Fiber Composition in Sows' Diets Modifies Clostridioides difficile Colonization in Their Offspring.

Dietary fiber has a potential to modulate the gut microbiota in sows. We hypothesized that a maternal diet rich in either high- or low-fermentable fiber during gestation and lactation influences Clostridioides difficile gut colonization in suckling piglets. Twenty sows were fed gestation and lactation diets enriched with either high-fermentable sugar beet pulp (SBP) or low-fermentable lignocellulose (LNC) fibers. C. difficile, toxin B (TcdB), fecal score, microbial abundance (16S-rDNA sequencing) and metabolites were measured in the feces from the sows and their piglets. C. difficile concentration was higher in piglets from the sows fed LNC than SBP along the study (P ≤ 0.05). Higher prevalence of C. difficile was noted in three-week-old piglets from sows fed LNC vs. SBP (45% vs. 0%, P = 0.001). TcdB prevalence was higher in six-day-old piglets from the sows fed LNC vs. SBP (60% vs. 17%, P = 0.009). In sows, fecal microbial metabolites were higher in SBP than LNC, while C. difficile concentration showed no difference. Higher microbial diversity Shannon index was noted in sows from SBP vs. LNC one week before parturition and at the parturition (P ≤ 0.05). Piglets from SBP vs. LNC tended to have higher microbial diversity Shannon index at two and three weeks of age. Diets enriched with high-fermentable fiber compared to low-fermentable fiber in sows reduced C. difficile colonization in their piglets. Susceptibility to colonization by C. difficile in neonatal piglets can be modulated by the sows' diet, supporting the hypothesis of the early microbial programming in the offspring and the importance of the sow-piglet couple.

Performance, health, bacterial metabolites and intestinal histomorphology in does and growing rabbits fed diets with increasing lignocellulose-to-cellulose proportions.

Adequate levels of dietary fibre can reduce the risk of intestinal health disorders in rabbits after weaning. Therefore, it was the aim of the study to investigate the impact of different lignocellulose and cellulose addition to rabbits' diets on performance and intestinal traits in lactating does and weaned rabbits. A total of 60 rabbit does (4.41 ± 0.45 kg body weight) were fed isonitrogenous diets with lignocellulose and cellulose inclusion rates [%] of 0/8, 2/6, 4/4, 6/2 and 8/0, resulting in dietary lignin-to-cellulose ratios of 0.26, 0.30, 0.32, 0.34 and 0.40, respectively. The diets were fed from 4 d ante partum (a.p.) until day 28 post partum (p.p. weaning). After weaning, 40 young rabbits from each feeding group were fed the same diets for 12 d, when 10 rabbits per group were sacrificed. Body weight gain (BWG), feed intake and feed conversion ratio were obtained. Gastrointestinal content and gut tissue were obtained from the growing rabbits and pH, short-chain fatty acids (SCFA) concentration and gut morphology were investigated. Digestive disorders were scored, and the sanitary risk index was calculated. Does fed the highest lignocellulose addition increased their feed intake [g · kg BW-1 · d-1] in the first two weeks p.p. (p < 0.001) and increased BWG (p < 0.001) but without impact on their offsprings' performance. Weaned rabbits fed the highest lignocellulose inclusion showed a reduced feed intake and increased sanitary risk index. Additionally, a higher number of rabbits showed intestinal disorders for a short time (1-2 d, p < 0.001). In the colon content, a higher pH and a lower dry matter content were observed in this group. Caecal contents of 40-d old rabbits showed lower total SCFA, acetate and propionate and higher i-valerate and L-lactate concentrations after feeding diets with high lignocellulose contents. The diet did not influence the acetate-to-propionate ratio. No histomorphological changes were observed in the jejunum, while crypt depth and width were reduced in the caecum of weaned rabbits fed diets with lignocellulose addition. It can be concluded that different proportions of lignocellulose to cellulose addition to rabbits' diets have an impact on health and performance with differences between does and weaned rabbits. While a high lignocellulose inclusion proved beneficial for does, for weaned rabbits, a lower addition should be preferred.

A Preliminary Survey of the Distribution of Segmented Filamentous Bacteria in the Porcine Gastrointestinal Tract.

Segmented filamentous bacteria (SFB) are present in various animal species including pigs. The aim of this work was to analyze the occurrence of SFB in different parts of the gastrointestinal tract of piglets of different ages. A total of 377 DNA extracts from stomach, jejunum, ileum, cecum and colon digesta, and from feces collected on different time points, originating from 155 animals, were screened by qPCR method with primers specific for the SFB. SFB sequences were detected in 74 of 377 samples (19.6%) from 155 animals in total. SFB were most abundant in ileum (50.0%), cecum (45.0%), and colon (37.0%), followed by feces (14.6%). SFB prevalence in sows was 12.9% (13/101) and 75.9% (41/54) in individual piglets. Of the 41 SFB-positive piglets, only two samples were from pre-weaning animals, while the rest of samples originated from post-weaning piglets. SFB sequences are abundant in post-weaning piglets, but not in suckling or adult animals. They are most abundant in the ileum and cecum of pigs. Further studies are warranted to reveal the role of SFB in pigs.

Effects of cereal and protein source on performance, apparent ileal protein digestibility and intestinal characteristics in weaner piglets.

Rye and rapeseed meal (RSM) are alternatives to wheat and soybean meal (SBM) in pig nutrition. The inclusion of rye and RSM instead of wheat and SBM may increase dietary fibre content in the feed, which may affect digestive physiology and intestinal function. However, research on the combined feeding of rye and RSM in piglets is still scarce. The aim of this study was to investigate the effects of rye and RSM containing feed on growth performance, protein and amino acid digestibility, physical characteristics of the digesta and the intestinal morphology in weaner piglets. A total of 88 piglets aged 28 d were randomly allotted to four different feeding groups at weaning: wheat/SBM, wheat/RSM, rye/SBM, rye/RSM. Cereals were included at 48%, SBM at 25% and RSM at 30%. Body weight gain and feed intake were recorded weekly. After 33 d, piglets were euthanised for assessment of digesta and intestinal morphology. Rye did not affect performance, whilst RSM resulted in reduced weight gain (p = 0.024) and feed intake (p = 0.037). Apparent ileal digestibility of crude protein and amino acids was decreased by rye and RSM (p < 0.05). Rye caused an acidification (p = 0.045) and increased viscosity (p = 0.013) of small intestinal digesta, a lower dry matter content of jejunal and colonic digesta (p < 0.05) and an increased relative weight of the colon tissue (p < 0.001). In conclusion, performance was not reduced when rye was used as an alternative to wheat in diets for weaner piglets. The inclusion of RSM instead of SBM to piglets' diet lowered animal performance under our conditions. Further investigations are warranted to characterise the quality of rapeseed meal and the optimum inclusion level in the diet for young pigs.

Formula Feeding Predisposes Neonatal Piglets to Clostridium difficile Gut Infection.

Spontaneous outbreaks of Clostridium difficile infection (CDI) occur in neonatal piglets, but the predisposing factors are largely not known. To study the conditions for C. difficile colonization and CDI development, 48 neonatal piglets were moved into isolators, fed bovine milk-based formula, and infected with C. difficile 078. Analyses included clinical scoring; measurement of the fecal C. difficile burden, toxin B level, and calprotectin level; and postmortem histopathological analysis of colon specimens. Controls were noninfected suckling piglets. Fecal specimens from suckling piglets, formula-fed piglets, and formula-fed, C. difficile-infected piglets were used for metagenomics analysis. High background levels of C. difficile and toxin were detected in formula-fed piglets prior to infection, while suckling piglets carried about 3-fold less C. difficile, and toxin was not detected. Toxin level in C. difficile-challenged animals correlated positively with C. difficile and calprotectin levels. Postmortem signs of CDI were absent in suckling piglets, whereas mesocolonic edema and gas-filled distal small intestines and ceca, cellular damage, and reduced expression of claudins were associated with animals from the challenge trials. Microbiota in formula-fed piglets was enriched with Escherichia, Shigella, Streptococcus, Enterococcus, and Ruminococcus species. Formula-fed piglets were predisposed to C. difficile colonization earlier as compared to suckling piglets. Infection with a hypervirulent C. difficile ribotype did not aggravate the symptoms of infection. Sow-offspring association and consumption of porcine milk during early life may be crucial for the control of C. difficile expansion in piglets.

A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions.

Characterisation of caecal microbial diversity of lactating does and their offspring given diets with different neutral detergent soluble to insoluble fibre ratios.

The effect of neutral detergent soluble fibre (NDSF) to neutral detergent fibre (NDF) dietary ratio (0.29, LR and 0.43, HR) on the caecal ecosystem of lactating does and their offspring was studied. From the 17th day of lactation, each diet was given to four does, allowing for free access to their litters. Does were sampled at 17 and 28 days of lactation, and also two pups per litter at 17 (milk-fed only), 28 (milk and solid fed) and 49 days of age. DGGE was used to study bacterial caecal biodiversity, and total bacterial concentration and relative proportions of Ruminococcus albus and Butyrivibrio fibrisolvens were quantified by real time PCR. In does, diet did not affect (P > 0.10) diversity indexes, total bacterial concentration or relative abundance of B. fibrisolvens, but at 28 days of lactation the proportion of R. albus was higher with LR (interaction Diet × Time, P = 0.037). Caecal communities of pups of 17 days were grouped by litter, but the influence of the mother was reduced at 28 days with solid feed intake, and at 49 days rabbits clustered by diet. Caecal biodiversity increased from 17 to 28 days, and was reduced at 49 days (Shannon index of 3.60, 3.71 and 3.57, respectively; P = 0.049). Total bacterial concentration and relative abundance of R. albus and B. fibrisolvens increased with solid feed intake from 17 to 28 days (P < 0.01), remaining unaffected thereafter. Access of pups to solid feed from 17 days of age modulates the development and composition of the caecal microbiota at weaning.

Clostridioides difficile-mesocolonic oedema in neonatal suckling piglets develops regardless of the fibre composition in sow's diets.

Maternal dietary factors have been reported to influence Clostridioides difficile colonisation in the offspring. Twenty suckling piglets from sows fed diets supplemented with high-fermentable sugar beet pulp (SBP) or low-fermentable lignocellulose (LNC) fibres during gestation and lactation were dissected in the first week after birth. Postmortem analysis included clinical mesocolon and faecal scoring, concentration of C. difficile and respective toxins in colon digesta and faeces, immunoglobulins in serum and inflammatory markers in serum and colon tissues. Sow colostrum was assessed for nutrients, immunoglobulins and biogenic amines. Toxin-neutralising IgG antibodies were measured in colostrum and serum of the sows, and in colon digesta and serum of the piglets. Mesocolonic oedema of different severity was present in most of the piglets from both sows' feeding groups. Concentrations of C. difficile, toxins and calprotectin in colon digesta and faecal contents did not differ between the study piglets. Calprotectin correlated positively with mesocolon score (rho = 413, P = 0.07). Piglets from sows fed LNC vs SBP tended to have higher IgA (P = 0.089), IgG (P = 0.053), total Ig (P = 0.053), albumin (P = 0.075) and total protein content (P = 0.007) in serum. Colon tissues of piglets from the SFB vs LNC had upregulated expression of ZO-1 (P = 0.021), PCNA (P = 0.015) and TGF-ß (P = 0.014). Titers of anti-toxin-IgG-antibodies in serum and colostrum and in piglet colon digesta and serum did not differ between sows from both dietary groups, but they all showed strong positive correlations. In conclusion, dietary sugar beet pulp or lignocellulose fed to sows did not influence the concentrations of C. difficile and toxins titers in colon digesta and faeces of neonatal piglets.

Evaluation of High Doses of Phytase in a Low-Phosphorus Diet in Comparison to a Phytate-Free Diet on Performance, Apparent Ileal Digestibility of Nutrients, Bone Mineralization, Intestinal Morphology, and Immune Traits in 21-Day-Old Broiler Chickens.

The supplementation of feed with phytases enables broilers to utilize more efficiently phosphorus (P) from phytic acid (IP6), the main storage form of P in plants. The current study evaluated the addition of 500, 1000, and 3000 FTU of phytase per kg to a phytate-containing diet with low P level (LP) fed to broilers from 1 to 21 days of age and compared it to a hypoallergenic phytate-free diet (HPF). There was a linear improvement in performance parameters with increasing levels of phytase in the LP diet (p < 0.001). Apparent ileal digestibility of crude protein, P, and some amino acids, increased with phytase. Crude ash, P, and the calcium content of tibia bones linearly increased with increasing levels of phytase (p < 0.001). Crypt depth (related to body weight) in the jejunum epithelium linearly decreased with phytase addition (p < 0.001). Cecal crypt depth decreased with phytase supplementation (p = 0.002). Cecum tissue showed lower counts of CD3-positive intraepithelial lymphocytes in broilers receiving the phytase in comparison to LP (p < 0.001), achieving similar counts to HPF-fed broilers. Although results from the current study seem to point out some mechanisms related to the immune response and mucosal morphology contributing to those overall beneficial effects, no clear differences between different phytase doses could be demonstrated in these specific parameters.

Porcine and Chicken Intestinal Epithelial Cell Models for Screening Phytogenic Feed Additives-Chances and Limitations in Use as Alternatives to Feeding Trials.

Numerous bioactive plant additives have shown various positive effects in pigs and chickens. The demand for feed additives of natural origin has increased rapidly in recent years to support the health of farm animals and thus minimize the need for antibiotics and other drugs. Although only in vivo experiments can fully represent their effect on the organism, the establishment of reliable in vitro methods is becoming increasingly important in the goal of reducing the use of animals in experiments. The use of cell models requires strict control of the experimental conditions so that reliability and reproducibility can be achieved. In particular, the intestinal porcine epithelial cell line IPEC-J2 represents a promising model for the development of new additives. It offers the possibility to investigate antioxidative, antimicrobial, anti- or pro-proliferative and antiviral effects. However, the use of IPEC-J2 is limited due to its purely epithelial origin and some differences in its morphology and functionality compared to the in vivo situation. With regard to chickens, the development of a reliable intestinal epithelial cell model has attracted the attention of researchers in recent years. Although a promising model was presented lately, further studies are needed to enable the standardized use of a chicken cell line for testing phytogenic feed additives. Finally, co-cultivation of the currently available cell lines with other cell lines and the development of organoids will open up further application possibilities. Special emphasis was given to the IPEC-J2 cell model. Therefore, all publications that investigated plant derived compounds in this cell line were considered. The section on chicken cell lines is based on publications describing the development of chicken intestinal epithelial cell models.

Porcine Colostrum Protects the IPEC-J2 Cells and Piglet Colon Epithelium against Clostridioides (syn. Clostridium) difficile Toxin-Induced Effects.

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet's intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring.