Araceae root and citrus fibers tend to decrease Escherichia coli adhesion and myeloperoxidase levels in weaned piglets.

Introduction: Weaning is a stressful experience in the piglet's life, and it often coincides with impaired gut health. Post-weaning diarrhea in piglets is frequently caused by enterotoxigenic Escherichia coli (E. coli). The first step of an E. coli infection is the adhesion to host-specific receptors present on enterocytes, leading to pro-inflammatory immune responses. The aim of this study was to examine if specific fiber fractions in the piglet diet can prevent E. coli adhesion and subsequent immune responses. Methods: The trial included 200 piglets (Danbred × Piétrain): 10 piglets/pen × 10 pens/dietary treatment × 2 dietary treatments. From weaning until 14 days (d14) post-weaning, piglets were fed a control diet or test diet with 2 kg/ton of a mixture of specific fiber fractions derived from Araceae root and citrus. Afterwards, 1 piglet per pen was euthanized, a section was taken at 75% of small intestinal length and E. coli colonization on the mucosal epithelium was quantified by scraping and conventional plating. From the same small intestinal section, histo-morphological indices were assessed, and mucosal scrapings were analyzed for gene expression of pro- and anti-inflammatory cytokines, and NF-kB. Analyses of specific intestinal bacteria and SCFA were performed on samples of intestinal content (small intestine, caecum, colon). Fecal samples were taken to measure myeloperoxidase (MPO), calprotectin and PAP/RAG3A as biomarkers for intestinal inflammation. Results and discussion: Piglets fed the fiber mixture tended to have decreased E. coli colonization to the mucosal epithelium (5.65 vs. 4.84 log10 CFU/g; P = 0.07), less E. coli in the caecum (8.91 vs. 7.72 log10 CFU/g; P = 0.03) and more Lachnospiraceae in the colon (11.3 vs. 11.6 log10 CFU/g; P = 0.03). Additionally, the fiber mixture tended to increase cecal butyric acid (10.4 vs. 19.1 mmol/kg; P = 0.07). No significant effect on histo-morphological indices and on gene expression of pro- and anti-inflammatory cytokines and NF-kB was observed. The fecal MPO concentration tended to decrease (20.2 vs. 10.4 ng/g; P = 0.07), indicating less intestinal inflammation. In conclusion, this study showed that specific fiber fractions from Araceae root and citrus in piglet weaner diets may decrease the risk of pathogen overgrowth by reducing E. coli adhesion and intestinal inflammation.

Effect of live yeast Saccharomyces cerevisiae supplementation on the performance and cecum microbial profile of suckling piglets.

One mechanism through which S. cerevisiae may improve the performance of pigs is by altering the composition of the gut microbiota, a response that may be enhanced by early postnatal supplementation of probiotics. To test this hypothesis, newborn piglets (16 piglets/group) were treated with either S. cerevisiae yeast (5 x 109 cfu/pig: Low) or (2.5 x 1010 cfu/piglet: High) or equivalent volume of sterile water (Control) by oral gavage every other day starting from day 1 of age until weaning (28±1 days of age). Piglet body weight was recorded on days 1, 3, 7, 10, 17, 24 and 28 and average daily gain (ADG) calculated for the total period. At weaning, piglets were euthanized to collect cecum content for microbial profiling by sequencing of the 16S rRNA gene. ADG was higher in both Low and High yeast groups than in Control group (P<0.05). Alpha diversity analyses indicated a more diverse microbiota in the Control group compared with Low yeast group; the High yeast being intermediate (P < 0.01). Similarly, Beta diversity analyses indicated differences among treatments (P = 0.03), mainly between Low yeast and Control groups (P = 0.02). The sparse Partial Least Squares Discriminant Analysis (sPLS-DA) indicated that Control group was discriminated by a higher abundance of Veillonella, Dorea, Oscillospira and Clostridium; Low yeast treated pigs by higher Blautia, Collinsella and Eubacterium; and High yeast treated pigs by higher Eubacterium, Anaerostipes, Parabacteroides, Mogibacterium and Phascolarctobacterium. Partial Least Squares (PLS) analysis showed that piglet ADG was positively correlated with genus Prevotella in High yeast group. Yeast supplementation significantly affected microbial diversity in cecal contents of suckling piglets associated with an improvement of short chain fatty acid producing bacteria in a dose-dependent manner. In conclusion, yeast treatment improved piglet performance and shaped the piglet cecum microbiota composition in a dose dependent way.

Supplementation with live yeast increases rate and extent of in vitro fermentation of nondigested feed ingredients by fecal microbiota.

Two studies were conducted to investigate the effect of live yeast (LY) on the in vitro fermentation characteristics of wheat, barley, corn, soybean meal (SBM), canola meal, and distillers dried grains with solubles (DDGS). In Study 1, LY yeast was added directly to in vitro fermentations inoculated with feces from lactating sows, whereas as in study 2, feces collected from lactating sows fed LY as a daily supplement was used. Selected feedstuffs were digested and the residue added to separate replicated (n = 3) fermentation reactions. Study 1 was conducted in two blocks, whereas study 2 was conducted using feces collected after a period of 3 (Exp. 1) or 4 wk (Exp. 2) of LY supplementation. Accumulated gas produced over 72 h was modeled for each substrate and the kinetics parameters compared between LY and control groups. The molar ratio of the volatile fatty acids (VFAs) produced in vitro were also compared at 12 and 72 h of incubation. In study 1, in vitro addition of yeast increased (P < 0.001) the rate of gas production (Rmax). However, a yeast × substrate effect (P < 0.05) observed for total gas accumulated (A), time to half asymptote (B), and time required to reach maximum rate of fermentation (Tmax) suggested that yeast-mediated increases in extent and rate of fermentation varied by substrate. Greater total gas production was observed only for corn and SBM, associated with greater B and Tmax. Supplementation with LY appeared to increase A and Rmax although with variation between experiments and substrates. In Exp. 1, LY decreased (P < 0.05) B and Tmax. However, a yeast × substrate effect (P < 0.05) was observed for only A (for wheat, barley, corn, and corn DDGS) and Rmax (wheat, barley, corn, and wheat DDGS). In Exp. 2, LY increased (P < 0.0001) A and decreased B. However, an interaction (P < 0.05) with substrates was observed for Rmax (except SBM) and Tmax. With exception of the DDGS samples, LY supplementation increased (P < 0.05) VFA production at 12 and 72 h of incubation. Yeast increased (P < 0.05) the molar ratios of acetic acid and branch-chain fatty acids at 12 h of incubation; however, this response was more variable by substrate at 72 h. In conclusion, LY supplementation increased the rate and extent of in vitro fermentation of a variety of substrates prepared from common feedstuffs. Greater effects were observed when LY was fed to sows than added directly in vitro, suggesting effects on fermentation were not mediated directly.

The safe enterocin DD14 is a leaderless two-peptide bacteriocin with anti-Clostridium perfringens activity.

Enterococcus faecalis 14, a strain previously isolated from meconium, displayed activity against four Clostridium perfringens isolates when co-cultured on agar plates. The anti-Clostridium activity was ascribed to the production of enterocin DD14, which was subsequently purified. The minimum inhibitory concentration (MIC) of enterocin DD14 against one collection strain and one clinical C. perfringens strain was determined at 50 µg/mL. Furthermore, using the intestinal epithelial cell line IPEC-1, it was shown that E. faecalis 14 was not cytotoxic after 24 h of contact, and no cytotoxicity was observed when IPEC-1 cells were incubated with pure enterocin DD14 for 4 h. Enterocin DD14 was characterised using mass spectrometry and was shown to consist of two small proteins of 5200.74 Da and 5206.41 Da, respectively. The two peptides (DD14A and DD14B) have highly similar amino acid sequences and no signal peptide, which classifies enterocin DD14 as a class IIb leaderless two-peptide bacteriocin. The genes encoding DD14A and DD14B were sequenced and were shown to be 100% identical to other previously described enterocins MR10A and MR10B, in contrast to the producing strains, which are different. Consequently, the present in vitro study supports the potential of this E. faecalis 14 strain and/or its purified enterocin DD14 as putative anti-C. perfringens compounds in chickens.

Dietary live yeast and mannan-oligosaccharide supplementation attenuate intestinal inflammation and barrier dysfunction induced by Escherichia coli in broilers.

The effects of live yeast (LY) and mannan-oligosaccharide (MOS) supplementation on intestinal disruption induced by Escherichia coli in broilers were investigated. The experimental design was a 3×2 factorial arrangement with three dietary treatments (control, 0·5 g/kg LY (Saccharomyces cerevisiae, 1·0×1010 colony-forming units/g), 0·5 g/kg MOS) and two immune treatments (with or without E. coli challenge from 7 to 11 d of age). Samples were collected at 14 d of age. The results showed that E. coli challenge impaired (P<0·05) growth performance during the grower period (1-21 d) and the overall period (1-35 d) of broilers, increased (P<0·05) serum endotoxin and diamine oxidase levels coupled with ileal myeloperoxidase and lysozyme activities, whereas reduced (P<0·05) maltase activity, and compromised the morphological structure of the ileum. Besides, it increased (P<0·05) the mRNA expressions of several inflammatory genes and reduced occludin expression in the ileum. Dietary treatment with both LY and MOS reduced (P<0·05) serum diamine oxidase and ileal myeloperoxidase levels, but elevated villus height (P<0·10) and the ratio of villus height:crypt depth (P<0·05) of the ileum. It also alleviated (P<0·05) E. coli-induced increases (P<0·05) in ileal Toll-like receptor 4, NF-κ B and IL-1 ß expressions. Moreover, LY supplementation reduced (P<0·05) feed conversion ratio of birds during the grower period and enhanced (P<0·05) the community diversity (Shannon and Simpson indices) of ileal microbiota, whereas MOS addition counteracted (P<0·05) the decreased ileal IL-10 and occludin expressions in challenged birds. In conclusion, both LY and MOS supplementation could attenuate E. coli-induced intestinal disruption by alleviating intestinal inflammation and barrier dysfunction in broilers. Moreover, LY addition could improve intestinal microbial community structure and feed efficiency of broilers.

Alternatives to Antibiotics to Prevent Necrotic Enteritis in Broiler Chickens: A Microbiologist's Perspective.

Since the 2006 European ban on the use of antibiotics as growth promoters in animal feed, numerous studies have been published describing alternative strategies to prevent diseases in animals. A particular focus has been on prevention of necrotic enteritis in poultry caused by Clostridium perfringens by the use of microbes or microbe-derived products. Microbes produce a plethora of molecules with antimicrobial properties and they can also have beneficial effects through interactions with their host. Here we review recent developments in novel preventive treatments against C. perfringens-induced necrotic enteritis in broiler chickens that employ yeasts, bacteria and bacteriophages or secondary metabolites and other microbial products in disease control.

Effects of dietary yeast strains on immunoglobulin in colostrum and milk of sows.

The ban of antibiotic growth promoters in pig diet required the development of alternative strategies and reinforced the importance of maternal immunity to protect neonates from intestinal disorders. Milk from sows fed active dry yeasts during gestation and lactation exhibited higher immunoglobulin (Ig) and protein content in milk at day 21 of lactation. In this study, we investigated whether the administration of Saccharomyces cerevisiae strains of various origins (Sc01, Sc02, Sb03) to sows during late gestation and lactation could induce higher Ig content in colostrum and milk. Results show that yeast supplementation did not increase significantly sow body weight at days 112 of gestation and 18 of lactation as well as piglet body weight gain from birth to weaning. In contrast, the IgG level in colostrum was increased in comparison with the control group when sows were supplemented with Sc01 at both 0.05 and 0.5% (p<0.05) and Sb03 at 0.5% (p<0.01). During the lactation, the level of milk IgG remained significantly higher in comparison with the control group when sows were supplemented with Sc02 at 0.05% and 0.5% and with Sb03 at 0.5%. Furthermore, in comparison with the control sows, the level of milk IgA was significantly maintained in sows supplemented with the 3 yeast strains at 0.05%. The incidence of piglet diarrhoea was decreased in groups Sc01 at both 0.05% and 0.5% and Sc02 at 0.05%. Thus, these results show that the 3 yeast strains display immunostimulatory effects on maternal immunity, but only Sc01 supplementation at 0.05% allowed jointly the increase of IgG level in colostrum, the maintenance of IgA level in milk and the decrease of piglet diarrhoea incidence. This stimulation of maternal immunity could be associated with a better systemic (colostrum IgG) and local (milk IgA) protection of neonates and suggests that dietary yeasts may have stimulated the local gut immune system of sows.

Saccharomyces cerevisiae modulates immune gene expressions and inhibits ETEC-mediated ERK1/2 and p38 signaling pathways in intestinal epithelial cells.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. ETEC infections cause pro-inflammatory responses in intestinal epithelial cells and subsequent diarrhea in pigs, leading to reduced growth rate and mortality. Administration of probiotics as feed additives displayed health benefits against intestinal infections. Saccharomyces cerevisiae (Sc) is non-commensal and non-pathogenic yeast used as probiotic in gastrointestinal diseases. However, the immuno-modulatory effects of Sc in differentiated porcine intestinal epithelial cells exposed to ETEC were not investigated. METHODOLOGY/PRINCIPAL FINDINGS: We reported that the yeast Sc (strain CNCM I-3856) modulates transcript and protein expressions involved in inflammation, recruitment and activation of immune cells in differentiated porcine intestinal epithelial IPEC-1 cells. We demonstrated that viable Sc inhibits the ETEC-induced expression of pro-inflammatory transcripts (IL-6, IL-8, CCL20, CXCL2, CXCL10) and proteins (IL-6, IL-8). This inhibition was associated to a decrease of ERK1/2 and p38 MAPK phosphorylation, an agglutination of ETEC by Sc and an increase of the anti-inflammatory PPAR-γ nuclear receptor mRNA level. In addition, Sc up-regulates the mRNA levels of both IL-12p35 and CCL25. However, measurement of transepithelial electrical resistance displayed that Sc failed to maintain the barrier integrity in monolayer exposed to ETEC suggesting that Sc does not inhibit ETEC enterotoxin activity. CONCLUSIONS: Sc (strain CNCM I-3856) displays multiple immuno-modulatory effects at the molecular level in IPEC-1 cells suggesting that Sc may influence intestinal inflammatory reaction.

Intrauterine growth restriction delays feeding-induced gut adaptation in term newborn pigs.

BACKGROUND: Neonates born after intrauterine growth restriction (IUGR) show higher mortality and morbidity and greater feeding problems postnatally. OBJECTIVES: We tested the hypothesis that IUGR affects the intestinal structure, function, microbiology and proinflammatory cytokine expression during the immediate neonatal period. METHODS: We firstly compared organ weights and intestinal digestive enzyme activities between control and IUGR newborn piglets delivered by cesarean section at full term or prematurely (91% gestation). Next, we compared intestinal structure, function and microbiota in spontaneously delivered control and IUGR term piglets during the period 0-5 days of age, when intestinal adaptation is normally very rapid. RESULTS: At the time of birth, organ weights and intestinal enzyme activities were not notably affected by IUGR, neither for preterm nor term pigs, except that IUGR was associated with a relatively long and thin intestine. Between birth and 5 days of age, the normal developmental pattern in the ileum and colon appeared to be delayed in term IUGR piglets, as indicated by lower ileal density (weight per unit length) and villous area (-20 to -30%), higher expression of the peptide transporter PEPT1 (+150% on day 2) and enhanced bacterial adhesion and translocation during days 2-5 (all p < 0.05). Further, expression of the proinflammatory cytokine IL-6 was modified in the intestine of term IUGR piglets at birth without any change in IL-1ß. CONCLUSION: IUGR is associated with a longer and thinner intestine at birth, and during the immediate postnatal period, intestinal adaptation and bacterial colonization are altered in IUGR piglets born at full term.

Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes.

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5'monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-L-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.

EP45 accumulates in growing Xenopus laevis oocytes and has oocyte-maturation-enhancing activity involved in oocyte quality.

The capacity of oocytes to fully support meiotic maturation develops gradually during oocyte growth. Growing oocytes accumulate proteins and mRNAs required for this process. However, little is known about the identity of these factors. We performed a differential proteomic screen comparing the proteomes of growing stage-IV oocytes, which do not undergo meiotic maturation in response to progesterone, with fully grown stage-VI ones, which do. In 2D gels of stage-VI oocytes, we identified a group of four protein spots as EP45 (estrogen-regulated protein 45 kDa), which belongs to the family of serine protease inhibitors and is also known as Seryp or pNiXa. Western blot analysis after mono- and bi-dimensional electrophoreses confirmed the accumulation of certain forms of this protein in oocytes between stages IV and VI. EP45 mRNA was not detectable in oocytes or ovaries, but was expressed in the liver. A low-mobility isoform of EP45 was detected in liver and blood, whereas two (occasionally three or four) higher-mobility isoforms were found exclusively in oocytes, suggesting that liver-synthesized protein is taken up by oocytes from the blood and rapidly modified. Alone, overexpression of RNA encoding either full-length or N-terminally truncated protein had no effect on meiotic resumption in stage-IV or -VI oocytes. However, in oocytes moderately reacting to low doses of progesterone, it significantly enhanced germinal-vesicle breakdown, showing a novel and unsuspected activity of this protein. Thus, EP45 accumulates in growing oocytes through uptake from the blood and has the capacity to act as an 'oocyte-maturation enhancer' ('Omen').

Intrauterine growth restriction modifies the developmental pattern of intestinal structure, transcriptomic profile, and bacterial colonization in neonatal pigs.

Neonates with intrauterine growth restriction (IUGR) are prone to suffer from digestive diseases. Using neonatal pigs with IUGR, we tested the hypothesis that IUGR may induce alterations in the developmental pattern of intestinal barrier and thereby may be responsible for IUGR-associated increased morbidity. Piglets with a birth weight near the mean birth weight (+/-0.5 SD) were identified as normal birth weight (control) and piglets with a mean -2 SD lower birth weight (-30%) were defined as piglets with IUGR. The developmental pattern of intestinal structure, transcriptomic profile, and bacterial colonization was investigated from birth to d 5 postnatal. At birth, intestinal weight and length, ileal and colonic weight per unit of length, and villous sizes were lower (P < 0.05) in piglets with IUGR than in same-age control piglets. These IUGR-induced intestinal alterations further persisted, although they were less marked at d 5. Counts of adherent bacteria to ileal and colonic mucosa were greater (P < 0.05) in 2-d-old piglets with IUGR than in same-age control piglets. Dynamic analyses of the transcriptomic profile of the intestine revealed molecular evidence of IUGR-induced intestinal growth impairment that may result from a change in the cell proliferation-apoptosis balance during the first days of life, while a protective process would occur later on. In addition, changes in the expression of several genes suggest a pivotal role of both glucocorticoids and microbiota in driving IUGR intestinal development during the neonatal period.

Fatal effects of a neonatal high-protein diet in low-birth-weight piglets used as a model of intrauterine growth restriction.

BACKGROUND: Although full-term infants suffering intrauterine growth restriction (IUGR) are routinely fed high-protein (HP) formulas to ensure catch-up growth, the effects of HP intake are poorly understood. An IUGR piglet model provides an opportunity to investigate these effects. METHODS AND RESULTS: Twelve IUGR piglets were artificially fed HP formulas (50% more protein in comparison to sow milk) from the 2nd day of life (d2) until d28. Unexpectedly, all HP piglets developed poor growth, severe hypotonia and polypnea between d10 and d16. One third died spontaneously. This syndrome was investigated to understand its pathophysiology and to adopt a strategy to restore health. Blood and urine biochemistry and amino acid concentrations were investigated in 10 HP piglets and 8 piglets that were fed a normal-protein (NP) formula. In comparison to NP piglets, HP piglets showed significant hypokalemia (2.7 +/- 0.6 vs. 3.6 +/- 0.6 mmol/l; p < 0.01), hypophosphatemia (1.5 +/- 0.2 vs. 3.0 +/- 0.3 mmol/l; p > 0.01), hypercalcemia (3.0 +/- 0.3 vs. 2.5 +/- 0.2 mmol/l; p < 0.01), hyperammonemia (365 +/- 4 vs. 242 +/- 15 micromol/l; p < 0.05), elevated blood urea (6.5 +/- 0.4 vs. 1.3 +/- 0.4 mmol/l; p < 0.01) and elevated taurine concentrations (50.2 +/- 8.5 vs. 17.7 +/- 2.7 micromol/l; p < 0.01). CONCLUSIONS: These altered parameters indicated inadequate potassium and phosphorus dietary supplies in HP piglets. When the HP formula was supplemented with monocalcium phosphate and monopotassium phosphate (HP-sup), serum biochemistry was normalized in piglets fed this formula (n = 8). This experimental strategy restored growth in IUGR piglets fed HP-sup, without a toxic effect. The current findings suggest that use of an HP formula without a proportional increase in its phosphorus and potassium content induces pathology similar to the refeeding syndrome in IUGR piglets.

IUGR does not predispose to necrotizing enterocolitis or compromise postnatal intestinal adaptation in preterm pigs.

UNLABELLED: IUGR and preterm birth are leading causes of neonatal morbidity. We tested the hypothesis that IUGR predisposes to gut maladaption and necrotizing enterocolitis (NEC) using preterm pigs as models for preterm infants. First, full-term normal birth weight (NW) and IUGR ( approximately 65% of NW) pigs were compared. IUGR reduced intestinal weight per length, proportion mucosa, villous area, and sucrase activity at 2 d after birth (p < 0.05) but did not change relative organ weights. Next, groups of preterm pigs were fed formula or colostrum, starting at birth or after 2-3 d of total parenteral nutrition (TPN). Neonatal mortality (not related to NEC) was increased in IUGR versus NW preterm pigs (28 vs 10%, p < 0.01). NEC incidence was similar between IUGR and NW but higher after formula than colostrum feeding (46 vs 12%, p < 0.01) and higher after TPN than without TPN (61 vs 34% for formula pigs, p < 0.01). After feeding, relative intestinal mass and length were higher in IUGR versus NW pigs (+25-80%, p < 0.05) while brush border enzyme activities were similar. An enhanced gut trophic response to enteral feeding may help to improve postnatal intestinal adaptation and NEC resistance in preterm IUGR newborns. ABBREVIATIONS: :