ETEC Challenge Research Articles

PSLBI-20 Dynamic response of piglets fed different sources and levels of fiber during an enterotoxigenic Escherichia coli (ETEC) challenge in the post-weaning transition

Abstract Dietary fiber can stimulate the growth of volatile fatty acid-producing bacteria, hence promoting gut health. A stimbiotic can accelerate the settlement of a fiber-fermenting microbiome after weaning. The present study aimed to investigate how different fiber types and a stimbiotic can support microbiome function and resilience of piglets facing an F18 ETEC challenge. Newly weaned piglets (n = 216) were blocked by body weight (BW) and randomly assigned to 1 of 4 dietary treatments (n = 9), including a corn-soy and wheat middlings control diet (CTL), high fiber diet (HF), CTL plus 0.01% stimbiotic (CS; Signis; AB Vista, UK), and HF plus 0.01% stimbiotic (HFS). The HF diets included sugar beet pulp at 5.0%, 3.8%, 2.5% and soy hulls at 4.0%, 3.0% and 2.0% for phases 1 (d 0 to 9), 2 (d 0 to 16), and 3 (d 16 to 22), respectively. A standard diet was fed during phase 4 (d 22 to 41). On d 5 post-weaning, all pigs were orally inoculated with 10 mL of F18 ETEC (108 CFU/mL). Twelve piglets were selected from different pens, and a fecal sample was collected on d 5 before challenge (0 d post-inoculation; dpi), d 12 (dpi 7) and d 29 (dpi 24) for analysis of microbial populations by qPCR and fecal calprotectin. Statistical analyses consisted of a multivariate analysis by age, with fiber, stimbiotic, and their interaction as fixed effects. There was no main effect of fiber or stimbiotic on final BW, and d 0 to 40 average daily gain (ADG) or gain to feed (G:F; P > 0.10). Overall, pigs fed CS had a greater intake than HFS (P < 0.05) with CTL and HF intermediate. Piglets fed CS and HF tended to have greater ADG from d 0 to 22 compared with pigs fed CTL (P < 0.10). Piglets fed CS tended to have heavier d 22 BW than CTL (P < 0.10), with HF and HFS intermediate. On dpi 0, butyrate-producing bacteria Lachnospiraceae and Ruminococcaceae were increased in piglets fed a stimbiotic (P < 0.05). On dpi 0 and 7, piglets fed HF had increased abundance of Lachnospiraceae (P < 0.05), whereas no difference was observed on dpi 24 (P > 0.10). Total E. coli did not vary between groups (P > 0.10). On dpi 0, calprotectin concentration was similar between treatments (averaged 25 ng/g feces; P > 0.10) Calprotectin was less in piglets fed a stimbiotic, at 7 vs 22 ng/g feces on dpi 7, and 8 vs 19 ng/g feces on dpi 24 in the stimbiotic and no stimbiotic group, respectively (P < 0.05). There was no fiber or fiber × stimbiotic interaction for calprotectin (P > 0.10). These results demonstrate that feeding high fiber may accelerate the settlement of the piglet microbiome profile as early as 5 d post-weaning. Feeding a stimbiotic may support the resilience of piglets facing an F18 ETEC challenge by promoting the growth of butyrate-producing bacteria, which alleviates the gastrointestinal tract inflammation response and ultimately boost growth performance.

Ningxiang Pig-Derived Parabacteroides distasonis HNAU0205 Alleviates ETEC-Induced Intestinal Apoptosis, Oxidative Damage, and Inflammation in Piglets.

Weaning is a critical stage in the growth and development of piglets, often inducing stress reactions. This study aims to investigate the effects of Parabacteroides distasonis (PBd) derived from Ningxiang pigs on growth performance, intestinal apoptosis, oxidative damage, and inflammation in ETEC-challenged weaned piglets. A total of 22 Duroc × Landrace × Yorkshire (DLY) piglets, 24 days old with similar body weights, were randomly divided into three groups: Control (n = 7), ETEC (n = 7), and PBd + ETEC (n = 8). The results show that, compared to the Control group, ETEC challenge led to decreased growth performance, reduced villus height in the duodenum and jejunum, increased crypt depth in the duodenum, a decreased villus-height-to-crypt-depth ratio, increased expression of apoptosis-related genes (Caspase-8 and Caspase-9), increased expression of oxidative damage-related genes (Nrf2, GSH-PX, mTOR, and Beclin1), increased expression of inflammation-related genes (Myd88, P65, TNF-α, and IL-6), and reduced the contents of SCFAs in the colonic chyme (acetate, propionate, butyrate, valerate, and total SCFAs). Compared to the ETEC group, the PBd + ETEC group alleviated the reduction in growth performance, mitigated intestinal morphological damage, and reduced the expression of the aforementioned apoptosis, oxidative damage, and inflammation-related genes with the increase in SCFAs. In conclusion, PBd derived from Ningxiang pigs effectively reduces ETEC-induced intestinal damage in weaned piglets, improves intestinal health, and increases the content of SCFAs in the colonic chyme, thereby enhancing growth performance.

Ningxiang pigderived Enterococcus hirae regulates the inflammatory function and enhances the protection of piglets against ETEC challenge.

This study investigated the effects of Enterococcus hirae (Eh) derived from Ningxiang pigs on growth performance, diarrhea incidence, and immune responses in ETEC-challenged piglets. The results showed that compared to the CON group, ETEC infection significantly reduced the average daily gain (ADG) and average daily feed intake (ADFI), increased rectal temperature, and resulted in a diarrhea rate of up to 24%. Additionally, ETEC infection significantly increased the spleen index and the expression of inflammatory cytokines in the spleen, serum and intestine, with decreasing serum sIgA and colonic SCFAs of piglets. Compared to the ETEC group, orally Eh significantly increased ADFI in ETEC-infected piglets, reduced the diarrhea rate to 11.53%, reduced the spleen index and the expression of inflammatory cytokines in the spleen, serum and intestine, with decreasing serum sIgA and colonic SCFAs of ETEC-infected piglets. Furthermore, correlation analysis revealed that the levels of SCFAs (particularly acetate) were significantly negatively correlated with the expression levels of inflammatory cytokines in colonic and splenic tissues, suggesting that acetate may be a key metabolite in the anti-inflammatory effects of Eh. These results indicate that Eh can enhance the protection of piglets against ETEC K88 via intestine-acetate-spleen axis, thereby alleviating diarrhea and improving growth performance in piglets.

386 Effects of Different Soy Protein Sources on Gut Microbiome Composition and Relationship with Performance in Pigs in Regular Nursery and F4 Enterotoxigenic Escherichia Coli (ETEC) Challenged Conditions

Abstract Provisoy, produced by hydro, thermal, and mechanical process using soybean meal (SBM) has been shown to improve both post-weaning pig growth and gut health compared with SBM. It was hypothesized that Provisoy could improve the utilization of both protein and fiber fractions along the gastrointestinal tract of a nursery pig resulting in a more balanced nutrient environment and robust microbiome in the hindgut compared with SBM. A total of 268 weaned pigs (body weight, 6.82 ± 0.85 kg) were allotted to either a regular nursery condition (5 pigs/pen, 10-11 pens/treatment) or subjected to ETEC challenge (3 pigs/pen, 12 pens/treatment, 1 pig per pen was orally gavaged with O149:K91:K88 strain on d 5 post-weaning). Pigs received one of the following diets: SBM, Provisoy or HP300 (soy protein from Hamlet Protein, used as a benchmark). A standard wheat-barley-SBM based diet was used, and test soy products were added to replace SBM in the treatment diets during the first three weeks of nursery program. All diets were isocaloric with the same standardized ileal digestible lysine level in a three-phase (38 days) nursery program. Fecal samples were collected on d 13 or d 14 post-weaning and subjected to DNA extraction and Nanopore shotgun sequencing. Data were center-log-ratio transformed and subjected to statistical analysis. Microbiome composition was affected by dietary treatment (P < 0.01) and challenge (P < 0.05), but their interaction was not significant. In regular nursery condition, both Provisoy and HP300 tended (P < 0.10) to have greater microbiome alpha-diversity compared with SBM, but no difference was observed between these two treatments. Only SBM fed pigs subjected to ETEC challenge had greater (P = 0.02) alpha-diversity compared with their non-challenged counterparts. Compared with the SBM treatment, pigs that received Provisoy in the regular condition had greater abundance of Coprococcus and Dialister, which were positively correlated with greater average daily gain (ADG; r = 0.53, P < 0.01, and r = 0.45, P = 0.01, respectively). Under ETEC challenge, an increase in abundance of Dialister was observed in pigs that received Provisoy treatment with a positive correlation with ADG (r = 0.42, P = 0.01). In conclusion, dietary inclusion of Provisoy or HP300 improved microbiome alpha-diversity compared with SBM. Provisoy helped promote the growth of beneficial bacteria in the hindgut of nursery pigs which were positively correlated with gain.

140 Effects of Dietary Inclusion of a Medium Chain Fatty Acid Blend and a Phytogenic Feed Additive on Growth Performance, Gut Health, and Mortality of Escherichia Coli-Challenged Pigs

Abstract An experiment was conducted to test the hypothesis that supplementing diets with a blend of medium chain fatty acids (MCFA) and a phytogenic feed additive (Fresta Protect, Delacon, Linz, Austria), together or separately, would improve growth performance and stool quality of nursery pigs challenged with Escherichia coli (ETEC). A total of 200 pigs [5.52 ± 0.17 kg body weight (BW)] were weaned at 17 to 19 days of age, allotted to 40 pens (5 pigs/pen), and allowed to acclimate for 2 days before initiation of dietary treatments. Pens were blocked by BW and randomly assigned to 1 of 5 treatments: negative control (NC), NC + 55 mg/kg carbadox (Positive control; PC), NC + 1.0% MCFA, NC + 0.1% phytogenic, or NC + 1.0% MCFA and 0.1% phytogenic. All pigs were administered an oral gavage of 5 mL ETEC (blend of K88+ and F18+) with a final titer of 1010 CFU/pig on day 5 and 6. Body weights were recorded on days 0, 4, 11, and 18, whereas feed intake was recorded for the overall trial (day 0 - 18). Fecal scores were assessed daily on an ordered likert scale to determine the joint probability of more normal stool quality. Data were analyzed using the lme4 package of R 4.1.2, and the statistical models included the fixed effect of treatment and random effect of BW block. Growth performance data were assumed to be normally distributed, whereas mortality and stool quality were analyzed using generalized linear mixed models specifying binomial and multinomial distributions, respectively. Contrasts were used to test for effects of dietary additive (carbadox, MCFA, phytogenic, or MCFA and phytogenic) compared with NC (Table 1). Results indicated post-challenge BW, post-challenge and overall average daily gain (ADG), and overall gain:feed (G:F) were greater (P < 0.05) for PC than for NC. In the pre-challenge phase, dietary inclusion of MCFA reduced (P < 0.05) final BW and ADG compared with NC, but overall G:F was greater (P < 0.05) than NC when the MCFA blend was fed. Inclusion of the phytogenic increased (P < 0.05) ADG in the post-challenge period and G:F in the overall trial compared with NC. The combination of MCFA blend and phytogenic resulted in increased (P < 0.05) post-challenge final BW, post-challenge ADG, and overall ADG compared with NC. All treatments, except MCFA blend, tended (P < 0.10) to reduce mortality compared with NC. The PC, MCFA blend, and combination treatments resulted in a greater (P < 0.05) probability of normal stool quality than NC. In conclusion, supplementation of the phytogenic and the combination of MCFA with phytogenic improved growth performance and tended to reduce mortality of nursery pigs under ETEC challenge. Furthermore, the combination improved stool quality.

Effects of mixed fibres and essential oils blend on growth performance and intestinal barrier function of piglets challenged with enterotoxigenic Escherichia coli K88.

This study was to evaluate the effects of supplementing mixed dietary fibres (MDF) and essential oils blend (EOB) either alone or in combination on growth performance and intestinal barrier function in weaned piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). Forty-two piglets (28 days old) were randomly allocated into six treatments in a 25-day experiment, and fed the basal diet (CON or ETEC) either with antibiotics (AT), MDF, EOB or MDF + EOB. On Day 22 of the experiment, pigs in CON and challenged groups (ETEC, AT, MDF, EOB and MDF + EOB) were orally administered sterile saline and ETEC containing 6 × 1010 CFU/kg body weightrespectively. On Day 26, all pigs were euthanized to collect samples. Before ETEC challenge, piglets in MDF and EOB had lower diarrhoea incidence (p < 0.01) than others. After ETEC challenge, piglets in ETEC had lower average daily gain and higher diarrhoea incidence (p < 0.05) than those of CON. Furthermore, compared to CON, ETEC group increased the serum lipopolysaccharideconcentration and diamine oxidaseactivity, and decreased mRNA levels of genes relating to barrier function (aquaporin 3, AQP3; mucin1, MUC1; zonula occludens-1, ZO-1; Occludin), and increased the concentration of cytokines (interleukin-1β/4/6/10, IL-1β/4/6/10) and secretory immunoglobulin A (sIgA) in jejunal mucosa (p < 0.05). However, these deleterious effects induced by ETEC were partly alleviated by MDF, EOB, MDF + EOB and AT. Additionally, compared to ETEC group, MDF increased Bifidobacterium abundance in cecal digesta and butyrate concentration in colonic digesta (p < 0.05). Also, EOB improved propionate concentration in cecal digesta, and MDF + EOB decreased IL-10 concentration in jejunal mucosa (p < 0.05) compared with ETEC. Conclusively, MDF and EOB either alone or in combination can improve growth performance and alleviate diarrhoea via improving intestinal barrier function of piglets after ETEC challenge, and all may serve as potential alternatives to AT for piglets.

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses.

Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311–mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311–mCherry challenge in Sprague–Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-STKO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-STKO than with E. coli BE311. Therefore, the E. coli BE311–mCherry and E. coli BE311-STKO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.

Long-Chain Polyunsaturated Fatty Acids Attenuate Enterotoxigenic Escherichia coli K88-Induced Intestinal Barrier Damage via PPARγ Regulation in IPEC-J2 Cells

ObjectivesEnterotoxigenic Escherichia coli (ETEC) impairs the intestinal barrier function and triggers inflammatory response in infants and young animals. Long-chain polyunsaturated fatty acids (LC-PUFA) have been reported to improve intestinal health and limit inflammation. The objective is to define the role of LC-PUFA on intestinal barrier integrity with ETEC induced inflammation in intestinal porcine epithelial cells (IPEC-J2). MethodsCultured IPEC-J2 cells were pretreated with 30 μM LC-PUFA and then incubated without ETEC challenge (NE) or with ETEC challenge (CE). Transepithelial electrical resistance (TEER) value, fluorescein isothiocyanate-dextran (FITC-dextran) flux, released cytosolic lactate dehydrogenase (LDH), and interleukin 8 (IL-8) measures were evaluated. Protein abundance of claudin-1, occludin, and nuclear peroxisome proliferator-activated receptor-gamma (PPARγ) were measured by Western blot analysis. ResultsTreatment of cells with docosahexaenoic acid (DHA) significantly increased TEER when compared to the bovine serum albumin (BSA; 14% NE and 25% CE, respectively) and compared to oleate control (25% NE and 27% CE, respectively) (P < 0.05). Further, DHA decreased FITC-dextran across the epithelial barrier by 56% (P < 0.05) and decreased IL-8 secretion by 26% compared to oleate, regardless of ETEC challenge (P > 0.05). In the presence of ETEC, eicosapentaenoic acid (EPA) protected against a decline in membrane claudin-1 compared to EPA non-challenged cells (P > 0.05). In addition, arachidonic acid (ARA) and DHA tended to increase membrane occludin (P < 0.1). Additionally, there was a 1.6-fold increase in nuclear PPARγ by treatments of both ARA and DHA compared to BSA, regardless of challenge (P < 0.05). Release of LDH, a measure of cell cytotoxicity, was decreased by the ARA (64%), EPA (27%), and DHA (34%) in ETEC treated cells compared to BSA ETEC treated cells (P < 0.05). ConclusionsThese data suggest that LC-PUFA protect tight junction proteins by modulating PPARγ, thereby improving epithelial integrity in ETEC-challenged IPEC-J2 cells. Funding SourcesUnited States Department of Agriculture Hatch Project.

PSXVI-3 Supplemental effects of Bacillus sp. in prevention of post-weaning diarrhea caused by enterotoxigenic F18+ Escherichia coli in nursery pigs

Abstract This study investigated the effects of Bacillus sp. on intestinal health and prevention of diarrhea by F18+ Escherichia coli (ETEC) in nursery pigs. Forty-eight weaned pigs (7.9 ± 0.5 kg BW) were allotted to 4 treatments with 12 replicates (NC: no-challenge; PC: challenge/no-treat; BMD: challenge/bacitracin; BAC: challenge/Bacillus sp. at 109 CFU/kg feed). All pigs were fed diets for 28 d. At d 7, challenged groups were orally inoculated with ETEC (1.8 × 1010 CFU) and NC group received sterile solution. Growth performance was analyzed weekly and pigs were euthanized on d 28 to measure intestinal health. Data were analyzed using SAS 9.4. There were no difference on growth performance among treatments during pre-challenge period. From d 7 to 14, PC tended to reduce (P = 0.067) ADG (352 to 440 g/d), whereas no effect was observed during the overall period. The PC increased (P &amp;lt; 0.05) fecal score at d 7 (post-challenge) (4.1 to 3.3) and d 8 (4.1 to 3.3), whereas BMD decreased (P &amp;lt; 0.05) it at d 9 (4.3 to 3.5) and d 11 (3.8 to 3.3). The PC increased (P &amp;lt; 0.05) MDA (0.56 to 0.30 μmol/mg protein) and tended to increase TNFα (P = 0.084; 0.89 to 1.04 pg/mg protein), whereas BMD and BAC reduced (P &amp;lt; 0.05) MDA (0.56 to 0.31, 0.29 μmol/mg protein) and TNFα (1.04 to 0.81, 0.77 pg/mg protein) in jejunal mucosa. The PC reduced (P &amp;lt; 0.05) α-diversity (Chao1: 32.3 to 19.4), whereas BAC increased it (Chao1: 19.4 to 30.7) whereas BMD increased (P &amp;lt; 0.05) it (Shannon: 1.85 to 2.44; Simpson: 0.55 to 0.70) of jejunal mucosa-associated microbiota. In conclusion, ETEC challenge caused diarrhea, increased immune response, and oxidative stress by disrupting mucosa-associated microbiota. The BMD and BAC reduced jejunal immune response and oxidative stress by restoring mucosa-associated microbiota.

Metabolomic Profile of Weaned Pigs Challenged with E. coli and Supplemented with Carbadox or Bacillus subtilis.

This study explored the metabolomic profiles in ileal mucosa and colon digesta in response to enterotoxigenic Escherichia coli F18 (ETEC) infection and dietary use of probiotics and low-dose antibiotics. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight) were randomly allotted to one of four treatments. Pigs in the negative control (NC) were fed a basal diet without ETEC challenge, whereas pigs in the positive control (PC), antibiotic, and probiotic groups were fed the basal diet, basal diet supplemented with 50 mg/kg of carbadox, or 500 mg/kg of Bacillus subtilis, respectively, and orally challenged with ETEC F18. All pigs were euthanized at day 21 post-inoculation to collect ileal mucosa and colon digesta for untargeted metabolomic profiling using gas chromatography coupled with time-of-flight mass spectrometry. Multivariate analysis highlighted a more distinct metabolomic profile of ileal mucosa metabolites in NC compared to the ETEC-challenged groups. The relative abundance of 19 metabolites from the ileal mucosa including polyamine, nucleotide, monosaccharides, fatty acids, and organic acids was significantly different between the NC and PC groups (q < 0.1). In colon digesta, differential metabolites including 2-monoolein, lactic acid, and maltose were reduced in the carbadox group compared with the probiotics group. In conclusion, several differential metabolites and metabolic pathways were identified in ileal mucosa, which may suggest an ongoing intestinal mucosal repair in the ileum of ETEC-challenged pigs on day 21 post-inoculation.

Manno-oligosaccharide attenuates inflammation and intestinal epithelium injury in weaned pigs upon enterotoxigenic Escherichia coli K88 challenge.

To explore the effect of manno-oligosaccharide (MOS) on intestinal health in weaned pigs upon enterotoxigenic Escherichia coli K88 (ETEC) challenge, thirty-two male weaned pigs were randomly assigned into four groups. Pigs fed with a basal diet or basal diet containing MOS (0·6 g/kg) were orally infused with ETEC or culture medium. Results showed that MOS significantly elevated the digestibility of crude protein and gross energy in both ETEC-challenged and non-challenged pigs (P < 0·05). MOS also elevated serum concentrations of IgA and IgM (P < 0·05), but decreased serum concentrations of TNF-α, IL-1β and IL-6 (P < 0·05) in ETEC-challenged pigs. Interestingly, MOS increased villus height and the ratio of villus height:crypt depth in duodenum and ileum (P < 0·05). MOS also increased duodenal sucrase and ileal lactase activity in ETEC-challenged pigs (P < 0·05). MOS decreased the abundance of E. coli, but increased the abundance of Lactobacillus, Bifidobacterium and Bacillus in caecum (P < 0·05). Importantly, MOS not only elevated the expression levels of zonula occludens-1 (ZO-1), claudin-1 and GLUT-2 in duodenum (P < 0·05) but also elevated the expression levels of ZO-1, GLUT-2 and L-type amino acid transporter-1 in ileum (P < 0·05) upon ETEC challenge. These results suggested that MOS can alleviate inflammation and intestinal injury in weaned pigs upon ETEC challenge, which was associated with suppressed secretion of inflammatory cytokines and elevated serum Ig, as well as improved intestinal epithelium functions and microbiota.

A Combination of Formic Acid and Monolaurin Attenuates Enterotoxigenic Escherichia coli Induced Intestinal Inflammation in Piglets by Inhibiting the NF-κB/MAPK Pathways with Modulation of Gut Microbiota.

This study determined the potential of formic acid plus monolaurin (FA + ML) as an alternative to antibiotics in diet when piglets are challenged with ETEC. Piglets fed the FA + ML diet had lower fecal score and rectal temperature after the ETEC challenge. In addition, FA + ML supplementation induced lower plasma TNF-α, IL-6, and IL-1β concentration postchallenge, downregulated the mRNA expression of TNF-α, IL-1β, IL-6, and TLR4 in the ileum and TLR4 and CFTR in the jejunum. Phosphorylation levels of NF-κB p65 and MAPK p38 were reduced in the ileum of piglets fed FA + ML diet. Supplementation of FA + ML increased the relative abundance of genera Lactobacillus especially Lactobacillus amylovorus species and decreased the genus abundances of Actinobacillus, unidentified Enterobacteriaceae, Moraxella. Collectively, the combination of formic acid and monolaurin in diets have the potential to be an antibiotic alternative to mitigate inflammatory response in piglets challenged with ETEC.

Enterococcus faecium NCIMB 10415 administration improves the intestinal health and immunity in neonatal piglets infected by enterotoxigenic Escherichia coli K88

BackgroundThis study aimed to investigate the effects of oral administration of Enterococcus faecium NCIMB 10415 (E. faecium) on intestinal development, immunological parameters and gut microbiota of neonatal piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). A total of 96 1-day-old sow-reared piglets were randomly assigned to 2 groups, with 48 piglets in each group. The piglets were from 16 litters (6 piglets each litter), and 3 piglets each litter were allocated to the E. faecium-supplemented (PRO) group, while the other 3 piglets were allocated to the control (CON) group. After colostrum intake, piglets in the PRO group were orally administrated with 3 × 109 CFU E. faecium per day for a period of one week. On day 8, one piglet per litter from each group was challenged (CON+ETEC, PRO+ETEC) or not (CON-ETEC, PRO-ETEC) with ETEC in a 2 × 2 factorial arrangement of treatments. On day 10 (2 days after challenge), blood and tissue samples were obtained from piglets.ResultsBefore ETEC challenge, there were no significant differences for the average daily gain (ADG) and fecal score between the two groups of piglets. After ETEC challenge, the challenged piglets had greater fecal score compared to the non-challenged piglets, whereas E. faecium administration was able to decrease the fecal score. Piglets challenged with ETEC had shorter villous height, deeper crypt depth, and reduced number of goblet cells in the jejunum and decreased mRNA abundance of claudin-1 in the ileum, whereas increased the percentage of lymphocytes, concentrations of IL-1β in the plasma and TNF-α in the ileal mucosa, as well as increased the mRNA abundances of innate immunity-related genes in the ileum tissue. These deleterious effects caused by ETEC were partly alleviated by feeding E. faecium. In addition, piglets in PRO-ETEC group had decreased the percentage of CD8+ T cells of the peripheral blood when compared to those in CON-ETEC group. Moreover, E. faecium administration increased Verrucomicrobia at phylum level and decreased Bilophila at genus level.ConclusionsThese results suggest that oral administration of E. faecium alleviated the intestinal injury and diarrhea severity of neonatal piglets challenged by ETEC, partly through improving the intestinal microbiota and immune response. This offers a potential strategy of dietary intervention against intestinal impairment by ETEC in neonatal piglets.

132 The impact of F18 ETEC challenge on intestinal integrity and immune response of nursery pigs, and the potential protective effects of direct-fed microbial blends

Abstract The objectives were to investigate the impact of an F18 ETEC challenge on localized immune responses and intestinal function of piglets, and to evaluate potential protective effects of direct-fed microbial blends (DFM1 and DFM2). Thirty-six pens of 3-wk old weaned piglets (2 pigs/pen; BW=6.42 ± 0.22 kg; L337 X Camborough, PIC, Hendersonville, TN) were assigned to one of four treatments: 1) NC: Non-challenged (n = 10), 2) PC:F18 ETEC challenged (n = 10), 3) PC+DFM1 (n = 8) or 4) PC+DFM2 (n = 8). Pigs were either sham-infected with saline or orally challenged with ETEC on d 7 (d 0 post-inoculation, dpi). On dpi 10, ileal mucosa was collected for disaccharidase quantification and whole ileal tissue was collected for immune marker and tight junction protein analysis via qRT-PCR. Data were analyzed using PROC MIXED of SAS (9.4) with dietary treatment as a fixed effect and pen as the experimental unit. There were no differences in lactase activity (P &gt; 0.10). Sucrase and maltase activity increased in PC vs. NC (P &lt; 0.05). Ileal mRNA abundance of zonula occludens-1 (ZO-1) decreased in PC and DFM1 compared with NC (P &lt; 0.05). DFM2 increased ZO-1 compared with PC (P &lt; 0.05). There was no difference in claudin-1 mRNA (P &gt; 0.10). Cluster of differentiation 14 (CD14) mRNA was elevated in challenged pigs compared with NC (P &lt; 0.05). Interleukin-10 (IL-10) tended to be elevated in both DFM treatments compared with PC (P &lt; 0.10). All challenged treatments tended to have elevated tumor necrosis factor-a (TNFa) abundance vs. NC (P &lt; 0.10). There were no differences in IL-1b, IL-6, or toll-like receptor 4 (TLR4) mRNA abundance (P &gt; 0.10). In conclusion, the ETEC challenge increased CD14, tended to increase TNFa, and decreased ZO-1 mRNA abundance, indicating activation of intestinal inflammation and impaired intestinal barrier integrity. DFM2 may improve intestinal barrier integrity impaired by ETEC as evidenced by increased ZO-1 mRNA.

Effects of dietary live yeast supplementation on growth performance, diarrhoea severity, intestinal permeability and immunological parameters of weaned piglets challenged with enterotoxigenic Escherichia coli K88.

This study aimed to investigate the effects of dietary live yeast (LY) supplementation on growth, intestinal permeability and immunological parameters of piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). Piglets weaned at 21 d were allocated into three treatments with six pens and six piglets per pen, receiving the control diet (CON), diets supplemented with antibiotics plus zinc oxide (ANT-ZnO) and LY (Saccharomyces cerevisiae strain CNCM I-4407), respectively, for a period of 2 weeks. On day 8, thirty-six piglets were selected as control without ETEC (CON), CON-ETEC, ANT-ZnO-ETEC and LY-ETEC groups challenged with ETEC until day 10 for sample collections. Piglets fed ANT-ZnO diet had the highest average daily gain and average daily feed intake (P<0·05) during the 1st week, but ADG of piglets fed the ANT-ZnO diet was similar as piglets fed LY diet during the second week. Piglets with LY-ETEC or ANT-ZnO-ETEC had markedly lower diarrhoea score (P<0·05) than piglets with CON-ETEC during the 24 h after ETEC challenge. Relative to piglets with CON, the counts of E. coli, urinary ratio of lactulose to mannitol, plasma IL-6 concentration, mRNA abundances of innate immunity-related genes in ileum and mesenteric lymph node tissues were increased (P<0·05), whereas the villous height of jejunum and relative protein expression of ileum claudin-1 were decreased (P<0·05) in piglets with CON-ETEC; however, these parameters did not markedly change in piglets with LY-ETEC or ANT-ZnO-ETEC. In summary, dietary LY supplementation could alleviate the severity of diarrhoea in piglets with ETEC, which may be associated with the improved permeability, innate immunity and bacterial profile.

Oral Administration of a Select Mixture of Bacillus Probiotics Affects the Gut Microbiota and Goblet Cell Function following Escherichia coli Challenge in Newly Weaned Pigs of Genotype MUC4 That Are Supposed To Be Enterotoxigenic E. coli F4ab/ac Receptor Negative.

The present study is important for improving our understanding of the protective role of probiotics against Escherichia coli infection in piglets. Structural disruption of the gut microbiota and impaired goblet cell function are collateral etiologic factors in enteric diseases. In this study, low, moderate, or high doses of a Bacillus licheniformis-B. subtilis mixture (BLS mix) were orally administered to MUC4-resistant piglets for 1 week before the F4-expressing ETEC strain (F4+ ETEC) challenge. Our findings suggest that oral administration of BLS mix reprograms the gut microbiota and enhances goblet cell function to ameliorate enteritis.

Vaccinating with conserved Escherichia coli antigens does not alter the mouse intestinal microbiome.

BackgroundEnterotoxigenic Escherichia coli (ETEC) causes diarrheal disease. Antigenic and structural heterogeneity among ETEC colonization factors has complicated vaccine development efforts. Identifying and characterizing conserved ETEC antigens that induce protective immunity is therefore of interest. We previously characterized three proteins (MipA, Skp, and ETEC_2479) that protected mice in an intranasal ETEC challenge model after vaccination. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. While the impact of inactivated viral vaccines and live-attenuated bacterial vaccines on the host microbiota have been examined, the potential impact of using subunit vaccines consisting of antigens that are also encoded by commensal organisms has not been investigated.FindingsWe addressed this issue by characterizing changes to mouse intestinal microbiomes as a function of vaccination. We failed to observe significant changes to mouse health, to mouse weight gain as a function of time, or to the diversity or richness of mouse intestinal microbiomes, as measured by analyzing alpha- and beta-diversity, as well as overall community structure, before and after vaccination.ConclusionsWe conclude that despite the conservation of MipA, Skp, and ETEC_2479 among Gram-negative bacteria, vaccination with these antigens fails to alter significantly the host intestinal microbiome.

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 1010 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.

Effect of added dietary threonine on growth performance, health, immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to Escherichia coli infection and challenged with E. coli K88ac.

Threonine (Thr) is important for mucin and immunoglobulin production. We studied the effect of added dietary Thr on growth performance, health, immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to E. coli K88ac (ETEC) infection and challenged with ETEC. Forty-eight 24-day-old weaned pigs were divided into two groups by their ETEC susceptibility using mucin 4 (MUC4) gene as a marker (2 MUC4(-/-) , not-susceptible, and 2 MUC4(+/+) , susceptible, pigs per litter). Within genotype, pigs were fed two different diets: 8.5 (LThr) or 9.0 (HThr) g Thr/kg. Pigs were orally challenged on day 7 after weaning and slaughtered on day 12 or 13 after weaning. Before ETEC challenge, HThr pigs ate more (p < 0.05). The diet did not affect post-challenge growth, but HThr tended to increase post-challenge feed efficiency (p = 0.087) and overall growth (p = 0.087) and feed efficiency (p = 0.055). Before challenge, HThr pigs excreted less E. coli (p < 0.05), while after challenge, diet did not affect the number of days with diarrhoea and ETEC excretion. MUC4(+/+) pigs responded to the challenge with more diarrhoea, ETEC excretion and anti-K88 IgA in blood and jejunal secretion (p < 0.001). HThr pigs had a higher increase of anti-K88 IgA values in jejunal secretion (p = 0.089) and in blood (p = 0.089, in MUC4(+/+) pigs only). Thr did not affect total IgA and IgM values, morphometry of jejunum, goblet cells count in colon, total mucin from jejunum and colon, but varied jejunal goblet cells counts (p < 0.05). In the first two post-weaning weeks, 8.5 g Thr/kg diet may be not sufficient to optimize initial feed intake, overall feed efficiency and intestinal IgA secretion and to control the gut microbiota in the first post-weaning week, irrespective of the pig genetic susceptibility to ETEC infection.

Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10(9) CFU/ml) or high (1 × 10(11) CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4(+) ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3(+) CD4(+) CD8(-) T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4(+) ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3(+) CD4(+) CD8(-) cells and Peyer's patch CD3(+) CD4(-) CD8(-) and CD3(-) CD4(-) CD8(+) cells. The percentage of ileal intraepithelial CD3(+) CD4(-) CD8(+) cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4(+) ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4(+) ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3(+) CD4(+) CD8(-) T cells, the expansion of Peyer's patch CD3(+) CD4(-) CD8(-) and CD3(-) CD4(-) CD8(+) cells, and the attenuation of F4(+) ETEC-induced increase in CD3(+) CD4(+) CD8(+) T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4(+) ETEC challenge, thus eliciting a strong proinflammatory response.