Enterotoxigenic Escherichia Coli F4 Research Articles

Dietary supplementation of blend of organic acids and monoglycerides alleviated diarrhea and systemic inflammation of weaned pigs experimentally infected with enterotoxigenic Escherichia coli F18

BackgroundThe emergence of antibiotic resistant microorganisms associated with conventional swine production practices has increased interest in acid-based compounds having antimicrobial properties and other biological functions as nutritional interventions. Despite the interest in organic acids and monoglycerides, few studies have examined the effects of the combination of these acid-based additives in weaned pigs under disease challenge conditions. Therefore, this study aimed to investigate the effects of dietary supplementation with blend of organic acids and/or medium-chain fatty acid monoglycerides on intestinal health and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC) F18 at 4-week of age.ResultsDietary supplementation of organic acids, monoglycerides, or both organic acids and monoglycerides (combination) reduced (P < 0.05) the diarrhea frequency of ETEC F18-infected pigs throughout the experimental period (d −7 to 21 post-inoculation). This is consistent with the reduced (P < 0.05) proportion of β-hemolytic coliforms in feces observed for the organic acid and combination treatments on d 10 post-inoculation. Supplementation of organic acids, monoglycerides, or combination also reduced (P < 0.05) bacterial translocation in mesenteric lymph nodes on d 21 post-inoculation. Pigs fed with monoglycerides or combination had lower (P < 0.05) white blood cells on d 5 post-inoculation, and pigs fed the combination also had lower (P < 0.05) lymphocytes than pigs in control group. Monoglyceride supplementation increased (P < 0.05) white blood cells and neutrophils compared with control group on d 14 post-inoculation. However, supplementation with organic acid blend, monoglyceride blend, or combination did not affect growth performance in this experiment.ConclusionsSupplementation with monoglycerides or organic acids alone or in combination improves the detrimental effects of ETEC F18 infection in weaned pigs, as indicated by reduced diarrhea, fecal shedding of β-hemolytic coliforms, and bacterial translocation, and thus enhancing disease resistance. Monoglycerides reduced the inflammatory response during peak infection, but their immunomodulatory and possible synergistic effects with organic acids need to be further investigated.

PSVIII-23 Attenuation of oxidative stress and inflammatory response improves gut integrity in piglets after infection

Abstract Enterotoxigenic Escherichia coli (ETEC) is a primary cause of post-weaning diarrhea in piglets, leading to serious health issues and economic loss in the swine industry. Increased oxidative stress and inflammation have detrimental effects on gut health. The present study aimed to investigate the regulation of oxidative stress and inflammatory response in a piglet model challenged with ETEC and to evaluate whether the chosen probiotic could improve intestinal function in piglets. A total of 24 weaned piglets susceptible to ETEC F4 infection were randomly divided into four groups: Group 1 (control), Group 2 (E. coli infection), Group 3 (E. coli infection received antibiotic avilamycin), and Group 4 (E. coli infection received B. licheniformis HG76). Group 2, Group 3 and Group 4 were infected with E. coli by an oral gavage of ETEC F4 (5 mL, 1 × 106 CFU/mL). Five days after infection, piglets in all groups were euthanized. The blood and intestine samples (a mid-jejunum segment) were collected, and serum was prepared. ETEC infection significantly increased serum and jejunal malondialdehyde (MDA) concentrations and serum diamine oxidase (DAO) activity (P &amp;lt; 0.05). ETEC infection also significantly decreased tight-junction proteins (ZO1, occludin), superoxide dismutase-1 (SOD), and heme oxygenase-1 (HO-1) expression (P &amp;lt; 0.05). Furthermore, ETEC infection significantly increased the expression of pro-inflammatory cytokines [interlueken (IL) -6, tumor necrosis factor (TNF) -α] in the intestine (P &amp;lt; 0.05). Antibiotic or probiotic supplementation mitigated oxidative stress and inflammation, as evidenced by decreased oxidative stress biomarkers, increased expression of antioxidant enzymes (SOD, HO-1) and downregulated the expression of proinflammatory cytokines (IL-6, TNF-α; P &amp;lt; 0.05). Antibiotic or probiotic supplementation also improved intestinal integrity, as demonstrated by decreased DAO activity and increased tight-junction proteins expression (P &amp;lt; 0.05). These results suggest that attenuation of oxidative stress and inflammatory response can improve piglet gut integrity after ETEC infection.

515 Effects of Bacillus licheniformis HG76 on barrier function and inflammation in the intestinal epithelia cells and gut health of weaned piglets under enterotoxigenic Escherichia coli F4 infection

Abstract Enterotoxigenic Escherichia coli (ETEC) F4 secretes enterotoxins and invades the small intestine, which damages the gut barrier function and stimulates the immune reactions characterized by inflammation. In the current study, the effects of B. licheniformis HG76 on gut health were evaluated with ETEC F4-induced IPEC-J2 cells and weaned piglets. The B. licheniformis HG76 was isolated from cecal contents of Rhode Island Red laying hens raised without using antibiotics. The results from the in vitro study showed that the ETEC F4 infection significantly induced IPEC-J2 cell death, and this effect was significantly attenuated by B. licheniformis HG76 treatment (P &amp;lt; 0.05). The gene expressions of zona occludens (ZO1) and tumor necrosis factor (TNF)-α were significantly increased during ETEC F4 infection with B. licheniformis HG76 treatment (P &amp;lt; 0.05). Meanwhile, the ETEC F4 infection significantly increased the mRNA abundance of toll-like receptors (TLR2, TLR4, and TLR5) (P &amp;lt; 0.05), but the B. licheniformis HG76 treatment alleviated this increase. For the in vivo study, 28 weaned piglets susceptible to ETEC F4 at 28 d of age were individually allotted to four experimental groups: NNC: unchallenged control group; NC: challenged control group; PC: antibiotic group (80 mg·kg-1 of avilamycin); and HG76: Bacillus group (1.0 × 109 CFU kg-1 diet of B. licheniformis HG76). The in vivo results showed during the post-challenge phase, ETEC F4 infection decreased average daily gain (ADG) and increased the severity of diarrhea at 44 h post-inoculum (hpi), respectively, in the NC group compared with the NNC group (P &amp;lt; 0.05). In addition, the infection decreased the villus height (VH) in the duodenum of pigs from the NC, PC and HG76 groups compared with the NNC group. The VH of jejunum and ileum in the pigs from the HG76 group were higher than other groups (P &amp;lt; 0.05). There was an increase (P &amp;lt; 0.05) of crypt depth (CD) in the jejunum and ileum of pigs from the HG76 group compared with those from the NNC group. The pigs in the NNC group had the greatest duodenal VH:CD ratio, but the least ileal VH:CD ratio among all the treatment groups. The Vmax value of sucrase in the HG76 group was greater than the NNC group (P &amp;lt; 0.05). When compared with the NC group, the relative mRNA abundance of interleukin 6 (IL6) was increased in the PC group (P &amp;lt; 0.05). In summary, these results suggest that B. licheniformis HG76 can protect the intestinal cells from ETEC F4 infection by inhibiting inflammation and maintaining barrier integrity, and the supplementation of B. licheniformis HG76 can improve the gut development and increase the digestive enzyme activity of ETEC F4 challenged piglets.

307 Awardee Talk: Effects of dietary valerate glycerides on weanling piglet diarrhea and immunity

Abstract The use of various feed additives for their non-nutrient, immunomodulatory qualities is under investigation for application in swine production, especially during the post-weaning period. Butyrate (C4) is a known modulator of intestinal physiology, produced endogenously by commensal microbes and found in other organic sources. However, the understanding of the role of valerate (C5) as a feed additive for pigs has not been well-evaluated. Two experiments were conducted utilizing glycerides of butyrate or valerate as feed additives for weanling piglets under enterotoxigenic Escherichia coli (ETEC) infection conditions. In both experiments, daily diarrhea scores were recorded to monitor the frequency of diarrhea. The first pilot experiment utilized 20 weanling piglets (21 to 24 d of age) housed individually and assigned to either control diet, or control diet supplemented with 0.10% butyrate glycerides or 0.10% valerate glycerides. This experiment also aimed to establish a coinfection disease model, administering two strains of ETEC at a dose of 0.5 × 109 CFU/1.5 mL for each strain after a 7-d adaptation period (F4 and F18; two highly prevalent strains in swine production). Overall, piglets had significantly (P &amp;lt; 0.05) decreased frequency of diarrhea in either treatment group compared with control, suggesting similar modulatory effects by dietary valerate glycerides and butyrate glycerides. In addition, pigs fed valerate glycerides tended (P = 0.061) to have less neutrophils and had significantly (P &amp;lt; 0.05) less serum TNF-α on d 4 post-inoculation (PI). Moving forward, a full-scale experiment was performed using 60 weanling piglets (21 to 24 d of age) to further investigate the effect of dietary valerate glycerides on piglet health under ETEC F18 infection conditions. Treatments included nursery basal diet (control), 0.075% or 0.10% monovalerin, or 0.1% trivalerin added to control. Pigs fed 0.1% trivalerin exhibited significantly (P &amp;lt; 0.05) reduced frequency of diarrhea and fecal shedding of ETEC F18. Serum samples were collected throughout the experimental period and six replicate pigs per treatment group were sacrificed on d 7 PI to collect intestinal samples for morphological analysis and intestinal mucosa RT-qPCR analysis. Pigs fed trivalerin had reduced (P &amp;lt; 0.05) serum TNF-α on d 0 before inoculation and d 6 PI compared with pigs in control. The mRNA expression of TNFA in ileal mucosa from pigs fed 0.1% monovalerin tended (P &amp;lt; 0.10) to be lower compared with control. In addition, ZO1 mRNA expression tended (P &amp;lt; 0.10) to be increased in jejunal mucosa from pigs fed 0.1% trivalerin. Overall, the second experiment indicated that dietary supplementation of valerate glycerides reduced diarrhea and fecal shedding of ETEC F18 in piglets infected with ETEC. In conclusion, both experiments provide evidence in support of supplementing 0.075 or 0.10% valerate glycerides could enhance disease resistance of weanling piglets experiencing ETEC-associated diarrhea.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 μg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

Survey on resistance occurrence for F4+ and F18+ enterotoxigenic Escherichia coli (ETEC) among pigs reared in Central Italy regions.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

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 2,500ppm) 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 150ppm Zn compared to 1,400 or 2,500ppm 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 150ppm Zn had lower (P = 0.01) ADG during d 5 to 7 compared to those fed 1,400 or 2,500ppm Zn, whereas control pigs' ADG were not affected by dietary Zn content. Challenged pigs fed 150ppm Zn also showed lower (P < 0.01) fecal dry matter (DM) on d 5 compared to control pigs fed 150ppm Zn and challenged pigs fed 1,400 or 2,500ppm Zn. Challenge increased (P < 0.01) ETEC shedding in all groups, but challenged pigs fed 150ppm Zn showed higher (P ≤ 0.05) fecal shedding of ETEC and toxins than when fed 1,400 or 2,500ppm. On d 3, C-reactive protein concentration in plasma was lower (P < 0.03) for pigs fed 1,400 and 2,500 compared to 150ppm Zn. Plasma haptoglobin and pig major acute phase protein were unaffected by dietary Zn content. On d 0, the serum Zn concentration was 586 ± 36.6 µg/L, which pigs fed 150ppm Zn maintained throughout the study. The serum Zn concentration increased (P ≤ 0.07) in pigs fed 1,400 or 2,500ppm Zn. The challenge decreased (P < 0.01) the serum Zn concentration in pigs fed 2,500ppm Zn. On d 5 and 7, serum Zn concentration was similar for challenged pigs fed 1,400 and 2,500ppm Zn, while control pigs fed 2,500ppm Zn had higher (P < 0.01) serum Zn concentration than 1,400ppm 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 150ppm 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.

Comprehensive analysis of circular RNAs in porcine small intestine epithelial cells associated with susceptibility to Escherichia coli F4ac diarrhea

BackgroundDiarrhea is one of the most common diseases in pig industry, which seriously threatens the health of piglets and causes huge economic losses. Enterotoxigenic Escherichia coli (ETEC) F4 is regarded as the most important cause of diarrhea in piglets. Some pigs are naturally resistant to those diarrheas caused by ETEC-F4, because they have no F4 receptors (F4R) on their small intestine epithelial cells that allow F4 fimbriae adhesion. Circular RNA (circRNA) has been shown to play an important regulatory role in the pathogenesis of disease. We hypothesized that circRNAs may also regulate the adhesion of piglet small intestinal epithelial cells to ETEC F4 fimbriae. However, the circRNA expression profiles of piglets with different Enterotoxigenic Escherichia coli F4 fimbriae (ETEC-F4ac) adhesion phenotypes are still unclear, and the intermediate regulatory mechanisms need to be explored. Hence, the present study assessed the circRNA expression profiling in small intestine epithelial cells of eight male piglets with different ETEC-F4 adhesion phenotypes and ITGB5 genotypes to unravel their regulatory function in susceptibility to ETEC-F4ac diarrhea. Piglets were divided into two groups: non-adhesive group (n = 4) with CC genotype and adhesive group (n = 4) with TT genotype.ResultsThe RNA-seq data analysis identified 13,199 circRNAs from eight samples, most of which were exon-derived. In the small intestine epithelial cells, 305 were differentially expressed (DE) circRNAs between the adhesive and non-adhesive groups; of which 46 circRNAs were upregulated, and 259 were downregulated. Gene ontology and KEGG enrichment analysis revealed that most significantly enriched DE circRNAs’ host genes were linked to cytoskeletal components, protein phosphorylation, cell adhesion, ion transport and pathways (such as adherens junction, gap junction) associated with ETEC diarrhea. The circRNA-miRNA-mRNA interaction network was also constructed to elucidate their underlying regulatory relationships. Our results identified several candidate circRNAs that affects susceptibility to ETEC diarrhea. Among them, circ-SORBS1 can adsorb ssc-miR-345-3p to regulate the expression of its host gene SORBS1, thus improving cell adhesion.ConclusionOur results provided insights into the regulation function of circRNAs in susceptibility to ETEC diarrhea of piglets, and enhanced our understanding of the role of circRNAs in regulating ETEC diarrhea, and reveal the great potential of circRNA as a diagnostic marker for susceptibility of ETEC diarrhea in piglets.

Promotion of intestinal epithelial cell apoptosis by enterotoxigenic Escherichia coli via PKA-mediated inhibition of mTORC1 activation

Promotion of intestinal epithelial cell apoptosis by enterotoxigenic Escherichia coli via PKA-mediated inhibition of mTORC1 activation

Antimicrobial Resistance and Virulence Factors Assessment in Escherichia coli Isolated from Swine in Italy from 2017 to 2021

Prudent antibiotic use in pigs is critical to ensuring animal health and preventing the development of critical resistance. We evaluated the antimicrobial resistance (AMR) pattern in commensal and enterotoxigenic Escherichia coli (ETEC) isolates obtained in 2017−2021 from pigs suffering from enteric disorders. Overall, the selected 826 E. coli isolates showed the highest level of resistance to ampicillin (95.9%), tetracycline (89.7%), cefazolin (79.3%), and trimethoprim/sulfamethoxazole (74.8%). The resistance rates of the isolates to ampicillin increased (p < 0.05), reaching 99.2% of resistant strains in 2021. Regarding isolates harboring virulence genes, ETEC F18+ were significantly more resistant to florfenicol, gentamicin, kanamycin, and trimethoprim/sulfamethoxazole than ETEC F4+ strains. E. coli lacking virulence factor genes were more resistant to amoxicillin with clavulanic acid and cefazolin, but less resistant to gentamicin (p < 0.01) than isolates harboring virulence factors. Throughout the study period, a significant number of ETEC F18+ isolates developed resistance to florfenicol, gentamicin, and kanamycin. Finally, ETEC 18+ significantly (p < 0.05) increased resistance to all the tested antibiotics. In conclusion, AMR varied for E. coli over time and showed high levels for molecules widely administered in the swine industry, emphasizing the need for continuous surveillance. The observed differences in AMR between commensal and ETEC isolates may lead to the hypothesis that plasmids carrying virulence genes are also responsible for AMR in E. coli, suggesting more research on genetic variation between pathogenic and nonpathogenic E. coli.

Dietary butyrate and valerate glycerides impact diarrhea severity and immune response of weaned piglets under ETEC F4-ETEC F18 coinfection conditions.

Enterotoxigenic Escherichia coli (ETEC) causes post-weaning diarrhea in piglets, significantly impacting animal welfare and production efficiency. The two primary ETEC pathotypes associated with post-weaning diarrhea are ETEC F4 and ETEC F18. During the post-weaning period, piglets may be exposed to both ETEC F4 and ETEC F18. However, the effects of coinfection by both strains have not been studied. Short chain fatty acid feed additives, such as butyrate and valerate, are being investigated for their potential to improve animal performance and disease resistance. Therefore, this pilot experiment aimed to test the effects of butyrate glycerides or valerate glycerides on growth performance, diarrhea incidence, and immune responses of piglets under ETEC F4-ETEC F18 coinfection conditions. Twenty piglets were individually housed and assigned to one of the three dietary treatments immediately at weaning (21 to 24 d of age). The dietary treatments included control (basal diet formulation), control supplemented with 0.1% butyrate glycerides or 0.1% valerate glycerides. After a 7-d adaptation, all pigs were inoculated with ETEC F4 and ETEC F18 (0.5 × 109 CFU/1.5 mL dose for each strain) on three consecutive days. Pigs and feeders were weighed throughout the trial to measure growth performance. Fecal cultures were monitored for hemolytic coliforms, and blood samples were collected for whole blood and serum analysis. Pigs fed valerate glycerides tended (P = 0.095) to have higher final body weight compared with control. The overall severity of diarrhea was significantly (P < 0.05) lower in both treatment groups than control. Pigs fed valerate glycerides tended (P = 0.061) to have lower neutrophils and had significantly (P < 0.05) lower serum TNF-α on day 4 post-inoculation. This pilot experiment established an appropriate experimental dose for an ETEC F4-ETEC F18 coinfection disease model in weaned piglets. Results also suggest that butyrate glycerides and valerate glycerides alleviated diarrhea and regulated immune responses in piglets coinfected with ETEC F4 and ETEC F18.

Evaluation of the diagnostic performance of a commercially available point-of-care test for post weaning diarrhoea in pigs-a pilot study

BackgroundPost weaning diarrhoea is expected to become an increasing problem in pig herds following the outphasing of medicinal Zinc Oxide. Currently, no equally effective substitute has been found and an increase in metaphylactic batch medication with antibiotics is expected. However, prudent use of antibiotics is needed to mitigate antibiotic resistance development and one option could be pre-treatment diagnostics. Employing a point-of-care test in a herd could provide fast diagnostics and help guide antibiotic treatment. Hence, the aim of this study was to evaluate the diagnostic performance of a commercially available point-of-care test for enterotoxigenic Escherichia coli (ETEC) F4, ETEC F18 and rotavirus in weaned pigs.ResultsIn total 115 diarrheic samples from two conventional herds were included in the evaluation of the Rainbow Piglet Scours test, which was compared to microbiological PCR analyses. The comparison yielded a diagnostic sensitivity, diagnostic specificity, positive and negative predictive value of 0.28, 0.99, 0.92 and 0.70 for ETEC F4, 0.40, 0.92, 0.91 and 0.45 for ETEC F18 and 0.67, 0.88, 0.91 and 0.61 for rotavirus.ConclusionsThe point-of-care test yielded a low diagnostic sensitivity and a high diagnostic specificity for ETEC F4, ETEC F18 and rotavirus. Due to the high level of false negatives, the test cannot be recommended for individual diagnostics on pig-level.

Impact of early inoculation of probiotics to suckling piglets on postweaning diarrhoea – A challenge study with Enterotoxigenic E. Coli F18

Impact of early inoculation of probiotics to suckling piglets on postweaning diarrhoea – A challenge study with Enterotoxigenic E. Coli F18

160 Effects of Bacillus Licheniformis PF9 on Growth, Nutrient Absorption, gut Barrier Integrity, Immunity, and Intestinal Microbiota of Weaned Piglets Challenged with Enterotoxigenic Escherichia Coli F4

Abstract This study aimed to evaluate the effects of Bacillus licheniformis PF9 on growth performance and gut health of weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4. Thirty-two weaned piglets susceptible of ETEC F4 were randomly allocated to 4 treatments 1) NNC, basal diet and non-challenged; 2) NC, basal diet; 3) PC, basal diet + 80 mg·kg-1 of Avilamycin; and 4) PF9, basal diet + 109 cfu/kg of Bacillus licheniformis PF9. ETEC F4 decreased average daily gain and increased feed conversion ratio of the NC group. An increase was found in FCR of the PC group during the post-challenge period (P &amp;lt; 0.05). ETEC F4 induced the severity of diarrhea at 3-, 36- and 40-hours post-inoculum (hpi) in the NC group (P &amp;lt; 0.05). Bacillus licheniformis relieved diarrhea severity at 3 hpi (P &amp;lt; 0.05). ETEC F4 reduced the duodenal, jejunal and ileal villus height of the NC group. A decrease in duodenal villus height in the PC group (P &amp;lt; 0.05). ETEC F4 reduced the relative mRNA level of Na+-glucose cotransporter 1 in the NC group (P &amp;lt; 0.05). Compared to the NC and NNC groups, Bacillus licheniformis increased relative mRNA levels of Aminopeptidase N, Occludin, Zonula occludens-1, and SGLT1 (P &amp;lt; 0.05). Bacillus licheniformis increased the relative mRNA level of excitatory amino acid transporter 1 (P &amp;lt; 0.05). Bacillus licheniformis showed a lower relative abundance of Bacteroidetes in the colon than piglets from the NNC group (P &amp;lt; 0.05). The non-challenged group had a higher relative abundance of Firmicutes in the ileum than the challenged piglets, meanwhile, a lower relative abundance of Proteobacteria in the ileum and colon in the NC group (P &amp;lt; 0.05). This study provides evidence that Bacillus licheniformis can be a potential antibiotics alternative to improve the gut health of piglets.

391 Young Scholar Award Talk: Supplementation of Oligosaccharide-Based Polymer Enhanced Growth and Disease Resistance of Weaned Pigs by Modulating Intestinal Integrity and Systemic Immunity

Abstract Oligosaccharides have been reported to possess ETEC receptor activity for bacterial adhesions; thus, additional supplementation may efficiently prevent enterotoxin-induced secretory diarrhea. Moreover, grafted polymers that combine multiple substances have been proposed for their potential synergistic effects on preventing human and animal diseases. Therefore, the objective of this experiment was to investigate dietary supplementation of oligosaccharide-based polymer on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC) when compared with in-feed antibiotics. Forty-eight pigs (7.23 ± 1.11 kg BW) were individually housed in disease containment rooms and randomly allotted to one of four treatments with 12 replicate pigs per treatment. The four dietary treatments were a nursery basal diet (CON), and 3 additional diets supplemented with 50 mg/kg antibiotics (Carbadox; CAR), 10 or 20 mg/kg of oligosaccharide-based polymer (OBP). The experiment lasted 18 d [7 d before and 11 d after the first inoculation (d 0)]. All pigs were orally inoculated with 3mL of F18 ETEC for 3 consecutive days from d 0 post-inoculation (PI). Growth performance and diarrhea severity were measured throughout the experiment. Blood, lymph nodes, spleen, and intestinal mucosa samples were collected during the peak infection period (d 2 and d 5 PI) and recovery period (d 11 PI). Pigs in CAR or OBP treatment groups had greater (P&amp;lt;0.05) body weight on d 5 or d 11 PI than pigs in the CON group, respectively. Supplementation of CAR or OBP enhanced (P&amp;lt;0.05) feed efficiency from d 0 to 5 PI and reduced (P&amp;lt;0.05) frequency of diarrhea throughout the experiment, compared with pigs in the CON group. Pigs in CAR or OBP groups had reduced (P&amp;lt;0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the CON group on d 2 and 5 PI. Pigs in OBP reduced (P&amp;lt;0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in CAR or OBP groups had reduced (P&amp;lt;0.05) total coliforms in the spleen on d 11 PI compared with pigs in the CON group. On d 5 PI, pigs in the OBP group had greater (P&amp;lt;0.05) gene expression of ZO1 in jejunal mucosa, but less (P&amp;lt;0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the CON group. On d 11 PI, supplementation of CAR or OBP up-regulated (P&amp;lt;0.05) gene expression of CLDN1 in jejunal mucosa, but OBP reduced (P&amp;lt;0.05) IL6 gene expression in ileal mucosa compared to pigs in the CON group. In conclusion, supplementation of oligosaccharide-based polymer improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics.

Assessment of the Effects of the Synbiotic Combination of Bifidobacterium longum subsp. infantis CECT 7210 and Oligofructose-Enriched Inulin Against Digestive Bacterial Infections in a Piglet Model.

The use of bifidobacteria as probiotics has proven to be beneficial in gastroenteric infections. Furthermore, prebiotics such as inulin can enhance the survival and growth of these bacteria. Two trials were performed to evaluate the effects of the administration of Bifidobacterium longum subsp. infantis CECT 7210 and oligofructose-enriched inulin against Salmonella enterica serovar Typhimurium or enterotoxigenic Escherichia coli (ETEC) F4. A total of 72 (Salmonella trial) and 96 (ETEC F4 trial) weaned piglets were used in a 2 × 2 design (with or without synbiotic, inoculated or not with the pathogen). After adaptation, animals were orally inoculated. Performance and clinical signs were evaluated. On days 4 and 8 (Salmonella trial) and 3 and 7 (ETEC F4 trial) post-inoculation (PI), one animal per pen was euthanized. Blood, digestive content and tissue samples were collected and microbiological counts, fermentation products, serum inflammatory markers and ileum histomorphometry analysis were performed. Both challenges had an impact on faecal consistency (p < 0.001), including the faecal shedding of Salmonella and increased numbers of enterobacteria and coliforms. The synbiotic administration did not have any effect on pathogen loads but induced changes in the fermentation profile, such as increased valeric acid in both trials as well as decreased acetic acid, except for Salmonella-challenged animals. The effect on propionate varied among trials, increasing in challenged synbiotic-treated pigs and decreasing in non-challenged ones in the Salmonella trial (P interaction = 0.013), while the opposed occurred in the ETEC F4 trial (P interaction = 0.013). The administration of the synbiotic increased intraepithelial lymphocytes (IEL; p = 0.039) on day 8 PI in the Salmonella trial and a similar trend occurred in non-challenged pigs in the ETEC F4 trial (P interaction = 0.086). The results did not provide evidence of reduced pathogen load with the synbiotic, although a modulation in fermentative activity could be identified depending on the challenge. Consistent increases were found in IEL, suggesting that this synbiotic combination has some immunomodulatory properties.

Evaluation of the prophylactic potential of non-enterotoxigenic Escherichia coli (non-ETEC) vaccine immunization and dietary mannan oligosaccharide competitive exclusion benefits against ETEC infections in weaned pigs

Enterotoxigenic Escherichia coli (ETEC) strains expressing F4 and F18 fimbriae are the most common causative agents of post-weaning diarrhoea (PWD) in pigs. The growing global restriction on the use of antibiotics in food animals has encouraged research into the development of nutritional and feeding strategies as well as vaccination against PWD. The aim of this study was to evaluate the efficacy of a live oral F4ac+ F18ac+ non-ETEC vaccine candidate (VAC) to stimulate gut and systemic cellular immunity in 4-week old pigs over 5 weeks following immunization. The onset and duration of protective immunity against on-farm occurring PWD, growth performance, diarrhoea scoring and mortality, as well as the phenotypic proportions of immune cells, were determined. Faecal and ileal samples were taken for determining the microbial composition or phenotyping of naïve/memory T cells. Also, the effect of prebiotic supplement mannan oligosaccharide (MOS) in the prevention of small intestinal colonization by ETEC, and its potential adjuvanticity in combination with the vaccine (VAC+MOS) were assessed. The pigs supplemented with MOS or that received VAC had significantly higher body weight (BW) (P&lt;0.05) on Day 14, whereas the VAC+MOS treated pigs had significantly lower BW on Day 35. Treatment with VAC+MOS resulted in considerably reduced clinical PWD, in particular the incidence and severity of diarrhoea and mortality. The total bacterial load in the ileum was much lower in the pigs from all 3 principal groups (MOS, VAC, and VAC+MOS) than in the control (CON) group (19 x 107, 17 x 107 and 12 x 107 vs. 23 x 108 CFU/mL, respectively) on Day 35. The pigs from the principal groups had significantly higher proportions of tested immune cells (P&lt;0.05) on Days 28 and 35. The localization and frequency of naive CD45RA+ and memory CD45RC+ T lymphocytes indicated their different distribution patterns within particular tissue structures, such as the villi, crypts, epithelium, lamina propria and areas (interfollicular follicular and Peyer’s patches) of ileal mucosa. This may indicate their different functions in intestinal immune responses to intraluminal microbes and their products, vaccinal immunogens and/or immunomodulators/adjuvants. To conclude, active mucosal immunity is needed to protect pigs against PWD. Hence, oral vaccination of pigs against both F4 and F18 ETEC, in combination with prebiotic supplementation represents a sustainable, practical and effective approach in PWD control.

Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity

BackgroundThere is a great demand for antibiotic alternatives to maintain animal health and productivity. The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1 antigen oligosaccharides-based polymer (Coligo) on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC), when compared with antibiotics.ResultsPigs in antibiotic carbadox or Coligo treatment groups had greater (P < 0.05) body weight on d 5 or d 11 post-inoculation (PI) than pigs in the control group, respectively. Supplementation of antibiotics or Coligo enhanced (P < 0.05) feed efficiency from d 0 to 5 PI and reduced (P < 0.05) frequency of diarrhea throughout the experiment, compared with pigs in the control group. Supplementation of antibiotics reduced (P < 0.05) fecal β-hemolytic coliforms on d 2, 5, and 8 PI. Pigs in antibiotics or Coligo groups had reduced (P < 0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI. Pigs in Coligo had reduced (P < 0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in antibiotics or Coligo groups had reduced (P < 0.05) total coliforms in spleen on d 11 PI compared with pigs in the control group. On d 5 PI, pigs in the Coligo group had greater (P < 0.05) gene expression of ZO1 in jejunal mucosa, but less (P < 0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the control group. Supplementation of antibiotics enhanced (P < 0.05) the gene expression of OCLN in jejunal mucosa but decreased (P < 0.05) IL1B and IL6 gene expression in ileal mucosa, compared with the control. On d 11 PI, supplementation of antibiotics or Coligo up-regulated (P < 0.05) gene expression of CLDN1 in jejunal mucosa, but Coligo reduced (P < 0.05) IL6 gene expression in ileal mucosa compared to pigs in the control group.ConclusionsSupplementation of Coligo improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics.

PSX-A-11 Late-Breaking: Effects of phytogenic supplementation on growth performance and intestinal barrier function in nursery pigs challenged with E. coli

Abstract The objective of this study was to investigate the health-promoting effects of red osier dogwood (ROD) extract as an alternative to antibiotics in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC). Twenty-eight weaned piglets (9.15±0.95 kg BW) confirmed to genetically susceptible to ETEC were individually assigned to one of the four dietary treatments in a completely randomized design. Experimental diets were, negative control (NC), corn-wheat soybean meal diet with no additives; positive control (PC), NC plus antibiotics; ROD1, NC plus 0.1% ROD extract; ROD2, NC plus 0.2% ROD extract. Piglets were orally challenged on d 7 with ETEC F4. Feed disappearance, body weight, fecal score, and rectal temperature were recorded. On d 14, piglets were euthanized to collect intestinal tissue samples for histomorphology and Ussing chamber analysis. Data were analyzed using the MIXED procedure of SAS using individual piglet as the experimental unit. There were no differences (P &amp;gt; 0.10) in histomorphology and intestinal permeability. Piglets fed the NC diet tended (P &amp;lt; 0.10) to have higher average daily gain, post-inoculation than those fed ROD1 or ROD2. Fecal score of piglets fed the PC diet tended to (P &amp;lt; 0.10) or was significantly lower (P &amp;lt; 0.05) than for piglets fed ROD1 or ROD2 on 0 and 2 days post-inoculation (dpi), respectively. On 0 dpi, piglets fed the ROD1 diet had significantly higher (P &amp;lt; 0.05) body temperature than those fed PC or ROD2. In conclusion, ROD extract supplementation might have some health-promoting effects on ETEC challenged piglets but could not improve gut health to the same extent as antibiotics.

Mint Oils: In Vitro Ability to Perform Anti-Inflammatory, Antioxidant, and Antimicrobial Activities and to Enhance Intestinal Barrier Integrity

The objectives of the study were to test the biological activities of peppermint and spearmint oils via (i) measuring in vitro anti-inflammatory effects with porcine alveolar macrophages (PAMs), (ii) determining the barrier integrity of IPEC-J2 by analyzing transepithelial electrical resistance (TEER), (iii) testing their antioxidant activities, and (iv) investigating the antimicrobial activity against enterotoxigenic Escherichia coli (ETEC) F18+. Briefly, (i) macrophages were seeded at 106 cells/mL and treated (24 h) with mint oils and lipopolysaccharide (LPS). The treatments were 2 (0 or 1 μg/mL of LPS) × 5 (0, 25, 50, 100, 200 µg/mL of mint oils). The supernatants were collected for TNF-α and IL-1β measurement by ELISA; (ii) IPEC-J2 cells were seeded at 5 × 105 cells/mL and treated with mint oils (0, 25, 50, 100, and 200 μg/mL). TEER (Ωcm2) was measured at 0, 24, 48, and 72 h; (iii) the antioxidant activity was assessed (0, 1, 50, 100, 200, 500, and 600 mg/mL) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and reducing power assays; (iv) overnight-grown ETEC F18+ were quantified (CFU/mL) after supplementing with peppermint and spearmint oils (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL). All data were analyzed using the MIXED procedure. Both mint oils significantly inhibited (p < 0.05) IL-1β and TNF-α secretion from LPS-stimulated PAMs. Mint oil treatments did not affect TEER in IPEC-J2. Spearmint and peppermint oils exhibited (p < 0.05) strong antioxidant activities in DPPH and reducing power assays. Both mint oils also dose-dependently inhibited (p < 0.05) the growth of ETEC F18+ in vitro. The results of the study indicated that both mint oils are great candidate feed additives due to their in vitro anti-inflammatory, antioxidant, and antimicrobial effects. Further research is needed to evaluate their efficacy in vivo.