Encapsulated benzoic acid supplementation in broiler diets influences gut bacterial composition and activity.

1. The present study investigated the effects of encapsulated benzoic acid (BA) supplementation in broiler feed on performance and gastrointestinal microbiota. 2. Eighty broilers were randomly divided into two groups. Birds in the control group were fed on maize-soybean-based diets. Birds in the treatment group were provided the same diet supplemented with 2 g/kg BA encapsulated in a vegetable oil matrix. 3. At the end of the trial (d 35), pH, bacterial composition and metabolites were determined in the crop, jejunum, ileum and caecum. 4. Growth performance variables and pH were not significantly different. 5. BA concentration decreased rapidly in the proximal gut. However, the treatment diet showed higher BA in the crop, jejunum, ileum and caecum. 6. Total lactate in the crop and D-lactate in the jejunum was higher in the BA treated group. Caecal total and branched chain fatty acids were decreased due to the treatment. 7. Lactobacilli populations were significantly altered by BA supplementation. A trend for increased lactobacilli was observed in the crop, while it became significant in the jejunum and ileum. Lactobacillus species responded differently to the treatment. Four of 5 measured Lactobacillus species, particularly in the ileum, followed the course observed for total lactobacilli; only Lactobacillus salivarius was not modified. 8. Correlation analysis showed that BA modified the intestinal microbiota. Lactobacilli correlated negatively to all studied clostridial clusters and enterobacteria. Clostridial clusters IV and XIVa were significantly increased in the jejunum, whereas only clostridial cluster XIVa was increased in the caecum. 9. Encapsulated BA modified the intestinal microbiota which can lead to the conclusion, that the main beneficial mode of action of BA in the gut appears to be the enhancement of lactic acid bacteria, which in turn may act as a vanguard against pathogens.

The effects of different thermal treatments and organic acid levels in feed on microbial composition and activity in gastrointestinal tract of broilers.

Thermal treatments of feed and organic acids are known to affect the gastrointestinal microbiota in chickens. The present study evaluated the effect of different thermal processes including pelleting (P), long-term conditioning at 85°C for 3 min (L), expanding at 110°C (E110), and 130°C for 3 to 5 s (E130) as well as organic acid (63.75% formic acid, 25.00% propionic acid, and 11.25% water) inclusion levels (0, 0.75, and 1.5%) on gastrointestinal microbiota in broilers. In total, 960 one-day-old chicks were randomly assigned to 8 replicates using a 3 × 4 factorial arrangement. At d 35, bacterial cell numbers in the crop, ileum, and cecum, and bacterial metabolites in the crop, gizzard, ileum, and cecum were determined. The inclusion of 1.5% organic acids increased cell numbers of all clostridial clusters in the crop. The organic acid supplementation increased the propionic acid concentration in the crop and gizzard and there was a decrease in lactic acid concentration. In the ileum, the 0% organic acid group had the highest numbers of Lactobacillus spp. and enterobacteria. Inclusion of 1.5% organic acids increased ileal acetate concentration. Increasing the feed processing temperature led to an increase of lactobacilli in the crop and ileum, whereas clostridia and enterobacteria seemed unaffected. Similarly, lactate concentrations increased in the ileum, but short-chain fatty acids remained identical. In the crop, an increase for acetate was found for the E130 group compared with all other thermal treatments. In conclusion, our study demonstrated that thermal treatments and organic acid supplementation to broiler diets more markedly influenced the bacterial status of the crop compared with the downstream segments and their effects decreased along the length of gastrointestinal tract. Whereas organic acids markedly modified bacterial composition and activity in the crop, expansion increased lactobacilli and lactate in the crop and ileum.

Dietary inclusion of feathers affects intestinal microbiota and microbial metabolites in growing Leghorn-type chickens.

Feather pecking in laying hens is a serious behavioral problem that is often associated with feather eating. The intake of feathers may influence the gut microbiota and its metabolism. The aim of this study was to determine the effect of 2 different diets, with or without 5% ground feathers, on the gut microbiota and the resulting microbial fermentation products and to identify keratin-degrading bacteria in chicken digesta. One-day-old Lohmann-Selected Leghorn chicks were divided into 3 feeding groups: group A (control), B (5% ground feathers in the diet), and C, in which the control diet was fed until wk 12 and then switched to the 5% feather diet to study the effect of time of first feather ingestion. The gut microbiota was analyzed by cultivation and denaturing gradient gel electrophoresis of ileum and cecum digesta. Short-chain fatty acids, ammonia, and lactate concentrations were measured as microbial metabolites. The concentration of keratinolytic bacteria increased after feather ingestion in the ileum (P < 0.001) and cecum (P = 0.033). Bacterial species that hydrolyzed keratin were identified as Enterococcus faecium, Lactobacillus crispatus, Lactobacillus reuteri-like species (97% sequence homology), and Lactobacillus salivarius-like species (97% sequence homology). Molecular analysis of cecal DNA extracts showed that the feather diet lowered the bacterial diversity indicated by a reduced richness (P < 0.001) and shannon (P = 0.012) index. The pattern of microbial metabolites indicated some changes, especially in the cecum. This study showed that feather intake induced an adaptation of the intestinal microbiota in chickens. It remains unclear to what extent the changed metabolism of the microbiota reflects the feather intake and could have an effect on the behavior of the hens.

Dose-dependent effects of dietary zinc oxide on bacterial communities and metabolic profiles in the ileum of weaned pigs.

Pharmacological levels of zinc oxide (ZnO) can improve the health of weaning piglets and influence the intestinal microbiota. This experiment aimed at studying the dose-response effect of five dietary concentrations of ZnO on small intestinal bacteria and metabolite profiles. Fifteen piglets, weaned at 25 ± 1 days of age, were allocated into five groups according to body weight and litter. Diets were formulated to contain 50 (basal diet), 150, 250, 1000 and 2500 mg zinc/kg by adding analytical-grade (>98% purity) ZnO to the basal diet and fed ad libitum for 14 days after a 7-day adaptation period on the basal diet. Ileal bacterial community profiles were analysed by denaturing gradient gel electrophoresis and selected bacterial groups quantified by real-time PCR. Concentrations of ileal volatile fatty acids (VFA), D- and L-lactate and ammonia were determined. Species richness, Shannon diversity and evenness were significantly higher at high ZnO levels. Quantitative PCR revealed lowest total bacterial counts in the 50 mg/kg group. Increasing ZnO levels led to an increase (p = 0.017) in enterobacteria from log 4.0 cfu/g digesta (50 mg/kg) to log 6.7 cfu/g digesta (2500 mg/kg). Lactic acid bacteria were not influenced (p = 0.687) and clostridial cluster XIVa declined (p = 0.035) at highest ZnO level. Concentration of total, D- and L-lactate and propionate was not affected (p = 0.736, p = 0.290 and p = 0.630), but concentrations of ileal total VFA, acetate and butyrate increased markedly from 50 to 150 mg/kg and decreased with further increasing zinc levels and reached low levels again at 2500 mg/kg (p = 0.048, p = 0.048 and p = 0.097). Ammonia decreased (p < 0.006) with increasing dietary ZnO level. In conclusion, increasing levels of dietary ZnO had strong and dose-dependent effects on ileal bacterial community composition and activity, suggesting taxonomic variation in metabolic response to ZnO.

Dietary fiber and its role in performance, welfare, and health of pigs.

Dietary fiber (DF) is receiving increasing attention, and its importance in pig nutrition is now acknowledged. Although DF for pigs was frowned upon for a long time because of reductions in energy intake and digestibility of other nutrients, it has become clear that feeding DF to pigs can affect their well-being and health. This review aims to summarize the state of knowledge of studies on DF in pigs, with an emphasis on the underlying mode of action, by considering research using DF in sows as well as suckling and weaned piglets, and fattening pigs. These studies indicate that DF can benefit the digestive tracts and the health of pigs, if certain conditions or restrictions are considered, such as concentration in the feed and fermentability. Besides the chemical composition and the impact on energy and nutrient digestibility, it is also necessary to evaluate the possible physical and physiologic effects on intestinal function and intestinal microbiota, to better understand the relation of DF to animal health and welfare. Future research should be designed to provide a better mechanistic understanding of the physiologic effects of DF in pigs.

Novel ex vivo screening assay to preselect farm specific pre- and probiotics in pigs.

A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.

Bar-coded pyrosequencing of 16S rRNA gene amplicons reveals changes in ileal porcine bacterial communities due to high dietary zinc intake.

Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.

Effect of a "diluted" diet containing 10% lignocellulose on the gastrointestinal tract, intestinal microbiota, and excreta characteristics of dual purpose laying hens.

Low performing dual purpose hens have different nutritional requirements compared to conventional hybrid hens. Lignocellulose is a low fermentable polymer, acting as a diet diluent and may influence physiological and digestive processes. This study investigated the effect of a 10% dietary lignocellulose dilution on the development of gastrointestinal organs, intestinal morphology, intestinal microbiota, and excreta characteristics of dual purpose hens. One-day-old female Lohmann Dual chicks were allocated to 12 pens and fed two different diets: A standard control diet (CON) and a treatment diet (LC), based on CON but diluted with 10% lignocellulose (ARBOCEL®). At 52 wk of age, gastrointestinal organs were extracted and weights determined. Colorectal tissue samples were chemically fixed and stained for histomorphological examinations. Cecal digesta samples were analyzed for bacterial metabolites and composition using gas chromatography, HPLC, photometry, and PCR. Excreta dry matter and viscosity was consistently assessed during the trial. LC-fed hens showed increased weights of the gizzard (P = 0.003), small (P < 0.001), and large intestine (P = 0.048) compared to hens fed CON. LC-fed hens had a larger colorectal villus area (P = 0.049), a higher mucosal enlargement factor of villi (P = 0.016) and crypts (P = 0.030) than CON-fed hens. The concentration of short-chain fatty acids (SCFAs) (P = 0.017) and ammonia (P = 0.013) was higher in CON-fed hens compared to LC-fed hens. Bacterial composition and activity was generally not affected by feeding the different diets. LC-fed hens had a higher excreta dry matter content than hens fed CON at 10 (P < 0.001), 17 (P < 0.001), and 22 (P = 0.002) wk of age. Correlation analyses revealed a negative relationship between the concentration of SCFAs in the cecum and the colorectal villus surface area (P < 0.01). In conclusion, the feeding of high levels of lignocellulose increased gastrointestinal organ weights and colorectal surface area in dual purpose laying hens. A higher intestinal surface area in combination with lower concentrations of SCFAs might indicate a compensatory reaction of hens fed LC enhancing the absorption of bacterial metabolites by increasing the intestinal mucosal surface.

Effect of feeding different levels of lignocellulose on performance, nutrient digestibility, excreta dry matter, and intestinal microbiota in slow growing broilers.

Lignocellulose is a constituent of plant cell walls and might be used as a fiber source in poultry nutrition. The current study investigated the impact of increasing dietary levels of lignocellulose on performance, nutrient digestibility, excreta DM, intestinal microbiota, and bacterial metabolites in slow growing broilers. At an age of 10 wk, 60 male broilers of an intercross line (New Hampshire × White Leghorn) were allocated to cages and fed isoenergetic and isonitrogenous diets containing 0.8% (LC1), 5% (LC2), or 10% (LC3) lignocellulose. After 23 D of feeding, broilers were killed and digesta samples of ileum and excreta analyzed for nutrient digestibility and DM. Cecal contents were analyzed for microbial composition and metabolites. Broiler performance was not affected by feeding dietary lignocellulose. LC3 fed broilers showed reduced ileal digestibility of protein compared to chickens fed LC1 (P = 0.003). Moreover, increasing levels of dietary lignocellulose reduced apparent digestibility of organic matter and gross energy (P < 0.001). Feeding of lignocellulose had no impact on the excreta DM of broilers. Increasing levels of dietary lignocellulose lowered cecal counts of Escherichia/Hafnia/Shigella (P = 0.029) and reduced the total concentration of short-chain fatty acids (P < 0.001), lactate (P < 0.05), and ammonia (P = 0.009). The molar ratio of cecal acetic acid was higher in LC3 fed broilers (P < 0.001), while the proportions of cecal propionic acid and n-butyric acid were higher in LC1 and LC2 fed chickens (P < 0.001). Correlation analyses indicated that dietary lignocellulose was negatively related to the total concentration of cecal bacterial metabolites (P < 0.001). In conclusion, the feeding of lignocellulose did not affect growth performance, but impaired nutrient digestibility of slow growing broilers. While minor changes in cecal microbial composition were detected, cecal bacterial metabolite concentrations were significantly reduced with increasing levels of dietary lignocellulose. These findings suggest that lignocellulose is not extensively degraded by bacteria residing in the large intestine of broilers.

Bacillus subtilis in broiler diets with different levels of energy and protein.

The present study evaluated the impacts of Bacillus subtilis (BAS) inclusion in broiler diets with standard nutrient content or nutrient deficiency (ND) on growth performance (GP) and nutrient digestibility. The 42 d experiment consisted of 6 experimental diets, a diet with standard nutrient content, and 2 diets with different levels of energy and protein deficiency, without or with BAS. At the end of experiment, apparent ileal digestibility coefficients (AIDC) of starch, crude protein (CP), and gross energy (GE) were determined. Furthermore, impacts of BAS supplementation in standard diets on gut histomorphology, bacterial metabolic activity, and composition were evaluated. Performance and AIDC data were subjected to ANOVA using GLM procedure with a 3 (nutrient levels) × 2 (BAS presence/absences) factorial arrangement of treatments. Gut histomorphology and microbiology data, obtained from broilers fed standard diets without (S) and with BAS (SB), were assessed by an independent Student's t-test. The ND in diets was effective enough to cause nutritional stress and negatively affect performance. Inclusion of BAS in both types of diet improved GP, which was due to the fact that adding BAS in these diets led to improvements in AIDC of CP, starch, and GE (P ≤ 0.05). Comparing only 2 experimental groups, S and SB, revealed no impact on bacterial composition and metabolism in the ileum and cecum, except a reduction in ileal lactobacilli number for SB group. Adding BAS to standard diet reduced crypt depth (CD) and increased villus length to CD ratio in the duodenum, whereas it had no impact on other histomorphological variables in the duodenum, jejunum, and ileum. In conclusion, supplementation of broiler diets with probiotic BAS can positively affect growth performance and nutrient digestibility and this positive impact might even be more pronounced in nutrient-deficient diets. However, the extent of the alleviating ability of BAS in nutrient-deficient diets as well as the biological mechanisms for such a phenomenon needs to be studied further.

Influence of lignocellulose and low or high levels of sugar beet pulp on nutrient digestibility and the fecal microbiota in dogs.

Lignocellulose is an alternative fiber source for dogs; however, it has not yet been studied as a feed ingredient for the nutrition of dogs. Eight adult Beagles were involved in the study, which consisted of 3 feeding periods of 8 to 12 wk each. All dogs received 3 different diets, which either had the same concentration of fiber sources (2.7% sugar beet pulp or lignocellulose) or were formulated for a similar concentration of approximately 3% crude fiber: 12% sugar beet pulp (highSBP; 3.1% crude fiber), 2.7% sugar beet pulp (lowSBP; 0.96% crude fiber), or 2.7% lignocellulose (LC; 2.4% crude fiber). Feces samples were collected at the end of each feeding period, and the apparent nutrient digestibility, daily amount, and DM content of feces and fecal cell numbers of relevant bacteria were analyzed. The daily feces amount was lower and the feces DM was higher when dogs were fed the LC diet and the lowSBP diet compared with the highSBP diet ( < 0.001). Apparent digestibility of CP, Na, and K was highest with the lowSBP diet followed by the LC and highSBP diets ( < 0.001). After feeding LC, the bacterial cell counts of spp., spp., and the cluster were reduced compared with feeding highSBP and even more reduced after feeding lowSBP ( < 0.001). The bacterial cell count of the cluster was lower in LC and lowSBP compared with highSBP ( < 0.001). The feces of dogs fed LC and lowSBP had lower concentrations of acetate ( < 0.001), propionate ( < 0.001), -butyrate ( = 0.015), total fatty acids ( < 0.001), and lactate ( < 0.001) compared with dogs fed highSBP. The concentration of -butyrate was higher in the feces of dogs fed with LC compared with dogs fed high and low sugar beet pulp (SBP; < 0.001). The pH of the feces of the LC-fed dogs was highest followed by lowSBP- and highSBP-fed dogs ( < 0.001). Depending on the concentration, the use of LC and SBP as fiber sources in dog feed has different impacts on the fecal microbiota and the apparent digestibility of nutrients. Therefore, different areas of application should be considered.

Protective effects of indigenous Escherichia coli against a pathogenic E. coli challenge strain in pigs.

To investigate the inhibitory effect of indigenous enterobacteria on pathogenic Escherichia coli, a challenge trial with postweaning pigs was conducted. A pathogenic E. coli strain was administered to all animals and their health was closely monitored thereafter. Faecal samples were taken from three healthy and three diarrhoeic animals. Samples were cultivated on MacConkey agar and isolates were subcultured. A soft agar overlay assay was used to determine the inhibitory activity of the isolates. A total of 1,173 enterobacterial isolates were screened for their ability to inhibit the E. coli challenge strain. Colony forming units of enterobacteria on MacConkey agar were not different between healthy and diarrhoeic animals in the original samples. Furthermore, numbers of isolates per animal were also not significantly different between healthy (482 isolates) and diarrhoeic animals (691 isolates). A total of 43 isolates (3.7%) with inhibitory activity against the pathogenic E. coli challenge strain were detected. All inhibitory isolates were identified as E. coli via MALDI-TOF. The isolates belonged to the phylotypes A, C and E. Many isolates (67.4%) were commensal E. coli without relevant porcine pathogenic factors, but toxin- and fimbrial genes (stx2e, fae, estIb, elt1a, fas, fan) were detected in 14 inhibitory isolates. Healthy animals showed significantly (P=0.003) more inhibitory isolates (36 of 482 isolates; 7.5%) than diseased animals (7 of 691 isolates; 1.0%). There were no significant correlations regarding phylotype or pathogenic factors between healthy and diseased animals. This study has shown that a small proportion of indigenous E. coli is able to inhibit in vitro growth of a pathogenic E. coli strain in pigs. Furthermore, healthy animals possess significantly more inhibitory E. coli strains than diarrhoeic animals. The inhibition of pathogenic E. coli by specific indigenous E. coli strains may be an underlying principle for the containment of pathogenic E. coli in pigs.

Efficacy of a single oral dose of a live bivalent E. coli vaccine against post-weaning diarrhea due to F4 and F18-positive enterotoxigenic E. coli.

F4- and F18-positive enterotoxigenic E. coli strains (F4-ETEC and F18-ETEC) are important causes of post-weaning diarrhea (PWD) in pigs. F4 (antigenic variant ac) and F18 (ab and ac) fimbriae are major antigens that play an important role in the early stages of infection. Herein, the efficacy of a live oral vaccine consisting of two non-pathogenic E. coli strains, one F4ac- and one F18ac-positive, was evaluated using F4ac-ETEC and F18ab-ETEC challenge models. A randomized, masked, placebo-controlled, block design, parallel-group confirmatory study with two different vaccination-challenge intervals (7 and 21 days) was conducted for each challenge model. The vaccine was administered in one dose, to ≥18-day-old piglets via drinking water. Efficacy was assessed by evaluating diarrhea, clinical observations, weight gain and fecal shedding of F4-ETEC or F18-ETEC. Anti-F4 and anti-F18 immunoglobulins in blood were measured. The vaccination resulted in significant reductions in clinical PWD and fecal shedding of F4-ETEC and F18-ETEC after the 7- and 21-day-post-vaccination heterologous challenges, except for after the 21-day-post-vaccination F4-ETEC challenge, when the clinical PWD was too mild to demonstrate efficacy. A significant reduction of mortality and weight loss by vaccination were observed following the F18-ETEC challenge. The 7-day protection was associated with induction of anti-F4 and anti-F18 IgM, whereas the 21-day protection was mainly associated with anti-F4 and anti-F18 IgA. The 7-day onset and 21-day duration of protection induced by this vaccine administered once in drinking water to pigs of at least 18days of age were confirmed by protection against F4-ETEC and F18-ETEC, and induction of F4 and F18-specific immunity. Cross protection of the vaccine against F18ab-E. coli was demonstrated for both the 7- and 21-day F18-ETEC challenges.

Intestinal lactose and mineral concentration affect the microbial ecophysiology along the gastrointestinal tract of formula-fed neonatal piglets.

Hyperprolificacy in modern pig breeds has led to increased use of artificial rearing and formula feeding of neonatal piglets, which may change their intestinal bacterial ecophysiology. Here, newborn piglets ( = 8 per group) were fed a bovine milk-based formula (FO) or allowed to suckle their mothers (sow milk [SM]) for 2 wk, and digesta samples from the stomach, jejunum, and colon were subsequently analyzed for enzyme activities, bacterial metabolites, and 16S rRNA transcripts of bacterial groups by quantitative real-time PCR. Jejunal lactase activity was lower and lactose concentration was greater in the jejunum and colon in the FO group compared with the SM group ( < 0.05). In the stomach, FO-fed pigs had a lower copy number of 16S rRNA transcripts for all analyzed bacterial groups ( < 0.05) except for the // group. In the jejunum, 16S rRNA transcripts of lactic acid bacteria and clostridial cluster I were lower ( < 0.05) in FO-fed pigs. In turn, transcript abundance of the group and clostridial cluster I was greater in FO-fed pigs in the colon ( < 0.05). In FO-fed piglets, concentrations of and lactate and total and individual short-chain fatty acids were higher in the colon ( < 0.05). Multivariate redundancy analysis revealed that the concentration of minerals (ash, Ca, Mg, K, Na, Mn, and Zn) were associated with reduced bacterial abundance and activity in the upper gastrointestinal tract, whereas lactose had the most pronounced effect on the colon microbiota. The present study revealed that, apart from lactose, the mineral concentration modifies the microbial communities in the gastrointestinal tract of FO-fed piglets.

Effect of different feed ingredients and additives on IPEC-J2 cells challenged with an enterotoxigenic Escherichia coli strain.

The intestinal porcine epithelial cell line IPEC-J2 was used as an in vitro model to assess effects of additives on the adhesion and cell toxic effects of a F4-positive (ETEC) and a F4-negative Escherichia coli (DSM 2840) strain. Bacterial adhesion was examined using flow cytometry in IPEC-J2 cells infected with bacteria stained with 5,6-carboxymethyl fluorescein diacetate succinimidyl ester. Measurement of transepithelial electrical resistance (TEER) was performed to characterize the impact on IPEC-J2 monolayer integrity. The feed additives were prepared as aqueous extract and tested in different dilutions and incubation times. The F4-positive ETEC strain had a high adhesion to IPEC-J2 cells and reduced TEER shortly after the in vitro infection. The nonpathogenic E. coli strain DSM 2840 showed only low adhesion capacity and no TEER impairment. Infection with ETEC with added test extracts showed a reduction of bacterial adhesion to IPEC-J2 cells by an autolyzed yeast product (p < 0.05). Bovine colostrum, an additive containing thyme extract and an organic acid mix did not interfere with the ETEC adherence. The TEER decrease of the IPEC-J2 monolayer after ETEC infection was not affected by the added substances. In conclusion, interference with epithelial adhesion might be a protective mechanism of the tested yeast extract, indicating that the cell culture model might be suitable as screening tool to complement in vivo challenge trials with piglets.

Effects of medium-chain fatty acids on the structure and immune response of IPEC-J2 cells.

Medium-chain fatty acids (MCFAs) have been suggested as an alternative to the use of antibiotics in animal nutrition with promising results. First, we studied the sensitivity of Salmonella Enteritidis and an enteropathogenic Escherichia coli strain against caprylic (C8), capric (C10) and lauric (C12) acids. A porcine in vitro model using the porcine cell line IPEC-J2 was used to test the effects of MCFAs on structural and immunological traits without and with a concomitant challenge with E. coli or S. Enteritidis. The three MCFAs exerted an inhibitory effect on bacterial growth, stronger for C12 than C8 or C10, S. Enteritidis being more sensitive than the E. coli strain. Flow cytometry showed a numeric concentration dependent increase in the adhesion of E. coli or S. Enteritidis to IPEC-J2 cells. Measurement of transepithelial electrical resistance after bacterial challenge showed negative effects of all MCFAs on IPEC-J2 cells at the highest concentrations. Immune parameters were affected by C8, since a concentration dependent effect starting at 5 mM was observed for mRNA expression of IL-6 and TLR-4 (up-regulated) and IL-8 (down-regulated). TLR-4 was up-regulated with C10 at 2 and 5 mM. The three MCFAs affected also the epithelial morphology through down-regulation of Occludin and up-regulation of Claudin-4 expression. In conclusion, the three MCFAs under study influenced bacterial growth rates and modified the gene expression to a different degree in the cell line IPEC-J2 but the effect on the morphological structure and response of the cells after bacterial challenge could not be assessed. Although these tests show a prior estimation of MCFAs effects in intestinal epithelium, in vivo confirmation is still needed.

Bovine milk-based formula leads to early maturation-like morphological, immunological, and functional changes in the jejunum of neonatal piglets.

Artificial rearing and formula feeding is coming more into the focus due to increasing litter sizes and limited nursing capacity of sows. The formula composition is important to effectively support the development of the gut and prevent intestinal dysfunction in neonatal piglets. In this study, newborn piglets ( = 8 per group) were fed a bovine milk-based formula (FO), containing skimmed milk and whey as the sole protein and carbohydrate sources, or were suckled by the sow (sow milk [SM]). After 2 wk, tissue from the jejunum was analyzed for structural (i.e., morphometry) and functional (i.e., disaccharidase activity, glucose transport, permeability toward macromolecules, and immune cell presence) changes and concomitant expression of related genes. Formula-fed piglets had more liquid feces ( < 0.05) over the entire experimental period. Although FO contained twice as much lactose (46% on a DM basis) as SM (21%) and no maltose or starch, the lactase activity was lower ( < 0.05) and glucose transport capacity was higher ( < 0.05) in FO-fed pigs. The relative proportion of intraepithelial natural killer cells and proinflammatory cytokine gene expression (, , and ) was higher in FO-fed pigs ( < 0.05). Piglets fed FO had deeper crypts, larger villus area, and higher expression of caspase 3 and proliferating cell nuclear antigen ( < 0.05). Epithelial permeability toward fluorescein isothiocyanate-dextran was higher and expression of claudin-4 was lower in FO-fed piglets ( < 0.05). The data suggest an early response to bovine milk-based compounds in the FO accompanied with early onset of functional maturation and impaired barrier function. Whether lactose, absence of species-specific protective factors, or antigenicity of foreign proteins lead to to the observed intestinal reactions requires further clarification.

Effects of the probiotic Enterococcus faecium NCIMB 10415 on selected lactic acid bacteria and enterobacteria in co-culture.

Enterococcus faecium NCIMB 10415 is used as a probiotic for piglets and has been shown to modify the porcine intestinal microbiota. However, the mode of action of this probiotic modification is still unclear. One possible explanation is the direct growth inhibiting or stimulating effect of the probiotic on other indigenous bacteria. Therefore, the aim of the present study was to examine the growth interactions of the probiotic with different indigenous porcine bacteria in vitro. Reference strains were cultivated with the probiotic E. faecium strain NCIMB10415 (SF68) in a checkerboard assay with 102 to 105 cells/ml inoculum per strain. Growth kinetics were recorded for 8 h and used to determine specific growth of the co-cultures. Additionally, total DNA was extracted from the co-cultures at the end of the incubation to verify which strain in the co-culture was affected. Co-cultivation with eight Enterococcus spp. tester strains showed strain-specific growth differences. Three of four E. faecium strains were not influenced by the probiotic strain. PCR results showed reduced growth of the probiotic strain in co-culture with E. faecium DSM 6177. Three of four Enterococcus faecalis strains showed reduced specific growth in co-culture with the probiotic strain. However, E. faecalis DSM 20478 impaired growth of the probiotic E. faecium strain. The growth of Lactobacillus johnsonii DSM 10533 and Lactobacillus reuteri DSM 20016 was enhanced in co-culture with the probiotic strain, but co-cultivations with Lactobacillus mucosae DSM13345 or Lactobacillus amylovorus DSM10533 showed no differences. Co-cultures with the probiotic E. faecium showed no impact on the growth rate of four different enterobacterial reference strains (2 strains of Salmonella enterica and 2 strains of Escherichia coli), but PCR results showed reduced cell numbers for a pathogenic E. coli isolate at higher concentration of the probiotic strain. As the in vitro effect of the probiotic E. faecium on enterococci was strain specific and the growth of certain Lactobacillus spp. was enhanced by the probiotic, these results indicate a direct effect of the probiotic on certain members of the porcine gastro intestinal microbiota.

Effects of dietary fat type and xylanase supplementation to rye-based broiler diets on selected bacterial groups adhering to the intestinal epithelium. on transit time of feed, and on nutrient digestibility.

Two experiments were conducted to examine the effects of different fat types, i.e., soybean oil (S) and beef tallow (T), in rye-based broiler diets, either unsupplemented (-) or supplemented (+) with xylanase (Avizyme 1300 at 1 g/kg diet), on selected bacterial groups adhering to the epithelium of crop, duodenum, jejunum, and ileum (Experiment 1, 16 d of age), on mean retention time (MRT) of digesta, and on digestibility of N and dry matter in successive segments of the digestive tract (Experiment 2, 24 d of age). Live weight of enzyme-treated and S-fed chickens was significantly higher than that for unsupplemented or T-fed birds, respectively, in both experiments. In Experiment 1, a reduction in bacterial colonization from crop to duodenum was followed by a continuous increase as far as the ileum. Xylanase supplementation significantly reduced enterobacteria and total anaerobe microbes with a similar trend for Gram-positive cocci and enterococci. The latter two groups were significantly enhanced in birds fed T. In Experiment 2, xylanase supplementation resulted in a decrease in MRT in several segments of the digestive tract. This effect was most pronounced in the small intestine, where MRT of 268, 217, 241, and 209 min in groups S-, S+, T-, and T+, respectively, were measured. Apparent digestibility of N and dry matter was slightly improved by xylanase supplementation in the jejunum and ileum. Nitrogen digestibility by the terminal ileum was 80.3, 83.7, 79.4, and 82.2% for the S-, S+, T-, and T+ groups, respectively, and dry matter digestibility amounted to 61.2, 65.5, 62.1, and 64.0%, respectively.

Effect of dietary zinc oxide on jejunal morphological and immunological characteristics in weaned piglets.

The aim of this study was to evaluate age-related changes and the effect of dietary Zn concentration on morphological and immunological characteristics in the gastrointestinal tract of piglets. A total of 96 purebred Landrace piglets were weaned at the age of 26 ± 1 d, and randomly allocated into 3 treatment groups fed with low (57 mg Zn/kg), medium (164 mg Zn/kg), and high (2425 mg Zn/kg) dietary Zn (ZnO). Piglets (4 males and 4 females per treatment group) were killed at 33 ± 1, 40 ± 1, 47 ± 1, and 54 ± 1 d of age. In the jejunum, villus height, crypt depth, and the number of goblet cells producing neutral, acidic, sulfated, and sialylated mucins were measured. Intraepithelial lymphocytes were characterized by flow cytometry and the gene expression of mucin 2 (MUC2), mucin 20 (MUC20), ß-defensin 3, and trefoil factor 3 (TFF3) was determined by reverse transcription quantitative PCR. Villus height and crypt depth in the jejunum showed age related differences (P < 0.01), whereas the dietary concentrations of Zn had no effect. The mucin types were modified mainly by age, and dietary Zn had no effect in the proximal jejunum. In the distal jejunum, age and Zn had effects on the mucin types. The abundance of sulfomucins decreased (P < 0.001) and sialomucins increased with age (P < 0.001), while high dietary ZnO reduced the sulfomucins (P < 0.001) and increased the sialomucins (P < 0.05) in the crypts. The phenotypes of lymphocytes in the epithelium of the proximal jejunum showed relatively constant percentages of T-cells, as well as natural killer cells. High dietary Zn treatment led to a reduced abundance of CD8(+) γδ T-cells (P < 0.05). The apportionment of different cytotoxic T-cell was age dependent. Although the percentage of CD4(-)CD8ß(+) increased (P < 0.01), the relative amount of CD4(+)CD8ß(+) decreased with age (P < 0.05). The expression of MUC2 and MUC20 was not influenced by age or dietary Zn concentration. High Zn intakes resulted in a reduced gene expression of ß-defensin 3 (P < 0.05), but did not affect the expression of TFF3. It is concluded that Zn in the form of ZnO appears to have specific effects on the innate and adaptive gut associated immune system of piglets. These might contribute to the positive effects of Zn on the prevention of postweaning diarrhea.