Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2-7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Zinc status and indicators of intestinal health in enterotoxigenic Escherichia coli F18 challenged newly weaned pigs fed diets with different levels of zinc.

This study investigated the impact of an enterotoxigenic Escherichia coli (ETEC) F18 challenge on newly weaned pigs when fed one of three Zn levels (150, 1,400, or 2,500 ppm) on performance, Zn status, ETEC shedding, and diarrhea. The ETEC challenge was hypothesized to have a more pronounced negative impact on pigs fed a diet containing 150 ppm Zn compared to 1,400 or 2,500 ppm Zn for 14 d after weaning. The study included 72 ETEC F18 susceptible pigs weaned at ~28 d of age (d 0 of the study). The pigs were distributed according to initial weight and litter to one of the three dietary Zn levels. Half of the pigs were challenged with ETEC on d 1 and 2. The challenge reduced (P ≤ 0.03) feed intake and average daily gain (ADG) during d 3 to 5. Challenged pigs fed 150 ppm Zn had lower (P = 0.01) ADG during d 5 to 7 compared to those fed 1,400 or 2,500 ppm Zn, whereas control pigs' ADG were not affected by dietary Zn content. Challenged pigs fed 150 ppm Zn also showed lower (P < 0.01) fecal dry matter (DM) on d 5 compared to control pigs fed 150 ppm Zn and challenged pigs fed 1,400 or 2,500 ppm Zn. Challenge increased (P < 0.01) ETEC shedding in all groups, but challenged pigs fed 150 ppm Zn showed higher (P ≤ 0.05) fecal shedding of ETEC and toxins than when fed 1,400 or 2,500 ppm. On d 3, C-reactive protein concentration in plasma was lower (P < 0.03) for pigs fed 1,400 and 2,500 compared to 150 ppm Zn. Plasma haptoglobin and pig major acute phase protein were unaffected by dietary Zn content. On d 0, the serum Zn concentration was 586 ±â€…36.6 µg/L, which pigs fed 150 ppm Zn maintained throughout the study. The serum Zn concentration increased (P ≤ 0.07) in pigs fed 1,400 or 2,500 ppm Zn. The challenge decreased (P < 0.01) the serum Zn concentration in pigs fed 2,500 ppm Zn. On d 5 and 7, serum Zn concentration was similar for challenged pigs fed 1,400 and 2,500 ppm Zn, while control pigs fed 2,500 ppm Zn had higher (P < 0.01) serum Zn concentration than 1,400 ppm Zn. On d 7, serum Zn concentration tended (P = 0.08) to be lower for pigs with diarrhea (fecal DM ≤ 18%). In summary, these results indicate that newly weaned pigs fed 150 ppm Zn are more susceptible to ETEC F18 colonization and its adverse consequences such as diarrhea and reduced growth, even though challenge did not increase acute phase proteins.

At weaning, the immune system of pigs is not fully developed, leaving them more susceptible to infections such as enterotoxigenic Escherichia coli (ETEC) F18. This study investigated the effect of dietary zinc content on newly weaned pigs infected with ETEC in terms of performance, serum zinc status, diarrhea, E. coli shedding, and systemic inflammation markers. Challenged pigs had reduced growth the first 3 d after the challenge when fed 150 ppm dietary Zn compared to non-challenged pigs. Moreover, the challenge increased E. coli fecal shedding and resulted in more liquid feces, and a greater risk of diarrhea in pigs fed 150 ppm zinc compared to 1,400 and 2,500 ppm zinc. The challenge was not able to induce a response in the acute phase proteins. The serum zinc concentration was lowest when feeding 150 ppm, and the ETEC challenge caused a reduction in the serum Zn concentration only when feeding 2,500 ppm zinc. These findings suggest that newly weaned pigs fed 150 ppm zinc are less capable of withstanding an ETEC challenge based on impaired growth performance and increased diarrhea and E. coli shedding.

Variability within L. albus and L. angustifolius Seeds in Dietary Fiber Components.

Lupin seeds have received increased attention due to their applications in the nutrition of humans and livestock. One of their special features is their high content of dietary fiber, which is influenced by the lupin species. No previous studies have focused on the variability in dietary fiber and its fractions within species so far. The aim of this study was to investigate the variability within L. albus and L. angustifolius (eight cultivars each) in the dietary fiber composition expressed as low-molecular-weight soluble dietary fiber (LMWSDF), soluble and insoluble non-cellulosic polysaccharides, cellulose, and Klason lignin. Additionally, we analyzed the proximate composition and the composition of amino acids and fatty acids. The results showed noticeable variability within both species not only in the total dietary fiber but also in all its fractions, especially in LMWSDF, cellulose, non-starch polysaccharides, and Klason lignin within L. angustifolius. This indicates that the cultivar choice should be based on the application for which it is used. Even though important nutrients, such as the most indispensable amino acids, are not highly variable within L. albus, dietary fiber variations can still have a marked influence on the nutritional value because of their influence on the digestibility of other nutrients.

Corrigendum: A westernized diet changed the colonic bacterial composition and metabolite concentration in a dextran sulfate sodium pig model for ulcerative colitis.

[This corrects the article DOI: 10.3389/fmicb.2023.1018242.].

Liquid fermented cereals with added Pediococcus acidilactici did not reduce post-weaning diarrhea in pigs - an Escherichia coli challenge study.

The effect of feeding fermented liquid feed (FLF) with added Pediococcus acidilactici to weaning piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4 on aspects of diarrhea, performance, immune responses, and intestinal epithelial barrier function was investigated. A total of 46 weaners (weaning at 27-30 days of age) were assigned to four treatments: (1) Non-challenged and dry feed (Non-Dry); (2) Challenged and dry feed (Ch-Dry); (3) Non-challenged and FLF (Non-Ferm); (4) Challenged and FLF (Ch-Ferm). All groups received the same feed, either dry (Non-Dry and Ch-Dry), or in liquid form (Non-Ferm and Ch-Ferm) in which the cereals with added P. acidilactici (106 CFU/g cereals) had been fermented for 24 h at 30°C. On day 1 and 2 post weaning, Ch-Dry and Ch-Ferm were orally inoculated with 5 mL × 109 CFU ETEC F4/mL, whereas the Non-Dry and Non-Ferm received the same amount of saline. Fecal samples and blood samples were collected through the study period. The microbial composition, concentration of microbial metabolites and nutrient composition indicated that the quality of the FLF was high. In the first week, ADFI of both non-challenged groups was significantly higher (p < 0.05) than that of the Ch-Ferm group. The two challenged groups had higher fecal levels of FaeG gene (ETEC F4 fimbriae) from day 2 to 6 post weaning (p < 0.01), and higher risk of having ETEC F4 present in feces from day 3 to 5 post weaning (p < 0.05) compared to non-challenged groups, indicating the validity of the ETEC challenge model. Generally, ADG of the two groups fed FLF were numerically higher than those fed dry feed. Neither challenge nor FLF affected diarrhea. No significant differences were measured between Ch-Ferm and Ch-Dry regarding the level of plasma haptoglobin and C-reactive protein, hematological parameters or parameters related to epithelial barrier. The data indicated a low level of infection caused by the ETEC challenge, while recovery from weaning stress could be observed. The study showed that a strategy like this can be a way of providing a high level of probiotics to pigs by allowing their proliferation during fermentation.

A westernized diet changed the colonic bacterial composition and metabolite concentration in a dextran sulfate sodium pig model for ulcerative colitis.

Introduction: Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic epithelium and has a blurred etiology. A western diet and microbial dysbiosis in the colon were reported to play a role in UC development. In this study, we investigated the effect of a westernized diet, i.e., increasing fat and protein content by including ground beef, on the colonic bacterial composition in a dextran sulfate sodium (DexSS) challenged pig study. Methods: The experiment was carried out in three complete blocks following a 2×2 factorial design including 24 six-week old pigs, fed either a standard diet (CT) or the standard diet substituted with 15% ground beef to simulate a typical westernized diet (WD). Colitis was induced in half of the pigs on each dietary treatment by oral administration of DexSS (DSS and WD+DSS, respectively). Samples from proximal and distal colon and feces were collected. Results and discussion: Bacterial alpha diversity was unaffected by experimental block, and sample type. In proximal colon, WD group had similar alpha diversity to CT group and the WD+DSS group showed the lowest alpha diversity compared to the other treatment groups. There was a significant interaction between western diet and DexSS for beta diversity, based on Bray-Curtis dissimilarly. The westernized diet and DexSS resulted in three and seven differentially abundant phyla, 21 and 65 species, respectively, mainly associated with the Firmicutes and Bacteroidota phyla followed by Spirochaetota, Desulfobacterota, and Proteobacteria. The concentration of short-chain fatty acids (SCFA) was lowest in the distal colon. Treatment had a slight effect on the estimates for microbial metabolites that might have valuable biological relevance for future studies. The concentration of putrescine in the colon and feces and that of total biogenic amines was highest in the WD+DSS group. We conclude that a westernized diet could be a potential risk factor and an exacerbating agent for UC by reducing the abundance of SCFA-producing bacteria, increasing the abundance of pathogens such as Helicobacter trogontum, and by increasing the concentration of microbial proteolytic-derived metabolites in the colon.

Antibacterial plant combinations prevent postweaning diarrhea in organically raised piglets challenged with enterotoxigenic Escherichia coli F18.

Antibiotics and zinc oxide restrictions encourage the search for alternatives to combat intestinal pathogens, including enterotoxigenic Escherichia coli (ETEC), a major cause of postweaning diarrhea (PWD) in pigs. PWD causes important economic losses for conventional and organic farming. This study investigated the effect of dietary supplementation with garlic and apple pomace or blackcurrant on infection indicators and the fecal microbiota of organic-raised piglets challenged with ETEC-F18. For 21 days, 32 piglets (7-weeks-old) were randomly assigned to one of four groups: non-challenge (NC); ETEC-challenged (PC); ETEC-challenged receiving garlic and apple pomace (3 + 3%; GA); ETEC-challenged receiving garlic and blackcurrant (3 + 3%; GB). ETEC-F18 was administered (8 mL; 109 CFU/ml) on days 1 and 2 postweaning. The 1st week, PC had lower average daily gain than those in the NC, GA, and GB groups (P < 0.05). NC pigs showed neither ETEC-F18 shedding nor signs of diarrhea. The PC group had higher diarrhea incidence and lower fecal dry matter than NC (≈5-10 days; 95% sEBCI). The GA and GB groups showed reduced ETEC-F18 and fedA gene shedding, higher fecal dry matter, and lower diarrhea incidence than the PC (≈5-9 days; 95% sEBCI). The NC, GA, and GB had normal hematology values during most of the study, whereas the PC had increased (P < 0.05) red blood cells, hemoglobin, and hematocrit on day 7. Haptoglobin and pig-MAP increased in all groups, peaking on day 7, but PC showed the greatest increase (P < 0.05). The fecal microbiota of PC pigs had reduced α-diversity (day 7; P < 0.05) and higher volatility (days 3-14; P < 0.05). Escherichia, Campylobacter, and Erysipelothrix were more abundant in the PC than in the NC, GB, and GA groups (log2FC > 2; P < 0.05), whereas Catenibacterium, Dialister, and Mitsoukella were more abundant in the NC, GB, and GA than in the PC group (log2FC > 2; P < 0.05). Prevotella and Lactobacillus were more abundant in the GB group (log2FC > 2, P < 0.05). In conclusion, dietary supplementation of GA and GB limited ETEC proliferation, reduced PWD, and beneficially impacted the fecal microbiota's diversity, composition, and stability.

Review on Preventive Measures to Reduce Post-Weaning Diarrhoea in Piglets.

In many countries, medical levels of zinc (typically as zinc oxide) are added to piglet diets in the first two weeks post-weaning to prevent the development of post-weaning diarrhoea (PWD). However, high levels of zinc constitute an environmental polluting agent, and may contribute to the development and/or maintenance of antimicrobial resistance (AMR) among bacteria. Consequently, the EU banned administering medical levels of zinc in pig diets as of June 2022. However, this may result in an increased use of antibiotic therapeutics to combat PWD and thereby an increased risk of further AMR development. The search for alternative measures against PWD with a minimum use of antibiotics and in the absence of medical levels of zinc has therefore been intensified over recent years, and feed-related measures, including feed ingredients, feed additives, and feeding strategies, are being intensively investigated. Furthermore, management strategies have been developed and are undoubtedly relevant; however, these will not be addressed in this review. Here, feed measures (and vaccines) are addressed, these being probiotics, prebiotics, synbiotics, postbiotics, proteobiotics, plants and plant extracts (in particular essential oils and tannins), macroalgae (particularly macroalgae-derived polysaccharides), dietary fibre, antimicrobial peptides, specific amino acids, dietary fatty acids, milk replacers, milk components, creep feed, vaccines, bacteriophages, and single-domain antibodies (nanobodies). The list covers measures with a rather long history and others that require significant development before their eventual use can be extended. To assess the potential of feed-related measures in combating PWD, the literature reviewed here has focused on studies reporting parameters of PWD (i.e., faeces score and/or faeces dry matter content during the first two weeks post-weaning). Although the impact on PWD (or related parameters) of the investigated measures may often be inconsistent, many studies do report positive effects. However, several studies have shown that control pigs do not suffer from diarrhoea, making it difficult to evaluate the biological and practical relevance of these improvements. From the reviewed literature, it is not possible to rank the efficacy of the various measures, and the efficacy most probably depends on a range of factors related to animal genetics and health status, additive doses used, composition of the feed, etc. We conclude that a combination of various measures is probably most recommendable in most situations. However, in this respect, it should be considered that combining strategies may lead to additive (e.g., synbiotics), synergistic (e.g., plant materials), or antagonistic (e.g., algae compounds) effects, requiring detailed knowledge on the modes of action in order to design effective strategies.

Parasite-Probiotic Interactions in the Gut: Bacillus sp. and Enterococcus faecium Regulate Type-2 Inflammatory Responses and Modify the Gut Microbiota of Pigs During Helminth Infection.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Potential relevance of pig gut content transplantation for production and research.

It is becoming increasingly evident that the gastrointestinal microbiota has a significant impact on the overall health and production of the pig. This has led to intensified research on the composition of the gastrointestinal microbiota, factors affecting it, and the impact of the microbiota on health, growth performance, and more recently, behavior of the host. Swine production research has been heavily focused on assessing the effects of feed additives and dietary modifications to alter or take advantage of select characteristics of gastrointestinal microbes to improve health and feed conversion efficiency. Research on faecal microbiota transplantation (FMT) as a possible tool to improve outcomes in pigs through manipulation of the gastrointestinal microbiome is very recent and limited data is available. Results on FMT in humans demonstrating the transfer of phenotypic traits from donors to recipients and the high efficacy of FMT to treat Clostridium difficile infections in humans, together with data from pigs relating GI-tract microbiota composition with growth performance has likely played an important role in the interest towards this strategy in pig production. However, several factors can influence the impact of FMT on the recipient, and these need to be identified and optimized before this tool can be applied to pig production. There are obvious inherent biosecurity and regulatory issues in this strategy, since the donor's microbiome can never be completely screened for all possible non-desirable microorganisms. However, considering the success observed in humans, it seems worth investigating this strategy for certain applications in pig production. Further, FMT research may lead to the identification of specific bacterial group(s) essential for a particular outcome, resulting in the development of banks of clones which can be used as targeted therapeutics, rather than the broader approach applied in FMT. This review examines the factors associated with the use of FMT, and its potential application to swine production, and includes research on using the pig as model for human medical purposes.

The Mode of Action of Chicory Roots on Skatole Production in Entire Male Pigs Is neither via Reducing the Population of Skatole-Producing Bacteria nor via Increased Butyrate Production in the Hindgut.

The effect of high levels of dietary chicory roots (25%) and intracecal exogenous butyrate infusion on skatole formation and gut microbiota was investigated in order to clarify the mechanisms underlying the known reducing effect of chicory roots on skatole production in entire male pigs. A Latin square design with 3 treatments (control, chicory, and butyrate), 3 periods, and 6 animals was carried out. Chicory roots showed the lowest numerical levels of skatole in both feces and plasma and butyrate infusion the highest. In the chicory group, an increased abundance of the skatole-producing bacterium Olsenella scatoligenes compared to the control group (P = 0.06), and a numerically higher relative abundance of Olsenella than for the control and butyrate groups, was observed. Regarding butyrate-producing bacteria, the chicory group had lower abundance of Roseburia but a numerically higher abundance of Megasphaera than the control group. Lower species richness was found in the chicory group than in the butyrate group. Moreover, beta diversity revealed that the chicory group formed a distinct cluster, whereas the control and butyrate groups clustered more closely to each other. The current data indicated that the skatole-reducing effect of chicory roots is neither via inhibition of cell apoptosis by butyrate nor via suppression of skatole-producing bacteria in the pig hindgut. Thus, the mode of action is most likely through increased microbial activity with a corresponding high incorporation of amino acids into bacterial biomass, and thereby suppressed conversion of tryptophan into skatole, as indicated in the literature.IMPORTANCE Castration is practiced to avoid the development of boar taint, which negatively affects the taste and odor of pork, and undesirable aggressive behavior. Due to animal welfare issues, alternatives to surgical castration are sought, though. Boar taint is a result of high concentrations of skatole and androstenone in back fat. Skatole is produced by microbial fermentation in the large intestine, and therefore, its production can be influenced by manipulation of the microbiota. Highly fermentable dietary fiber reduces skatole production. However, various theories have been proposed to explain the mode of action. In order to search for other alternatives, more efficient or less expensive, to reduce skatole via feeding, it is important to elucidate the mechanism behind the observed effect of highly fermentable dietary fiber on skatole. Our results indicate that highly fermentable dietary fiber does not affect skatole production by reducing the number of skatole-producing bacteria or stimulating butyrate production in the large intestine.

Impact of Bacillus spp. spores and gentamicin on the gastrointestinal microbiota of suckling and newly weaned piglets.

Administrating antibiotics to young piglets may have short- and long-term consequences on the gut microbiota. We hypothesised that these consequences may be alleviated by concurrent probiotic administration. The study objective was to investigate the effect of administrating gentamicin and a mixture of Bacillus (B.) licheniformis, B. subtilis and B. amyloliquefaeceans spores on the gut microbiota of piglets pre- and post-weaning. Twenty-four sows and their litters were randomly allocated to four treatment groups receiving; a) Bacillus spore mixture (six B. subtilis, two B. amyloliquefaeceans, and one B. licheniformis) fed to sows and piglets (PRO); b) gentamicin (5 mg per day) administered to piglets on day 4, 5, and 6 of age (AB); c) Bacillus spore mixture fed to sows and piglets, and gentamicin to piglets (PRO+AB); or d) no administration of probiotics or antibiotics (CTRL). Faecal and digesta samples were collected repeatedly during the study. Selected samples were subjected to 16S rRNA gene sequencing, culture counts, and organic acid, biogenic amine and tissue gene expression analysis. Treatment had a significant effect on the faecal microbial community composition on day 28 and 42, and colonic community on day 28. Faecal species richness (observed and estimated) and Shannon index, and colonic species richness, were higher in AB compared to PRO piglets on day 28, and were not significantly different from day 42. PRO piglets had the highest faecal concentration of iso-butyric acid on day 7 and a higher butyric acid concentration compared to CTRL piglets. We conclude that gentamicin and Bacillus spores influence the gut microbial diversity of piglets, although administration of gentamicin did not result in dysbiosis as hypothesised.

Effects of alpha-(1,2)-fucosyltransferase genotype variants on plasma metabolome, immune responses and gastrointestinal bacterial enumeration of pigs pre- and post-weaning.

In pigs, the alpha-(1,2) fucosyltransferase (FUT1) gene has been highlighted for its properties in controlling the intestinal expression of enterotoxigenic E. coli (ETEC) F18 receptors; a pathogen causing edema disease and post-weaning diarrhoea. In this study, we hypothesized that pigs with different genotypes (ETEC F18 resistant (FUT1AA) versus susceptible (FUT1AG)) differed in following systemic and enteric responses: growth performance, plasma metabolic profiles, expression of candidate genes for intestinal mucosal homeostasis and immunity, number of selected bacteria and the concentration of short-chain fatty acids (SCFA) in faeces and digesta in piglets pre and post-weaning, and on the ETEC F18 adherence ex vivo. Genotype had the strongest impact on plasma metabolomic profile on day 7 and 28 of age. FUT1AG piglets had higher level of N-methyl-2-pyrrolidinone, hippuric acid, oxindole, and 3-oxo-5-beta-chol-7-en-24-oic acid on day 7, and a higher level of guanosine on day 28 than that in the FUT1AA piglets. FUT1AA piglets had a higher level of betaine on day 7 and 3-methylguanine on day 28. On day 34 of age, the FUT1AA pigs had higher levels of S-2-hydroxyglutarate, L-phenylalanine, tauroursodeoxycholic acid and an undetermined PC/LysoPC, while Ile Glu Phe Gly peptide and genistein 5-O-glucuronide, and PC (18:0/0:0) were at higher levels in the FUT1AG piglets. FUT1 genotype did not affect the growth performance and expression of candidate genes. FUT1AG piglets had a higher number of haemolytic bacteria in faeces and in digesta than that in FUT1AA at 34 days of age. The colonic acetic acid concentration was highest in FUT1AG piglets. FUT1 genotype may influence not only the expression of E. coli F18 receptors but could potentially impact the gut homeostasis and metabotype of piglets pre and post-weaning. Further investigations on the relation between FUT1 genotype and these aspects including the intestinal commensal microbiota will expand the knowledge on factors affecting the intestinal ecosystem.

Development of a species-specific TaqMan-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet.

Olsenella scatoligenes is the only skatole-producing bacterium isolated from the pig gut. Skatole, produced from microbial degradation of l-tryptophan, is the main contributor to boar taint, an off-odor and off-flavor taint, released upon heating meat from some entire male pigs. An appropriate method for quantifying O. scatoligenes would help investigating the relationship between O. scatoligenes abundance and skatole concentration in the pig gut. Thus, the present study aimed at developing a TaqMan-MGB probe-based, species-specific qPCR assay for rapid quantification of O. scatoligenes. The use of a MGB probe allowed discriminating O. scatoligenes from other closely related species. Moreover, the assay allowed quantifying down to three target gene copies per PCR reaction using genomic DNA-constructed standards, or 1.5 × 103 cells/g digesta, using O. scatoligenes-spiked digesta samples as reference standards. The developed assay was applied to assess the impact of dietary chicory roots on O. scatoligenes in the hindgut of pigs. Olsenella scatoligenes made up < 0.01% of the microbial population in the pig hindgut. Interestingly, the highest number of O. scatoligenes was found in young entire male pigs fed high levels of chicory roots. This indicates that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of this skatole-producing bacterium in the pig hindgut. Accordingly, the abundance of O. scatoligenes in the hindgut does not seem to be an appropriate indicator of boar taint. The present study is the first to describe a TaqMan-MGB probe qPCR assay for detection and quantification of O. scatoligenes in pigs.

Butyrylation of Maize and Potato Starches and Characterization of the Products by Nuclear Magnetic Resonance and In Vitro Fermentation.

Intake of butyrylated starches may increase colonic butyrate supply, which can be of public health and clinical benefit by maintaining colonic health. The objective was to investigate if an organocatalytic method with tartaric acid as a catalyst could be applied to produce butyrylated products from different starch sources and to characterize their chemical structure and fermentation capability by using solid-state 13C MAS NMR (magic angle spinning nuclear magnetic resonance) spectroscopy and an in vitro fermentation model, respectively. Low-amylose and high-amylose potato starch (LAPS and HAPS) and low-amylose and high-amylose maize starch (LAMS and HAMS) were subjected to organocatalytic butyrylation. This resulted in products with an increasing degree of substitution (DS) measured by heterogenous saponification and back titration with the HCl (chemical method) depending on reaction time. NMR analysis, however, showed that the major part of the acylation was induced by tartarate (75⁻89%) and only a minor part (11⁻25%) by butyrate. Generally, the chemical method overestimated the DS by 38% to 91% compared with the DS determination by NMR. Increasing the DS appeared to lower the in vitro fermentation capability of starches independent of the starch source and, therefore, do not seem to present a feasible method to deliver more butyrate to the colon than lower DS products.

The microbial community of the gut differs between piglets fed sow milk, milk replacer or bovine colostrum.

The aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n 36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance of Lactococcus in the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance of Lactobacillus in the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

The dose-response relationship between the amount of straw provided on the floor and gastric ulceration of pars oesophagea in growing pigs.

The aim of the present study was (1) to determine the dose-response relationship between the amount of straw provided on the floor and oesophageal ulceration in pigs kept under typical Danish production conditions (18 pigs/pen, 0.7m2/pig, partly slatted floor, ad libitum access to feed), (2) to reveal whether straw ingestion explains the effect of straw provision on the stomach health and (3) to elucidate the effect of straw ingestion on the stomach conditions. Data were collected at slaughter (approximately 100kg body weight) on pigs provided with straw amounts in the range 10g to 500g wheat straw/pig/day from 30kg body weight and fed a wheat-based pelleted feed added 15% non-heated and non-pelleted rolled barley. Aims (1) and (2) included 712 pigs kept in 42 pens, whereas (3) was studied on a subset of 37 pigs with either none or obvious amounts of straw in the stomach. The amount of straw provided affected stomach health in a curvilinear manner. Provision of up to approximately 300gstraw/pig/day progressively decreased the risk of oesophageal ulceration and scarring. At larger amounts of straw the ulceroprotective effect of straw was reduced which requires further investigation. Straw ingestion appeared to be an essential intermediary factor for the improvement of stomach health. The number of pigs without straw in the stomach decreased linearly with the logarithm of the amount of straw provided, which explained the effect of straw provision on stomach health. The structure of the stomach contents increased as revealed by the increased weight and dry matter content and decreased sedimentation of the digesta. Provision of approximately 300gstraw/pig/day on the floor may be a potential strategy to reduce, but not inevitably eliminate, oesophageal ulceration in pigs in commercial pig production.

Development of Bacillus subtilis mutants to produce tryptophan in pigs.

OBJECTIVES: To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. RESULTS: A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l. Genome sequence analysis revealed that M1 had three single nuclear polymorphisms (SNPs) and M2 had two SNPs compared to the wild type strains. In both mutants SNPs were found in genes regulating tryptophan synthesis. Reverse transcription PCR confirmed up-regulation of the tryptophan synthesis genes in both mutants, the expression was up to 3 times higher in M2 than in M1. CONCLUSIONS: Tryptophan-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications.

Draft Genome Sequence of Megasphaera sp. Strain DJF_B143, an Isolate from Pig Hindgut Unable to Produce Skatole.

The butyrate-producing Megasphaera spp. predominate in the pig hindgut and may play important roles in gut health. Moreover, one Megasphaera isolate has been reported to produce the boar taint compound, skatole. Here, we provide a 2.58-Mbp draft genome of a pig hindgut isolate, Megasphaera sp. DJF_B143, unable to produce skatole.

Draft Genome Sequence of Olsenella scatoligenes SK9K4T, a Producer of 3-Methylindole (Skatole) and 4-Methylphenol (p-Cresol), Isolated from Pig Feces.

Olsenella scatoligenes SK9K4(T) is a strictly anaerobic bacterium isolated from pig feces that produces the malodorous compounds 3-methylindole (skatole) and 4-methylphenol (p-cresol). Here, we report the 2.47 Mbp draft genome sequence of SK9K4(T), exploring pathways for the synthesis of skatole and p-cresol from the amino acids tryptophan and tyrosine, respectively.