contents from the same piglets as inoculum. There were significant differences between inocula in terms of overall fermentation characteristics and composition, and between substrates. There was also a significant interaction between inocula and substrates, suggesting local differences in microbial activity. For the two diets with and without addition of these fermentable ingredients, there were significant differences in terms of kinetics, but less so in terms of end products. It was concluded, that inocula from ileum and colon should be used, to obtain a more accurate assessment of potential feed ingredients that will stimulate fermentation in piglet GIT. Unrelated to this in vitro work, an in vivo production trial was carried out in collaboration. In this experiment the effect of varying the proportions of dietary protein (CP) and carbohydrates (CHO) in weaner diets was examined in relation to feed intake, digestibility, growth performance and several gut parameters (e.g., bacteriology, fermentation end products, villus height/ crypt depth). Directly after weaning, piglets from different litters were selected and equally distributed over four dietary treatments ; 1=lowCP & lowCHO; 2=lowCP & highCHO; 3=highCP & lowCHO; 4=highCP & highCHO. At day 7 post-weaning, two piglets per treatment were selected and sacrificed to collect chyme from the ileum and colon. The chyme samples analyzed at Wageningen University (WU) for DM, VFA, NH3 and lactic acid. There was a significant difference between ileal and colonic chyme, with higher values for the VFA, NH3, and branched chain proportions (BCP) in the colonic chyme. Lactic acid and the molar proportions of acetic acids were significantly higher in the ileal chyme. The dietary treatments did not have a significant effect on end products. However, in the case of colonic chyme, the piglets receiving diets with high CHO (Diets 2 & 4) tended to have higher acetic acid, butyric acid and total VFA concentrations, and lower concentrations of NH3 and BCP. End products (acetic, butyric, iso-butyric, total VFA) of colonic chyme tended to be more affected by fermentable CHO-level in the case of high crude protein diets, whilst for NH3, a reverse effect was observed. This would suggest that when both dietary CP and fermentable CHO are in short supply, extra fermentable CHOs lead to a strong decrease in NH3, which is most likely, a result of increased incorporation of N into GIT microbial biomass. Overall, it was concluded that the dietary contrasts used in the current experiment were not sufficient to significantly affect fermentation processes in the ileum, and only led to small effects in the colon. In another experiment at INRA in Rennes, France, to test 4 weaning diets, also differing mainly in their protein and fermentable carbohydrate proportions. Samples of digesta were analyzed at WU for DM, VFA, NH3, and lactic acid. In all cases, GIT site had a significant effect on fermentation end products, with increasing concentrations from ileum to colon. Also, the BCP showed a similar pattern with highest values in the colon. However, the only significant effect found between diets was for NH3. There were some significant differences in the interaction for Diet x GITm for BCP. Diet R1 gave the lowest BCP at the ileal level, but was highest for the colon. This could indicate that the ratio between protein and CHO availability is sufficient to supply the fermentation processes in the ileum, whilst in the colon there seems to be a shortage of CHOs for fuelling hindgut fermentation. Overall, it would be recommended that increased fermentable CHO in weaning piglet diets, with a concomitant reduction in proteinaceous ingredients will improve piglet health at the critical time of weaning. However, it would seem that an in vitro assessment of a range of potential ingredients prior to the design of new diets may lead to more consistent results in the field. This is an important finding for feed manufacturers with an interest in weaning piglet diets.

Zinc deficiency in rats has been shown to alter the villous-crypt architecture and enzyme activities of the small intestine. This model proved valuable in demonstrating the positive effects of probiotic on the restoration of intestinal alterations. Therefore, we conducted an experiment with this model of zinc deficiency in weaned piglets in order to develop it for studying alternatives to in-feed antibiotics. Two weaning diets supplemented (control) or not supplemented (Zn-) with zinc were formulated. The diets were pair-fed for three to four weeks to 32 piglets weaned at 21 days of age and placed in individual plexiglass-covered cages. They had free access to demineralised water. Feed intake, bodyweight and consistency of faeces were recorded throughout the trial. At the end, the piglets were sacrificed. Blood was collected for zinc and alkaline phosphatase (a Zn-dependent metalloenzyme) determination. Organs and the gastrointestinal tract and its segments (full and empty) were weighed. Mucosal tissues from the small intestine were sampled for histomorphometry and determination of enzyme (maltase, amino-peptidase A, dipeptidyl peptidase IV and alkaline phosphatase) activity. Jejunal digesta were sampled for bacteriology (total counts of anaerobes and aerobes, lactobacilli, enterococci and coliforms). Plasma levels of zinc and alkaline phosphatase were two and three-fold lower in the Zn-deficient group, showing the depression of the circulating pool of zinc and consequences on a Zn-dependent metalloenzyme. No diet effects were observed for feed intake, growth performance or consistency of faeces throughout the trial or on the weights of organs and gastrointestinal tract, except a tendency for a lighter pancreas in the Zn-deficient piglets. However, the weight of fresh digesta in the stomach was higher, and that in the caecum and colon lower, with the Zn-deficient diet. No significant effect was observed on the villous-crypt architecture and enzyme activities of the mucosa of the small intestine, except a tendency for a smaller villous area in the Zn-deficient piglets. The intestinal microflora was little influenced by the diet. In conclusion, feeding a Zn-deficient diet for three to four weeks to weaned piglets had little effect on the villous-crypt architecture, enzyme activities and the microflora of the small intestine in this study. This limits the interest of the model for investigating alternatives to in-feed antibiotics in the weaned piglet.

Immune development was studied in conventional (farm reared) and isolator (SPF) reared piglets to 28 days of age. Pairs of farrowing sows were selected from a commercial pig farm and at 24 hours, half of each litter was removed from sow and transferred to an SPF isolator. The other half of the litter remained with the sow and the piglets were not given access to creep feed. The isolator piglets were fed a bovine-based milk replacer on an hourly-based cycle. Piglets (n=6 per group) from both experimental groups were killed at 2, 5, 12, 20 and 28 days of age. Tissue samples were collected from the small intestine, spleen and mesenteric lymph node for phenotypic analysis, by flow cytometry and immunohistology, and functional analysis, by in vitro cytokine production and cell proliferation/survival analysis. The intestines of newborn pigs are essentially devoid of immune cells and remain so in germ free animals until the introduction of a bacterial population. In conventionally reared pigs, immune cells start to appear in the intestine within a few days. The number of cells presented represents the culmination of a number of processes including, cell emigration, cell proliferation and cell death. Cells isolated from the lamina propria of piglets raised in a SPF isolator (high hygiene) showed an increase in the relative survival of CD4+ cells from 5-12 days of age. Possibly indicating that these cells were more reactive than equivalent cells isolated form pigs reared under conventional (low hygiene) conditions. Rearing environment had little influence on the overall numbers of CD14+, CD14+CD16+ , MHC Class II+ CD16+ and MHC Class II+ APC. However there was a significant decrease in MHC Class II expression at 20 days of age in the farm-reared piglets. The results showed that that co-localisation of both CD14+CD16+MHC II+MIL11+ and CD16+MHC II+MIL11+ events were significantly greater than predicted. In contrast the observed CD16+MIL11+ association was less that expected and there was no difference from expected for CD14+CD16+MIL11+. Taken together these results would suggest that there were significant interactions between CD16+MHC II+ and CD14+CD16+MHC II+ cells and MHC II+ and endothelium. These changes in interaction were not related to age or rearing environment. There was a rapid increase in IL-2 production in the first few days of life and this is consistent with the rapid influx of T cells with the peak coinciding with the arrival of CD+ T cells. In contrast IL-4 production was high at all time points suggesting that a small number of T lymphocytes present in the cultures were able to produce high levels of IL-4 or that other cell types present may have been able to produce IL-4. The considerable environmental changes that the high-hygiene isolator-reared group experienced had little effect on the progress of this extensive phenotypic T cell development. As other work has shown that germ-free animals show a total lack of immune cells in this environment and our animals shared the same farm environment and maternal colostrum during their first day of life, this early life exposure to microflora appears to be sufficient to drive the subsequent immune maturation. Parallel studies performed on samples collected from these pigs (WP 2; participant number 3) would suggest that qualitatively there were no significant differences in the intestinal microbial populations between farm and isolator reared piglets during the first 12 days of life. This would suggest that the differences observed between isolator and farm reared piglets at 5 days of age were not an effect of microbial but rather removal from the sow and change of diet. In contrast lamina propria IL-4 production was slightly reduced levels in the isolator-reared animals which might be indicative of a lack of maturity or altered Th1:Th2 balance. The results have shown that there are significant differences in the cell populations found in the intestinal lamina propria of young (less that 28 days) as opposed to older piglets. The results strongly support the conclusion that at ages commonly used in european pig weaning practices, the piglets immune system may not be sufficiently developed to respond to the challenges of weaning. This would suggest that either a later a weaning age should be adopted or we should seek to enhance the development of the piglets' mucosal immune system. The results rearing piglets under different hygiene conditions have supported the conclusion that this developmental process is influenced by gut microbial flora. Research programmes to develop novel immune potentiators or to provide "natural" alternatives to anti-microbials (eg FEED FOR PIG HEALTH) should provide a rational scientific basis for the formulation of future pig weaning practices.

Lymphocyte populations in the intestinal mucosa are inducer and effector cells for the mucosal immune response. The aim of the studies was to follow the lymphocyte kinetics in the period around weaning. The experimental approach was the labelling of all cells in the S-phase of the cell cycle using the thymidine analogue bromodesoxyuridine (BrdU). BrdU can be detected by immunohistochemistry. Using a double labelling system proliferating cells and their phenotype can be analysed. 30 minutes before sampling piglets received a single intravenous dose of BrdU (5 mg/kg body weight). Thus all cells in the S-phase of the cell cycle were labelled. The sections were evaluated qualitatively. In all gut locations studied (jejunum, jejunal Peyer's patches, ileum, colon) proliferating CD3, CD4 and CD8 T cells were detected. The frequency of proliferating T cells was higher in the lamina propria of the mucosa. In the T cell areas of the Peyer's patches many proliferating T cells were detected. There was no difference in piglets that had received different diets. Highly fermentable carbohydrates caused a remarkable change of the intestinal flora, however the cell proliferation within the mucosa of these piglets was not affected by the changes in the intestinal lumen. In summary, the mucosal lymphocyte populations are regulated by immigration and as these results demonstrate by a local proliferation.

Characteristic dietary feature at weaning is a switch from milk to plant-based diet, i.e. from a non-fibrous to a fibrous diet. The study aimed at evaluating the effects of such an abrupt dietary switch on gastric emptying in pigs maintained on a milk substitute after weaning. Eighteen piglets were kept on a milk substitute for five weeks after weaning and were then either submitted to a dietary switch to wheat- or barley-based diets or kept on the milk substitute (n = 6 piglets per group). All piglets were fasted during one day before the switch and daily food intake was then linearly increased to reach initial values within three days. Gastric emptying was measured by ×-scintigraphy before and after the switch. Corpo-antral peristalsis was also evaluated by the use of high frequency scintigraphic frames. Gastric emptying of the wheat-based diet was accelerated on days 1 to 3 after the switch but was similar to the milk substitute thereafter. This acceleration was concomitant to an enhanced frequency of corpo-antral waves on day 2 and day 3. On the contrary, gastric emptying of the barley-based diet tended to be enhanced on day 2 but was delayed on days 4 and 5, without any change in frequency of corpo-antral waves. We concluded that a switch from a non-fibrous to a fibrous diet alters gastric emptying differently depending on the type of dietary fibre.

Fasting by newly weaned piglets, is considered to be a significant contributor to the post-weaning syndrome, which includes diarrhoea and even death of piglets. An in vivo experiment was conducted to monitor changes in fermentation end products in the faeces of weaning piglets by the inclusion of selected fermentable carbohydrates in the diet. The experiment was repeated in three replicates of 16 piglets. Specially raised piglets (neither antibiotics nor creep feeding) were weaned abruptly at four weeks of age. Each replicate was conducted over a period of ten days. The piglets were offered one of two dietary treatments: control diet (CON), or fermentable carbohydrate enriched diet (CHO); and were subjected to one of the two fasting treatments (fasting for two days in the beginning of the experimental period and non-fasting). Faecal samples were collected per rectum every day during the experimental period, and visually examined for indications to diarrhoea. Piglets were slaughtered at the end of the experimental period and digesta samples collected from different parts of gastrointestinal tract (GIT): first and second halves of the small intestine, caecum and colon. The dry matter, volatile fatty acid (VFA) profile, and ammonia concentrations were analysed from the faecal and digesta samples. Daily feed intake was also recorded. The consistency (DM) of the digesta did not indicate any differences between treatments; hence, 48 hour post-weaning fasting did not seem to affect the occurrence of diarrhoea under these experimental conditions. Fasting also had no effect on fermentation end products by day 10. This was unexpected, as it had been anticipated that the fasted animals would show reduced performance characteristics. This may have been due to the comparatively controlled conditions of the experimental facilities. However, as a result of these findings, it was also hypothesized that the propensity to fast in response to stress may be an individual characteristic of the animal, and future experiments will take this into account.

The recent ban of in-feed antibiotics in animal production brought scientists to find alternatives to in-feed antibiotics. Ussing chamber is an in vitro technique in which intestinal tissue is collected and immediately mounted as a flat sheet between two half-chambers, establishing a luminal and a serosal side. This technique allows the measurement of actively transported ions as well as the permeability of the tissues, two parameters relevant to evaluate gut health. Ussing chambers have been used to describe the changes of intestinal physiology occurring at weaning. We observed that weaning induces acute alteration of the epithelial barrier function and electrolyte transport in the first days. Long-lasting modifications (15 days after weaning) have also been observed. Alternatives to in-feed antibiotics can be evaluated by studying intestinal physiology of animals treated with those alternatives. A benefic effect of pro- and pre-biotics on rat intestine, human biopsies or epithelial cells monolayers has been demonstrated using Ussing chambers. In pigs, the influence of the source of dietary ingredients or the physical form of the diet seems limited. A few results obtained in pigs with pre- or probiotics appear less convincing than in laboratory animals. Another way to use Ussing chambers is to incubate the intestinal tissue with different substances added directly into the chambers. Some substances can be detrimental to the intestine, inducing electrolyte secretion or decreasing barrier function. However, some substances showed a beneficial effect. Although this approach has limits and should be combined with in vivo measurements, it constitutes a rapid way to evaluate the effect of substances on intestinal physiology and can also, at least partly, elucidate the mechanisms of actions of those alternatives.

This study was assigned to confirm if: - A delayed weaning results in a better performance, gut integrity and welfare of weaned piglets; - Antibiotic-free, creep-feed intake improves the gut integrity; - Developing a pig specific ELISA for determination of intestinal fatty acid binding protein (I-FABP) as a potential serological marker for intestinal damage and assessment of welfare in both intensive and extensive management systems. The first two objectives were targeted in an experiment involving in total 16 litters (160 sucking piglets at 2 days of age). Eight litters were offered creep feed (barley-based) from day 12 of age, and the remaining not. A half of those animals (fed without and with creep feed) was weaned at 4 weeks of age, and the other half at 7 weeks of age. After weaning all piglets were offered the same creep feed. At day 1 pre-weaning, and days 3 and 6, post-weaning seven piglets from each group were euthanized to obtain intestinal tissue slices for studies of macromolecular transport in Ussing chambers. Besides, the morphological indices (villous heights and crypt depths) were measured in these intestinal slices. Although prolonging the sucking period of piglets from 4 to 7 wk of age resulted in a milder weaning stress (=lower cortisol levels in blood) and slightly longer epithelial villi, no significant differences due to the weaning age could be found in the gut integrity (as measured in terms of net fluid absorption from the intestinal lumen or paracellular transport and I-FABP levels). Despite the consumption of creep feed by piglets up to 4 weeks of age was rather limited, the intestinal paracellular fluxes and villous heights were greater (P<0.05) in comparison to their littermates, which were deprived of the creep feed. Both groups did not differ in cortisol levels (=weaning stress) and the gut integrity (=net fluid absorption and I-FABP levels). Post-weaning growth retardation could be observed in piglets, which received no access to creep feed during 7-week sucking periods. Another objective of this study was to develop a pig specific ELISA for determination of I-FABP as a potential serological marker for intestinal damage and rapid assessment of welfare in both intensive and extensive management systems. As demonstrated in human and rat studies, this biomarker is exclusively expressed in intestinal epithelial cells, is a small intracellular protein, has a high level of expression, and is detectable in the circulation and urine. For this purpose were used 6 young boars (20-25 kg BW). They were anaesthetized to place catheters in the carotid artery + blood flow probe (around the arteria mesenterica cranialis, AMC). After initial blood sampling (t=0), three jejunal segments (10-20cm) were ligated, and again blood sampling was carried out (t=30 min and further in 15min intervals). At t=60min was a clamp placed around AMC (to get 90% ischemia). At t=60, 120 and 150min the ligated loops were removed, and the mucosal layer was removed. Afterwards, the measurements of macromolecular permeability using horse radish peroxidase (HRP) were carried out in Ussing chamber for 2 h. After 90 min clamping, the intestines were re-perfused and blood flow re-established as normal. The EDTA-plasma was tested with ELISA test kit for humans with rabbit polyclonal antibodies, and purified human I-FABP as standard. During 60min of surgery plasma I-FABP appeared to be stable (=no intestinal damage). At 90% ischemia, a rapid increase of I-FABP during 30min, and plateau after 60min (at t=120min). Reperfusion at 150min had no impact on plasma I-FABP (whereas in rodents ischemic injury is exacerbated). In conclusion, we found that a rise of I-FABP in plasma coincides with increased intestinal permeability due to ischemia. Alike in humans, plasma I-FABP concentrations in pigs can be used as sensitive biomarker of (mild) damage of the intestinal mucosa. The porcine I-FABP cDNA sequence has been determined and it appeared to be of a high identity with homologous genes from other species. This sequence allows us to clone and purify recombinant porcine I-FABP in order to raise specific antibodies and develop an assay for porcine I-FABP. A key innovative feature is that the established values of I-FABP can serve as as a rapid diagnostic tool for monitoring "on site" the pig intestinal health status and welfare.

Initially, an in vitro experiment was conducted to examine differences in fermentability of four carbohydrate-rich feed ingredients, and two weaning piglet diets with and without these ingredients, using both the ileal contents and the faeces of unweaned piglets as inocula. In the first part of experiment, cumulative gas production was measured in time; using faecal inocula mixed from nine specially raised crossbred piglets (no creep feed or antibiotics) at three weeks of age. Inulin, lactulose, unmolassed sugar beet pulp, wheat starch and the two complete diets, were used as substrates, and fermented in vitro for 72 hrs. Gas production was measured mainly as an indicator of the kinetics of fermentation. Fermentation end products including total gas, VFA, ammonia, and organic matter loss, were also measured. For those fermentations of individual ingredients, samples were also collected for PCR / DGGE analyses both before, and after the fermentation process, to study changes in the composition of the bacterial community. This procedure was repeated one week later, using ileal contents from the same piglets as inoculum. There were of course, significant differences between the substrates, but also, and more importantly, between the inocula, both in terms of the overall fermentation characteristics, and the bacterial composition. The latter was an important finding, because it showed that, in vitro at least; the substrate used could lead to a significant shift in the bacterial population, both in terms of detectable composition, and the activity. To determine whether this in vivo finding could also be detected in vivo an animal experiment was conducted to examine changes in fermentation end products in the gastrointestinal tract (GIT) of weaning piglets by the addition (or not) of fermentable carbohydrates in the diet. Specially raised piglets (neither antibiotics nor creep feeding) were weaned abruptly at four weeks of age. Piglets were randomly allotted to either a control diet (CON) or a diet enriched with fermentable carbohydrates (CHO; added inulin, lactulose, unmolassed sugar beet pulp, and wheat starch). Piglets were slaughtered on the 1st, 4th and 10th days after weaning. Digesta samples were collected from different parts of the GIT (first half of small intestine, second half of small intestine, caecum, and colon) and analyzed for dry matter, volatile fatty acids (VFA) and ammonia. Feed intake, growth and feed conversion ratio were also recorded. Data analysis showed that there was no difference in production performances between the treatment groups. Concentrations of VFA and ammonia were significantly different between diets, GIT sites of fermentation(P < 0.001), and the slaughtering day (P < 0.05). It was concluded that the addition of fermentable carbohydrates of varying fermentabilities (both in terms of rates and end-products) stimulated the production of fermentation end products in the four different areas of the GIT studied in weaning piglets. Quantitatively, the results were not the same as for in vitro differences, though this was not expected, and statistical correlations still need to be carried out. The qualitative agreement was very encouraging however.

Litters weaned under commercial conditions undergo drastic social changes such as terminated access to sow's milk, transport to new pens, mixing with foreign litters, access to unfamiliar feeds (dry/slurry) and water nipples. This is manifested by apathy or aggression, no or little interest in new feed intake, growth check, and greater mortality. It is speculated that solid pellets are less preferably consumed than slurry (particularly in the first week after weaning), and it may lead to villous atrophy, reduced gut integrity, and increased pro-inflammatory cytokine expression. Therefore, we studied the effects of an isocaloric weaner diet fed either as dry pellets or slurry (1:2.5 wt/vol) on the in vitro gut permeability, morphology and pro-inflammatory cytokines with 30 crossbred piglets. They were weaned at 28 days of age and fed over 7 days with gradually increasing daily rations, i.e., from 0.5x maintenance requirements (MR) for NE at weaning to 2.0xMR for NE at day 5 post-weaning. The samples of proximal and mid jejunum were obtained at weaning (n=6), on day 3 (n=12) and on day 7 post-weaning (n=12) to measure villous height and crypt depth, mRNA expression level of interleukin 8, and permeability in Using chambers (via unidirectional flux of 14C-GlySar and 3H-mannitol as markers for trans- and paracellular transport, respectively). Piglets fed dry pellets had significantly greater (P<0.05) transcellular transport compared to those fed slurry. Paracellular transport and morphology (the villous length and crypt depth) were similar for both dietary physical forms, although numerically longer villi (by 15%) and deeper crypts (by 7%) were found in piglets fed dry pellets. Age effects on morphological indices could be detected: - Crypt depth on day 3 was decreased, and on day 7 increased numerically compared to the day at weaning; - Crypt depth on day 7 was significantly increased compared to day 3; - Villous length on day 3 decreased (P<0.05) compared to the day at weaning; - On day 7 as compared to weaning, the slurry diet caused a further decrease of villous length, whereas in piglets fed the dry, pelleted diet villi were partly restored to a similar height as on the day at weaning; - Paracellular transport increased (P<0.05) on day 3 and partially decreased on day 7 post-weaning compared to the day at weaning. The transcellular transport increased (P<0.05) on day 3 and day 7 compared to the day at weaning. Pearson's correlates among the permeation biomarkers, morphological measures and immunological indices of the gut during 7 days post-weaning in piglets were found to be rather low (<0.5).

This study aimed at investigating intestinal physiology of piglets at weaning. Sixty piglets weaned at 21 days were 2day-food-deprived then tube-fed using two different diets (a conventional diet vs. a wheat-enriched diet). They were slaughtered at day 0, day 2, day 5, day 8 or day 15 post-weaning. Jejunum, ileum and colon were mounted in Ussing chambers. In addition, jejunum of four growing pigs was studied 35 days after weaning. Secretory function was assessed by basal short-circuit current (Isc) and secretagogue-stimulated Isc. Glucose absorption was measured by the increase in Isc after addition of glucose. Epithelial barrier function was measured by transmucosal resistance (R) and horseradish peroxidase (HRP) fluxes across the epithelium. There were no significant differences between the animals fed the two diets for any of the parameters studied. As already described, a transient villus atrophy was observed. At the same time, we observed an increased basal Isc in jejunum and colon, increased glucose absorption and a dramatic drop of R in jejunum. These parameters had returned to pre-weaning values by day 5. Weaning was also followed by long-lasting modifications. In jejunum, responses to the secretagogues and glucose absorption were decreased the second week after weaning and were not different between day 15 and day 35. Ileal transmucosal resistance exhibited an increase on day 5 and stayed stable thereafter. HRP flux in jejunum dropped the second day and stayed at this low level throughout the experiment. We conclude that weaning induces transient dramatic changes in intestinal physiology but is also a period of maturation of the intestine.

Immune development was studied in conventional (farm reared) and isolator (SPF) reared piglets to 28 d of age. Pairs of farrowing sows were selected from a commercial pig farm and at 24 hours, half of each litter was removed from sow and transferred to an SPF isolator. The other half of the litter remained with the sow and the piglets were not given access to creep feed. The isolator piglets were fed a bovine-based milk replacer on an hourly-based cycle. Piglets (n=6 per group) from both experimental groups were killed at 2, 5, 12, 20 and 28 days of age. Tissue samples were collected from the small intestine, spleen and mesenteric lymph node for phenotypic analysis, by flow cytometry and immunohistology, and functional analysis, by in vitro cytokine production and cell proliferation/survival analysis. Gross morphological examination of the intestines from isolator and farm reared showed that those from the farm-reared animals were thicker but this was not reflected in any difference in the number of immune cells isolated. Work carried out under the current project has confirmed the progressive nature of this developmental process, and highlighted that lack of maturity of the pig gut immune at ages that are commonly used in European weaning practices. In the present study the use of flow cytometry for the characterisation of lamina propia cells has enabled us to identify the sub-populations involved. Whilst CD3+ T cells dominated throughout the first 28 days of life, there were significant changes in the major sub-populations. In pigs less than 12 days the lamina propria is dominated by T cells expressing either low levels of CD8 (CD8low) or negative for CD4 and CD8 (CD4-CD8- double negative). Interestingly from 12 - 28 days CD4+CD8+ cells dominate and this cell type has been associated with anti-viral activity (Saalmuller et al .1999). The CD8low cells also carried initially high levels of CD25 and low levels of CD45RC, representing a recently activated cell population. However, unlike the CD4 cell population, although CD25 was lost by day 12, low levels of CD45RC remained on this T cell subset. Unfortunately, for technical reasons, a further phenotypic characterization was not possible for the CD4-CD8- negative cell type. Other work (see below) demonstrated that LP T cells were capable of IL-2 secretion until day 21, this capacity was subsequently lost, whereas IL-4 secretion continued. From this time point onwards, the lamina propria environment was characterized by resting cells of advanced memory status, secreting IL-4 and not Il-2, possibly regulated or regulating or both. A further unusual cell type characteristic of the gut of very young pigs was a CD2+CD3- cell population, expressing uniformly high levels of CD25. The function and identity of these CD2+CD3-CD25+++ cells is as yet unknown, other work with appropriate markers (not shown) appears to preclude their identity as NK or B cells. However, their phenotype may be consistent with the existence of extra-thymic T cell development in the pig gut. In general, polyclonal T cell activation of both spleen and lamina propria cells resulted in decreased numbers of CD4+ single positive cells and increased numbers of CD4+CD8+ double positive cells, consistent with induced expression of CD8aaon activated CD4+ cells. The present study describes of antigen presenting cell populations during the first four weeks of life. The results showed that there were significant changes in the myeloid subsets with age, with a loss of CD14+ and increase in CD16+ and MHC II+. Interestingly even during this early stage there were clear interactions between CD16+MHC II+ and CD14+CD16+MHC II+ cells and MHC II+ and endothelium. These results would strongly indicate the potential for cellular cooperation within the lamina propria at this time and might highlight a possible mechanism whereby the organisation of lamina propria immune cells is established. The results have shown that there are significant differences in the cell populations found in the intestinal lamina propria of young (less that 28 days) as opposed to older piglets. The results strongly support the conclusion that at ages commonly used in European pig weaning practices, the piglets immune system may not be sufficiently developed to respond to the challenges of weaning. This would suggest that either a later weaning age should be adopted or we should seek to enhance the development of the piglets' mucosal immune system.

Mucin is the main constituent of gastrointestinal mucus and is responsible for its physico-chemical and physiological properties. Previous studies have suggested that this glycoprotein represents a major component of undigested endogenous protein at the ileum. The aim of the study was to estimate the ileal output of this glycoprotein using three methods: direct ELISA, hexosamine-based method and ethanol precipitation. For setting up the ELISA assay, the glycoprotein was isolated from intestinal mucus scraping by cesium chloride density gradient ultracentrifugation and a rabbit's hyperimmune plasma was raised against the purified glycoprotein. Ileal outputs of hexosamine and glycoprotein were measured in weaned piglets fed a control diet (C) based on casein or diets which contained 50% of crude protein supplied by white (WCP) or black (BCP) chickpea. The hexosamine output was higher (P < 0.05) with WCP diet (2.3 and 1.5g.kg-1 of dry matter intake for glucosamine and galactosamine, respectively, than with diet C (1.1 and 0.7g.kg-1 of DMI). The hexosamine-based and ethanol precipitation methods, but not the ELISA, allowed showing significant differences between diet treatments (P < 0.05). Although hexosamine-based and ethanol precipitation methods for estimating ileal glycoprotein appeared to be more satisfactory than the developed ELISA to display diet effects in this study, it remains to determine whether the higher glycoprotein data variability observed with ELISA reflects the actual biological variability of the phenomenon or not.

The aim of the experiments was to follow the development of the intestinal immune system within the period around weaning. The piglets were reared under several environments that were controlled for different parameters. The cytokine profiles and lymphocyte subsets were studied in these pigs. The most important aspect of the study was that pigs in a highly standardised experimental setup were quite homogeneous so that the analysis of cytokine expression resulted in a clear pattern beginning with 2 days before weaning til day 11 after weaning. Interestingly the significant reduction of environmental antigens in the experiments carried out in collaboration showed a smaller number of cells expressing the inflammatory and non-inflammatory cytokines, but the overall pattern of isolator pigs was comparable to that of control animals. It was also examined whether the exposition of the intestinal mucosa to comensal bacteria (Lactobacillus sobrius) or bacterial toxins had an impact on the subsets of the dendritic cells in these tissue compartments. On hour after antigen application there was no change in the intestinal morphology but after cholera toxin application the subset of SIRPalpha positive dendritic cells had increased.

Experiments were conducted in weaned piglets with or without PHA lectins from Phaseolus vulgaris beans included in the diet in various levels and periods post weaning. PHA lectins adhere to the mature cells of the villi in the digestive tract thus reducing the absorptive capacity of the villi. This increases the amount of substrate in the small intestine and induces bacterial growth, especially proliferation of E. coli. In this respect PHA lectins increase the intestinal damage as caused by the weaning process. PHA was included as a model substance to induce and standardise the intestinal damage in newly weaned piglets. The development of the digestive tract was recorded on days 0, 7 and 14 post-weaning. In general, the parameters suggest a decrease in integrity and digestive capacity during the first week post weaning and a gradual recovery of the gut in the second week post weaning. Villus height, villus/crypt ratio, enzyme activity and fat digestibility were reduced on day 7 and increased up to day 14 post weaning. The development of the microflora in this period was confirmed by an increase in the number of DGGE bands in ileal chyme and feaces and a decrease in number of E. coli bacteria. The weaning process caused a transient decrease in colonisation resistance and digestive capacity of the digestive tract, as indicated by morphological, physiological and bacterial characteristics. Partial recovery took place from day 7 to day 14 post-weaning. In our experiments, villus height, enzyme activity and fat digestibility were the most responsive parameters. Feed intake was very low in the first week post weaning and correlated to the reduced villus height and digestive capacity. A gradual increase in feed intake in week 2 to avoid bacterial overgrowth improved faecal fat digestibility and increased the number of DGGE bands, suggesting an improved colonisation resistance. The inclusion of PHA lectins reduced the villus height and tended to reduce ileal fat digestibility and increase the number of E. coli bacteria in the faeces on day 7 post weaning. Voluntary feed intake of the piglets decreased with the inclusion of PHA lectins in the diet. This decrease relative to the control diet was 5-15% in the first week and 25-35% in the second week of PHA inclusion respectively. Feed intake decreased with increasing inclusion of Phaseolus vulgaris in the diet. Furthermore, the reduction in feed intake was relatively higher when lectins were included in week 3-4 post weaning compared to inclusion in week 2-3 post weaning. Daily gain of the piglets was drastically reduced by inclusion of PHA lectins by 30-50% and 50-70% in the first and second week of PHA inclusions respectively. This decrease resembles the lower feed intake with lectins. In addition, the feed conversion ratio was drastically increased with lectins in the diet. The faecal consistency was lower with inclusion of lectins in the second week of inclusion, but not in the first week. PHA lectins were included in some treatments to induce a relatively standardised damage of the digestive tract, as previously described. This destructive effect of lectins was confirmed by a reduced villus height, fat digestibility and sucrase-isomaltase activity. However, PHA lectins in the diet also reduced voluntary feed intake compared to the control diet. This indicates that PHA lectins may have a direct effect and an effect mediated by feed intake, since a lower feed intake by itself also influences intestinal parameters. Results of a growth experiment demonstrated a reduced growth performance and feed utilisation with PHA lectin inclusion in the diet. This indicates a lower digestion of the feed because of intestinal malfunctioning due to lectin inclusion. The lower faecal consistency is in agreement with this result.

An in vivo experiment was conducted to monitor changes in fermentation end products in the faeces of weaning piglets by the inclusion of selected fermentable carbohydrates in the diet. Specially raised piglets (neither antibiotics nor creep feeding) were weaned abruptly at four weeks of age. Piglets were randomly allotted to either a control diet (CON), or a diet enriched with fermentable carbohydrates (CHO). Faecal samples were collected per rectum every day during the experimental period. The dry matter, volatile fatty acids (VFA) and ammonia concentrations were analyzed from these samples. Daily feed intake was also recorded. There was no difference in concentrations of VFA in faeces, between the treatment groups. However, the ammonia concentration was significantly lower in the faeces of piglets fed with the CHO diet compared with the CON diet, (P < 0.05). This study concluded that the inclusion of fermentable carbohydrates with different fermentabilities in weaning diets significantly reduced the protein fermentation along the GIT as demonstrated by the reduced faecal concentration of ammonia. This has important implications for the health of newly weaned piglets.

Various sole or blended carboxylic acids or their salts are postulated as biologically safer alternatives than antibiotics. These products are claimed to improve the performance of piglets through an effect on the gut microflora and gut health. However, in healthy piglets, opportunistic pathogens exist as part of the intestinal ecology, and benefits seen in terms of the performance may not be detectable. To obtain a better discrimination of their potency in vivo, various infection models are postulated. This study aimed to evaluate the biological and physiological responses to sole or blended organic acidifiers for piglets being exposed to viro-bacterial infection when kept individually and groups. In total, 48 crossbred piglets after weaning at 28 days of age were placed in individual pens and allotted to four isoproteinic and isoenergetic dietary treatments: 1) a basal diet + 40 ppm avilamycin; 2) a basal diet; 3) a basal diet + 2% lactic acid; 4) a basal diet + 0.8% blended malic acid and Mg-citrate. Major ingredients used to formulate the basal diet were barley (24%), wheat (24%) soybean meal (16%), whey (15%), and fish meal (8%), whereas the contents of CP and ileal digestible lysine were 23.3 and 1.2%, respectively. The diets were offered ad libitum (controlled daily) in a dry form. On days 4 and 5 post-weaning, a half of the piglets from each dietary treatment was infected orally with RV 277 and ETEC serotype O149K91F4ac. The response of piglets to the dietary treatments and infection was monitored in terms of: visual monitoring of clinical health status, mortality, performance (feed intake, growth rate and feed conversion ratio),faecal consistency and faecal scour index, faecal shedding of haemolytic E.coli. Besides, on day 10 post-infection venous blood indices were assayed (heamogram), and afterwards the animals were euthanized to sample intestinal mucosa from the duodenum, jejunum and ileum, pancreas, and caecal digesta. Morphological indices (villus height and crypt depth in the jejunum and ileum) and activity of intestinal/pancreatic enzymes were determined. Each piglet was an experimental unit. The data were subjected to the ANOVA analysis of variance for the randomised block design. The significance of the differences between treatments was tested with the Student t-test at P < 0.05. As anticipated, the controlled oral infection with RV and ETEC was moderate (only 1 piglet died). However, in comparison to non-infected piglets, the infected piglets had a lower feed intake (FI) and average daily gain (ADG) at a worse feed conversion ratio (FCR), irrespective of the diet. The infected piglets had a greater (P<0.001) faecal scour index and significantly increased numbers of leukocytes number (+3.3 x 103/mm3), lymphocytes (+1.07 x103/mm3), neutrophiles (+1.9 x103/mm3) and eosinophiles (+0.07 x103/mm3). Besides, the challenging resulted in a higher (P<0.05) content of mucosal protein, whereas the activities of intestinal enzymes such as duodenal and ileal aminopeptidase N and ileal maltase were reduced. Pancreatic enzyme activities were unchanged, despite we found that 62% of the infected animals excreted haemolytic E.coli serotype K88 in faeces, and the infection increased (P<0.05) faecal shedding of haemolytic E. coli.The effects of dietary treatments on the performance, functionality the digestive tract and haemogram in infected or non-infected piglets were not statistically significant, and no interaction between these treatments and infection was found. Thus this may imply that the microbial load and the metabolic demands of the gut exposed to ETEC and RV might have been predominantly modulated by endogenous SCFA liberated from fermentable carbohydrates of this specific basal diet in the stomach and small intestine in amounts far exceeding the minimum inhibitory concentrations. In summary, the RV and ETEC infection negatively affected piglet's performance and health-related parameters, irrespective the diet. Antibiotic-free diets supplemented or not with these organic acidifiers, allowed obtaining comparable values as with avilamycin during the post-infective period.

We studied gut morphology, physiology and intestinal ecology during 15 d post-weaning in piglets in order to better understand gut disorders and to define gut health criteria. We investigated digestive tract characteristics, mucosal morphology, absorptive and secretory physiology, epithelial barrier function and bacterial populations along the intestines. After a 2 day-fast, the piglets were fed intragastrically six times daily, following a scheduled feed intake plan. The piglets were pair-fed either a wheat-based or a control diet and slaughtered kinetically. Blood was collected for gastrin and CCK assays. Gut weight characteristics were recorded. Mucosa from small and large intestines was studied for absorptive, secretory and barrier function in Ussing chambers, and for morphometry and digestive enzyme activities and gene expression. Pancreas was collected for protein, trypsin and lipase activity determination. At days 0, 8 and 15, intestinal contents were collected for counting various types of bacteria. Data were subjected to analyse of variance (diet factor removed because non significant). In order to have a global view of weaning diet adaptation, 71 variables (excluding bacterial counts) measured on d 5-15 were analysed by principal component analysis. The weight of stomach and large intestine relative to body weight were not affected by fasting at weaning, contrary to that of small intestine which was lighter at d2 than at day 0. After day 2, the relative weight of gut segments and digesta contents increased rapidly. The pancreas presented high protein and enzyme contents at d2, as compared to day 0. Between day 2 and day 5, all pancreatic parameters decreased sharply, but afterwards trypsin and amylase activities were stimulated. Villous atrophy was maximal at day 2 in the duodenum and at day 5 in the ileum. Atrophy was the greatest in the duodenum and villous area was not fully recovered at d15 post-weaning. Weaning diet intake favoured an increase in ileal villous size up to values recorded at day 0. Crypt size was not changed with fasting but it increased sharply thereafter. At day 15, crypt length and surface were superior to values measured at day 0 and day 2. Expression of intestinal enzymes was highly modulated by weaning. At day2, fasting had depressed intestinal enzyme activities, but not specific mRNA. Duodenal enzyme expression (mRNA and activities) of maltase increased after re-feeding, whereas that of lactase decreased. The basal short-circuit current in jejunum and colon, and glucose absorption in jejunum, were increased 2 day after weaning, whereas a dramatic drop of tissue resistance occurred in the jejunum. These parameters had returned to pre-weaning values by day 5. Jejunal responses to secretagogues and glucose absorption were decreased the second week after weaning. Ileal transmucosal resistance was higher on day 5 and stayed stable thereafter. Fluxes of macromolecules across the jejunal mucosa dropped on day 2 and stayed at this low level thereafter. Gastrin and CCK levels were low after the fasting period and increased thereafter. In jejunal digesta, similar counts of bacteria were observed at day 0 and day 8. Afterwards, counts of total anaerobes and aerobes, enterobacteria and enterococci tended to decrease. In colonic digesta, counts of all bacterial types were higher on day 8 compared to day 0. Afterwards, they decreased between day 8 and 15, except for lactobacilli that did not change. Principal component analysis was first performed with the 71 variables and then with the 12 non-redundant and most significant variables. The first three principal components explained 70% of the variability between piglets. Component 1 discriminated those slaughtered at day 5 and day 15 and could be interpreted as the axis of time post-weaning. It correlated with variables measuring gut weight characteristics. Component 2 was correlated with variables measuring structural and functional characteristics of ileal mucosa. The global data analysis indicated that the post-weaning period could be divided into two periods. The early phase consisted in an acute deterioration of the structure and activities of the duodenal mucosa, probably in relation with fasting. By contrast, duodenal crypts and levels of mRNA coding for enzymes were not affected by fasting. At the ileum, consequences of fasting were globally less marked. Pancreatic juice secretion in the duodenum was transiently lowered while pancreas protein and enzymes were transiently increased. The late post-weaning phase (day 5-15) corresponded to an adaptation of the gut to the weaning diet. The main factors of this adaptation were the re-feeding process and time, independently of the diet composition. Maturation was the major feature of the ileum. Duodenal structural characteristics, and more generally the weight characteristics of the gut could be considered as valuable gut health criteria. Variables measuring functional activities of the ileal mucosa could be considered as gut maturation criteria.

Several alternatives to in-feed antibiotics, such as zinc oxide, glutamine, organic acids (Ca-formate, formic acid and Na-butyrate) and probiotics have been tested on two different types of co-cultures: Caco-2 cells co-cultured with human PBMC (peripheral blood mononuclear cells) and IPEC-1 cells co-cultured with lymphocytes isolated from pig mesenteric lymph nodes (MLN). The effect of all these alternatives on membrane permeability was measured. The effect of zinc oxide (ZnO) against enterotoxigenic E. coli (ETEC) K88 induced cell damage was tested by measuring the transepithelial electrical resistance (TEER) and 14C-inulin transfer from apical to basolateral compartment. A protection against ETEC induced reduction of membrane permeability was observed already with a concentration of 0.2mM ZnO. Also formic acid (25-30mM) was able to protect against ETEC, whereas butyrate (up to 8mM) was ineffective. Experiments on Caco-2 cells and human co-cultures treated with Bifidobacterium animalis and Lactobacillus casei GG showed that these probiotics did not prevent the ETEC induced permeability decrease, even with probiotic concentration 100 times higher than ETEC. Experiments performed on porcine co-cultures infected with ETEC and treated with L. amylovorus-like and L. amylovorus DSMZ gave similar results to those obtained when IPEC-1 cells were cultured alone. In summary, the effects observed on TEER of co-cultures did not differ from that observed on intestinal cells cultured without lymphocytes.

The goal of our study is to analyse the adaptability of pancreatic secretion during the first days following weaning, and to estimate the effect of food intake level. Suckling piglets are fitted with two catheters introduced into the pancreatic duct (collection of juice) in one hand and the duodenum (reintroduction of juice) in the other hand. Piglets weaned at 33 days of age are fed with a starter diet at a high (TH group) or low (TB group) food intake level. Collections of juice start the day before weaning (day 1) and continue up five days after weaning (day 1 to day 5), during both basal and post-prandial periods. Juice volume and protein and trypsin flows increase (P<0,05) after weaning. Compared to the suckling piglets, basal values measured at day 5 are increased by 7 and 47 times (P<0,05) for protein and trypsin flows respectively. Meal consumption emphasizes this effect since during the first 30 minutes following the meal at day 5 protein and trypsin quantities are 17 and 412 higher (P<0,05) than preprandial secretions. Between day 1 and day 5, specific activity of trypsin increases 14 times (P<0,05) whereas specific activity of lipase decreases 3,6 times (P<0,05). Piglets fed with a lower food intake level have a secretion of proteins and trypsin 2 times lower during the basal period compared to the TH group, and a secretion of trypsin 80 times lower during the postprandial period at day 3 (P<0,05). When animals at equal levels of feed intake (i.e. L piglets at day 5 post-weaning and H piglets at day 3 post-weaning) were compared, pancreatic trypsin flow was 45-fold lower in piglets that suffered from a 3d-period of anorexia immediately after weaning, suggesting that a low level of feed intake during the days following weaning delayed the adaptation of pancreatic function. Pancreatic function adapts at weaning with an increase of secretion and a modification of juice composition. A lower level of food intake during the first days following weaning delays adaptability of the pancreatic function. In piglets that suffered from a long period of anorexia immediately after weaning the delay in adaptation evidenced during the re-feeding period may result in reduced digestive capacity and subsequently, in a decreased digestibility in nutrients. In terms of livestock production system, these results highlight the necessity to reduce the anorexia period in order to prevent, or at least to limit, pancreatic adaptation defaults.

There is some evidence that zinc oxide (ZnO) is able to protect against intestinal diseases. Despite the suggestions of some studies that ZnO may have antibacterial activity, mechanisms of ZnO protective effect have not yet been elucidated. The potential benefits of ZnO against the damages induced by enterotoxigenic Escherichia coli (ETEC) strain K88 on Caco-2 cells and the mechanisms related have been investigated. Cell permeability, measured as transepithelial electrical resistance (TEER) and 14C-inulin transfer, was unchanged by 0.01 and 1 mM ZnO treatments and moderately increased with 5 mM ZnO, as compared to untreated cells. ETEC (1 x 108 CFU/ml) was able to induce a TEER decrease and a 14C-inulin transfer increase after 2.5 h of infection in Caco-2 cells. Treatment with ETEC together with ZnO (already with 0.2 mM) prevented membrane integrity disruption. ETEC was able to adhere to enterocytes and, to some extent, to invade the cells. ZnO reduced bacterial adhesion and blocked invasively, in a dose dependent manner. ETEC infection upregulated the expression of pro-inflammatory cytokines interleukin (IL)-8, growth-related oncogene (GRO)-a and tumour necrosis factor (TNF)-a, and reduced that of anti-inflammatory cytokine transforming growth factor-b, as compared to uninfected cells. Addition of ZnO (0.2 or 1 mM) counteracted the alterations on cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to an antibacterial activity of ZnO, since the viability of ETEC grown in ZnO containing medium was unaffected. In conclusions, ZnO may protect intestinal cells against ETEC infection by inhibiting adhesion and internalisation of bacteria, preventing increase of tight junction permeability and modulating cytokine gene expressions.

Research shows that dietary zinc oxide (ZnO) at pharmacological level improves growth and/or reduces diarrhoea in weaned pigs. However, the theories to explain ZnO action conflict with experimental data, and it is not clear if other zinc sources supplied over requirements can improve growth or health of piglet. Thus, dietary strategies to supplement zinc within the EU rules are difficulty identified. Recent findings show also that dietary glutamate reduces villus atrophy after weaning and is an important fuel for the enterocyte. We hypothesized that zinc chelated with glutamate could have a different absorption or channelling into a metabolic pool, compared to the oxide. Our goal was to study the effects of two doses of ZnO or zinc glutamate on growth performance, gut characteristics, health and immunity of weaned pigs. On day 0, 60 weaned pigs (21 d age), based on live weight and litter, were assigned to 5 dietary treatments: control (C); zinc oxide or zinc-glutamate chelate (Zn-Glu), at 200 or 2500 mg Zn/kg. On d 1, the subjects were orally challenged with 1.5 ml of a 1010 CFU/ml E. coli K88 suspension. Zinc did not improve growth performance and feed intake, whatever were the source and the level of addition. In experiments with weaning pigs in which growth was positively related to high doses of zinc oxide, the period of observation was longer. Seven days could not have been sufficient to evaluate the influence of zinc supplementation. On d 4, both high zinc supplementations reduced the total E. coli bacteria and E. coli K88 faecal excretion (P<0.05). Whatever was the source, with Zn supplementations, a trend of reduction of K88-specific IgA was observed in serum on d 4 (P=0.10), but not on d 6, and in saliva on d 6 (P=0.07), but not on d 5. The contemporary reductions of faecal excretion and IgA production suggest that zinc has a local action, and that, likely, the reduced antigenic charge decreases the recruitment of the immune response. Differences were not detected for specific IgA content in the jejunum secrete. At the sacrifice, when the secrete was sampled, the immune stimulation from E. coli K88 could have been already declining. Effects of the diet were observed on small intestinal morphology, but not equal along different tracts. In the duodenal tract, the villus height was improved by zinc supplementations (P<0.01), whereas crypt depth was not affected. This confirms that zinc can have an important role in the protection of small intestinal structure. However, in the jejunum and ileum, the morphology was not associated with the increasing zinc additions. For both zinc sources, with high doses of zinc the concentration of this mineral in liver was the triple of the values observed for the control and for the low Zn doses. These results agree with other researches, and suggest that zinc availability was not affected by chelation with glutamate. High level of zinc from zinc oxide and zinc-glutamate chelate added to weaning pig diet for short term may have an important role in resistance to infection reducing the coliforms multiplication and E. coli K88 gut colonization.

The analysis of the ban at the farming level links the input/output changes at the farm level with the restructuring of the agricultural sector, i.e., farm enlargement and diversification or specialisation of farm production. According to farm accountancy data, the sources of size economies that drives farm enlargement are labour, building and machinery, and animal feed factors of production. These sources of size economies are not deeply affected by the ban. Only veterinary services are sources of size diseconomies. However, total veterinary costs usually do not exceed 10% of feed costs. The sources of scope economies that drive on-farm association of productions are mainly land as more land per animal reduces the cost of manure management and optimises the crop fertilisation. Scope economies are enhanced by tighter enforcement of water quality regulations. However labour and labour quality is a source of scope diseconomies as diversified production requires more competences and entails organisational difficulties. Farm enlargement and farm diversification conflict because labour is a source of size economies and a source of scope diseconomies. Hence the trade-offs between size and scope economies with the AGP ban is an empirical issue. Simulations using a cost-minimising model of the farm sector on French data suggest the potential development of pig production in extensive cattle farms with grassland. This prediction is only observed in a small extend as it also depends on local contextual factors that are not controlled by the model: manure elimination facilities, manure "market", pig production acceptability. The sanitary conditions in specialised farms compared to diversified farms remain an open question. As alternatives are only imperfect substitutes of in-feed antibiotics and farms with worst breeding and sanitary conditions will have to adapt quickly or disappear.

Cytokines play a central role in immune cell response but they also participate in the maintenance of tissue integrity. At weaning in pigs, changes in cytokine network may be expected in the gut since abrupt changes in dietary and environmental factors lead to important morphological and functional adaptation of the gut. In this study 45, 28 d-old piglets were used to measure the gene expression of six inflammatory cytokines by RT-PCR along the small intestine (SI) and the proximal colon at different time points (0, 1, 2, 5 and 8 d) post-weaning. Villous/crypt architecture and enzymatic activities of lactase and sucrase in the SI were also examined. Our results confirmed that weaning is associated with morphological and enzymatic changes in the SI. In addition, our data indicated that cytokine response in the gut could be divided in two periods: an early acute response (d0 to d2 post-weaning) and a late long lasting response (d2 to d8 post-weaning). Between d0 and d2, there was an increase in the levels of IL-1b, IL-6 and TNF-a mRNA. Significant up regulation of IL-1b mRNA was observed in most parts of the intestine whereas, IL-6 and TNF-a mRNA significantly increased only at specific sites of the intestine. Between d2 to d8, the levels of IL-1b, IL-6 and TNF-a mRNA rapidly returned to pre-weaning values, except for TNF-a mRNA that remained high in the distal SI. A significant decrease of IL-12p40 and IL-18 mRNA as compared to d0 was also noted. Taken together our results demonstrate that weaning in piglets is associated with an early and transient response in gene expression of inflammatory cytokines in the gut.

The assessment of dietary strategies in weaning pigs is difficult. In commercial farms, accurate control of experimental conditions is problematic, whereas in experimental stations, hygienic standards are often not comparable to the ones of normal herds. The oral challenge with K88 E.coli has been often used in feeding trials on piglets. The predisposition to this colibacillosis depends on the presence of intestinal receptors for the fimbriae of K88 E.coli, but this susceptibility is not considered when feeding strategies are tested on challenged pigs. We elaborated records relating to 372 piglets from 8 feeding trials. All the pigs were orally stimulated with K88 E.coli. The trials started at the weaning (14 to 21 d). After a variable period of adaptation, subjects were orally challenged with 1.5 ml of a 10x10 CFU/ml O149 K88ac E. coli suspension. Data were elaborated considering the trial and the susceptibility to K88 E.coli adhesion to small intestine villi, assessed by in vitro test on jejunum samples, and their interaction. Compared with records on negative pigs, oral stimulation in positive subjects reduced growth and feed intake and increased K88 E.coli faecal excretion, diarrhoea and anti K88 E.coli specific IgA secretion. Not all these parameters where affected in all the trials. Villus height increased in 1 out of 3 trials in positive pigs. The oral challenge with K88 E.coli is a valid tool to study the effect of dietary strategies on growth, health and immunity of weaning pigs, but the susceptibility to K88 E.coli villus adhesion should be considered.

In a large-scale experiment the influence of an anti microbial growth promoter (AGP), medium chain fatty acids (MCFA) and fishoil were included in diets for weaned piglets and compared to a negative control treatment without anti microbials. The inclusion of avilamycine (40 ppm) as growth promoter significantly improved daily gain (14%) and feed conversion ratio (5%) in comparison to the negative control treatment (Table 4). This result showed the efficacy of the antimicrobial growth promoter, even under relatively good sanitary housing conditions. The growth performance data for piglets fed the MCFA diets also were significantly improved compared to the control treatment, 19% for daily gain and 6% for feed conversion ratio. The results with MCFA were not significantly different from the treatment with the antimicrobial growth promoter suggesting that MCFA is a promising feed ingredient to be used as alternative for AGP. This is most likely due to the antimicrobial properties of MCFA. In an additional treatment, the effect of fish oil was tested in a diet without AGP. Fish oil was expected to have beneficial effects because of its high content of n-3 fatty acids, which may influence the immune system. In this experiment, however, the inclusion of fish oil did not significantly improve growth performance of the newly weaned piglets. This result does not exclude a beneficial effect of fish oil on the immuno competence of the piglet, but such an effect was not detectable in this experimental design. Inclusion of medium chain fatty acids in the diet, improved growth performances to the same extend as the AGP avilamycine. Consequently, the use of medium chain fatty acids as a potential alternative for AGP rewards further elucidation.

Probiotics are fundamental constituents of intestinal microflora, which may provide protection against inflammatory disease induced by pathogens. However, the mechanisms underlying their activities are still largely unknown. It has been investigated whether Bifidobacterium animalis and Lactobacillus casei GG protected the human intestinal Caco-2 cells from the infection of enterotoxigenic Escherichia coli (ETEC) strain K88. Both these probiotics were able to affect the ETEC adhesion. Moreover, they inhibit the strong neutrophil transmigration caused by ETEC infection. The decrease of neutrophil migration was associated with an inhibition of the ETEC induced up-regulation of chemoattractant cytokines interleukin (IL)-8, growth-related oncogene (GRO)-a and epithelial neutrophil-activating peptide (ENA)-78. Moreover, B. animalis and L. casei GG counteracted the increase of the pro-inflammatory cytokines IL-1b, interferon (IFN)-g and tumour necrosis factor (TNF)-a, and the decrease of the anti-inflammatory transforming growth factor (TGF)-b, that were caused by ETEC infection. The benefits were not ascribable to a bactericidal activity of probiotics, since the viability of ETEC was unaffected by growing the pathogen in medium containing B. animalis or L. casei GG. These results indicate that B. animalis and L. casei GG were able to protect the cells against ETEC infection by various mechanisms.

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