Citric Acid Promoting B Lymphocyte Differentiation and Anti-epithelial Cells Apoptosis Mediate the Protective Effects of Hermetia illucens Feed in ETEC Induced Piglets Diarrhea

Newborn piglets are prone to diarrhea after weaning as a result of changes in their environment and feed. Enterotoxigenic Escherichia coli (ETEC) K88 strain is a typical pathogen that causes diarrhea in such stage of piglets. Hermetia illucens larvae are widely used in livestock and poultry production because of their high nutritional value and immunoregulatory effects. This study aimed to evaluate the protective effects of H. illucens feed in protecting against ETEC induced diarrhea in piglets and to unravel the mechanisms of immune modulation and intestinal barrier maintenance. The results showed that after ETEC infection, citric acid in the serum of the groups fed on H. illucens larvae increased significantly, which stimulated macrophages to secrete cytokines that promote B lymphocyte differentiation, ultimately increasing the production of IgA and IgG in serum. Concomitantly, citric acid also had a positive effect on the intestinal barrier damaged due to ETEC infection by inhibiting the production of inflammatory cytokines, reducing the Bcl-2/Bax ratio, and promoting the expression of tight junction proteins. Correlation analysis showed that the increase of citric acid levels might be related to Massilia. Thus, citric acid derived from H. illucens larvae can improve the immune performance of weaned piglets and reduce ETEC-induced damage to the intestinal barrier in weaned piglets.

Trace Amounts of Antibiotic Altered Metabolomic and Microbial Profiles of Weaned Pigs Infected with a Pathogenic E. coli­­­­­­­

Background: Our previous study has shown that supplementation of trace amounts of antibioticexacerbated the detrimental effects of enterotoxigenic E. coli (ETEC) infection and delayed the recovery of pigs that may be associated with modified metabolites and metabolic pathways. Therefore, the objective of this study was to explore the impacts of trace levels of antibiotic (carbadox) on host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18. Results: The multivariate analysis highlighted a distinct metabolomic profile of serum and colon digesta between trace amounts of antibiotic (TRA) and label-recommended dose antibiotic (REC) on d 5 post-inoculation (PI). The relative abundance of metabolomic markers of amino acids, carbohydrates, and purine metabolism were significantly differentiated between the TRA and REC groups (q < 0.2). In addition, pigs in REC group had the highest (P < 0.05) relative abundance of Lactobacillaceae and tended to have increased (P < 0.10) relative abundance of Lachnospiraceae in the colon digesta on d 5 PI. On d 11 PI, pigs in REC had greater (P < 0.05) relative abundance of Clostridiaceae compared with other groups, whereas had reduced (P < 0.05) relative abundance of Prevotellaceae than pigs in control group.Conclusions: Trace amounts of antibiotic resulted in differential metabolites and metabolic pathways that may be associated with its slow responses against ETEC F18 infection. The altered gut microbiota profiles by label-recommended dose antibiotic may contribute to the promotion of disease resistance in weaned pigs.

Impacts of feeding organic-acid-based feed additives on diarrhea, performance, and fecal microbiome characteristics of pigs after weaning challenged with an enterotoxigenic strain of Escherichia coli

Post weaning diarrhea (PWD) caused by enterotoxigenic strains of E. coli (ETEC) remains a major problem in the industry, causing decreases in performance and survival of weaned pigs. Traditionally, antimicrobials have been used for its mitigation/control. This study tested the hypothesis that a combination of two organic acid (OA)-based commercial feed additives, Presan FX [an OA, medium-chain fatty acid (MCFA) and phenolic compound-based product] and Fysal MP (free and buffered OA based on formic acid), would reduce PWD and improve post-weaning performance in pigs challenged with an F4-ETEC. This combination was assessed against a Negative control diet without any feed additives and a diet containing amoxicillin. Combined with a reduction in temperature during the infection period, inoculation with F4-ETEC resulted in 81% of pigs developing diarrhea, but with no differences between treatments (P &gt; 0.05). However, between d 14-20 of the study and due to colonization by Salmonella serovars, pigs fed the combination of Presan FX and Fysal MP showed less (P = 0.014) diarrhea commensurate with a lower (P = 0.018) proportion of Salmonella numbers relative to total bacterial numbers. This caused less (P = 0.049) therapeutic antibiotic administrations relative to the diet with amoxicillin during this time. The diversity of bacteria within amoxicillin-treated pigs was lower (P = 0.004) than the diversity in control or Presan + Fysal MP-treated pigs (P = 0.01). Pair-wise comparisons showed that amoxicillin-treated pigs had altered (P &lt; 0.001) fecal microbial communities relative to both Presan FX + Fysal MP-treated pigs and control pigs. Amoxicillin-treated pigs were characterized by an increased abundance of bacterial families generally linked to inflammation and dysbiosis in the gastrointestinal tract (GIT), whereas Presan FX + Fysal MP-treated pigs had an increased abundance of bacterial families considered beneficial commensals for the GIT. Control pigs were characterised by an increased abundance of Spirochaetaceae associated with healthy piglets, as well as bacterial families associated with reduced feed intake and appetite. The combination of two OA-based feed additives did not reduce the incidence of F4 ETEC-associated diarrhea nor enhance performance. However, the combination markedly reduced diarrhea caused by Salmonella that occurred following the ETEC infection, commensurate with less therapeutic administrations relative to the diet with amoxicillin.

Lactobacillus rhamnosus LB1 Alleviates Enterotoxigenic Escherichia coli-Induced Adverse Effects in Piglets by Improving Host Immune Response and Anti-Oxidation Stress and Restoring Intestinal Integrity

Enterotoxigenic Escherichia coli (ETEC) is a common enteric pathogen that causes diarrhoea in humans and animals. Lactobacillus rhamnosus LB1 (formerly named Lactobacillus zeae LB1) has been shown to reduce ETEC infection to Caenorhabditis elegans and Salmonella burden in pigs. This study was to evaluate the effect of L. rhamnosus LB1 on the gut health of lactating piglets that were challenged with ETEC. Six-four piglets at 7 days of age were equally assigned into 8 groups (8 piglets per group): 1) control group (basal diet, phosphate buffer saline); 2) CT group (basal diet + 40 mg/kg colistin); 3) LL group (basal diet + 1 × 107 CFU/pig/day LB1); 4) HL group (basal diet + 1 × 108 CFU/pig/day LB1); 5) ETEC group: (basal diet + ETEC challenged); 6) CT + ETEC group (basal diet + CT + ETEC); 7) LL + ETEC group (basal diet + 1 × 107 CFU/pig/day LB1 + ETEC); 8) HL + ETEC group (basal diet + 1 × 108 CFU/pig/day LB1 + ETEC). The trial lasted ten days including 3 days of adaptation. Several significant interactions were found on blood parameters, intestinal morphology, gene, and protein expression. ETEC infection disrupted the cell structure and biochemical indicators of blood, undermined the integrity of the intestinal tract, and induced oxidative stress, diarrhoea, intestinal damage, and death of piglets. The supplementation of L. rhamnosus LB1 alleviated ETEC’s adverse effects by reducing pig diarrhoea, oxidative stress, and death, modulating cell structure and biochemical indicators of blood, improving the capacity of immunity and anti-oxidation stress of pigs, and restoring their intestinal integrity. At the molecular level, the beneficial effects of L. rhamnosus LB1 appeared to be mediated by regulating functional related proteins (including HSP70, Caspase-3, NLRP3, AQP3, and AQP4) and genes (including RPL4, IL-8, HP, HSP70, Mx1, Mx2, S100A12, Nrf2, GPX2 and ARG1). These results suggest that dietary supplementation of L. rhamnosus LB1 improved the intestinal functions and health of piglets.

Intestinal and Mucosal Microbiomic Response to Oral Challenge of Enterotoxigenic Escherichia Coli in Weaned Pigs

Enterotoxigenic Escherichia coli (ETEC) is closely associated with diarrhoea in children in resource-limited countries and of travellers’ diarrhoea. This study aims to investigate the change of ileal mucosal microbiome and ileal protein expression as well as their correlation in pigs by E. coli K88 (ETEC). Seven weaned male pigs were orally given 1 ×109 CFU of ETEC (ETEC, n = 7), and the other seven received saline (CON, n = 7). Ileal tissues were obtained 48 h after the ETEC challenge for both proteomic and mucosal microbiomic analyses. Nine proteins were altered in expression level in the ETEC group, including decreased expression of FABP1 and FABP6 involved in bile acid circulation. TLR-9 mediated pathway was also affected at transcription level with increased expression of SIGIRR and MyD88. Correlation analysis revealed correlations between the ileal proteins and mucosal bacterial taxa, including the positive correlation between Lactobacilli and PPP3CA (r = 0.9, p < 0.001), and negative correlation between Prevotella with CTNND1 (r = -0.7, p < 0.01). In conclusion, ETEC infection caused inflammation and impaired the circulation of bile acids, and the mucosal microbiome may affect the expression of intestinal proteins. Further studies are needed for exact roles of these affected processes in the pathogenesis of ETEC-triggered diarrhoea.

Transcriptome Analysis Identifies Strategies Targeting Immune Response-Related Pathways to Control Enterotoxigenic Escherichia coli Infection in Porcine Intestinal Epithelial Cells

Enterotoxigenic Escherichia coli (ETEC) is an important cause of post-weaning diarrhea (PWD) worldwide, resulting in huge economic losses to the swine industry worldwide. In this study, to understand the pathogenesis, the transcriptomic analysis was performed to explore the biological processes (BP) in porcine intestinal epithelial J2 cells infected with an emerging ETEC strain isolated from weaned pigs with diarrhea. Under the criteria of |fold change| (FC) ≥ 2 and P &lt; 0.05 with false discovery rate &lt; 0.05, a total of 131 referenced and 19 novel differentially expressed genes (DEGs) were identified after ETEC infection, including 96 upregulated DEGs and 54 downregulated DEGs. The Gene Ontology (GO) analysis of DEGs showed that ETEC evoked BP specifically involved in response to lipopolysaccharide (LPS) and negative regulation of intracellular signal transduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that immune response-related pathways were mainly enriched in J2 cells after ETEC infection, in which tumor necrosis factor (TNF), interleukin 17, and mitogen-activated protein kinase (MAPK) signaling pathways possessed the highest rich factor, followed by nucleotide-binding and oligomerization domain-like receptor (NLRs), C-type lectin receptor (CLR), cytokine–cytokine receptor interaction, and Toll-like receptor (TLR), and nuclear factor kappa-B (NF-κB) signaling pathways. Furthermore, 30 of 131 referenced DEGs, especially the nuclear transcription factor AP-1 and NF-κB, participate in the immune response to infection through an integral signal cascade and can be target molecules for prevention and control of enteric ETEC infection by probiotic Lactobacillus reuteri. Our data provide a comprehensive insight into the immune response of porcine intestinal epithelial cells (IECs) to ETEC infection and advance the identification of targets for prevention and control of ETEC-related PWD.

236 Performance and Antibiotic Use of Piglets Vaccinated with an E. coli F4/F18 Vaccination for the Prevention of F18-ETEC Post-weaning Diarrhea

Post-weaning Escherichia coli diarrhea (PWD) remains a major cause of economic losses for the pig industry. PWD, caused by enterotoxigenic E. coli (ETEC), typically provokes mild to severe watery diarrhea between 5 and 10 days after weaning. Recently, an oral live bivalent E. coli F4/F18 vaccine (Coliprotec® F4/F18; Elanco) is available on the European market, which reduces the impact of PWD provoked by F4-ETEC and F18-ETEC. The objective was to compare technical results of E. coli F4/F18 vaccination with previous standard therapeutic approach under field conditions. A 1100-sow farm with diagnosed problems of PWD due to F18-ETEC was selected. Control piglets received the standard treatment protocol with antimicrobials during the post-weaning phase. Vaccinated piglets were immunized at 18 days with the oral live bivalent E. coli F4/ F18 vaccine. At weaning, no standard group medication (ZnO and antibiotics) was applied for prevention of PWD. Piglets were fed a commercial dry feed. Several performance parameters were collected: weight at d0-47, ADWG, ADFI, FCR, TI100 and mortality. Statistical analysis was performed with JMP 14.0 – comparison of means. Oral E. coli F4/F18 vaccination significantly reduced the mortality rate (3.56% to 1.67%; P&lt; 0.05) and TI100 (10 to 0 days; P&lt; 0.05). All other performance parameters (ADWG, ADFI and FCR) were at the same level compared to pre-vaccination. Live E. coli F4/F18 vaccination against PWD resulted in similar technical performance parameters, in combination with a significant reduction in the mortality and medication use. In conclusion, control of PWD through vaccination is a good option in order to prevent piglets from the negative clinical outcomes of F18-ETEC infection during the post-weaning period.

238 Vaccination with an E. coli F4/F18 Vaccine for the Prevention of F4-ETEC Post-weaning Diarrhea Resulted in Reduced Post-weaning Mortality and Antibiotic Use

Post-weaning Escherichia coli diarrhea (PWD) remains a major cause of economic losses for the pig industry. PWD, caused by enterotoxigenic E. coli (ETEC), typically provokes mild to severe watery diarrhea between 5–10 days after weaning, which may result in mortality. Most common adhesins in ETEC are F4 and F18 fimbriae. Therapy to combat PWD typically consists of antibiotic treatment in combination with ZnO (3,000 ppm). Recently, an oral live bivalent E. coli F4/F18 vaccine (Coliprotec® F4/F18; Elanco) was approved on the European market, which reduces the impact of PWD provoked by F4-ETEC and F18-ETEC. The objective was to evaluate mortality and antibiotic use following E. coli F4/F18 vaccination under field conditions. A 160-sow farm (weaning at 26 days) with diagnosed problems of PWD due to F4-ETEC was selected. Piglets were vaccinated at 21 days with the oral live bivalent E. coli F4/F18 vaccine. At weaning, no standard group medication (ZnO and antibiotics) was applied for prevention of PWD. Several performance parameters were collected: treatment incidence (TI100), mortality and days in nursery. Vaccination (n = 3 groups) was compared to a historical control (n = 3 groups) Oral E. coli F4/F18 vaccination significantly reduced TI100 (18.6 ± 6.3 days to 2.4 ± 1.9 days; P &lt; 0.05) due to the reduction in days of antimicrobial group treatment. Mortality rate significantly reduced (11.2 ± 2.6% in control to 4.5 ± 1.5% in vaccinated group; P &lt; 0.05) following vaccination. Days in nursery (48.5 ± 0.3 days) remained constant throughout the trial. The results show that live E. coli F4/F18 vaccination against PWD has significantly impacted mortality, in combination with a reduction in medication use. In conclusion, control of PWD through oral vaccination is a successful option in order to prevent piglets from the negative clinical outcomes of F18-ETEC infection during the post-weaning period.

Chicken Egg Yolk Antibody (IgY) Protects Mice Against Enterotoxigenic Escherichia coli Infection Through Improving Intestinal Health and Immune Response

Chicken egg yolk antibody (IgY), considered as a potential substitute for antibiotics, has been used for preventing pathogens infection in food, human and animals. This study investigated effects of IgY on growth, adhesion inhibitory and morphology of enterotoxigenic Escherichia coli (ETEC) K88 in vitro, and evaluated the protective effects of IgY on intestinal health and immune response of mice infected with ETEC in vivo. Sixty pathogen-free C57BL/6J (4-6 weeks of age) mice were divided into six treatments: control (neither IgY nor ETEC infection), ETEC infection, ETEC-infected mice treated with 250 μL of high-dose (32 mg/mL), medium-dose (16 mg/mL) or low-dose (8 mg/mL) anti-ETEC IgY, or ETEC-infected mice treated with 250 μL of non-specific IgY (16 mg/mL). Anti-ETEC IgY inhibited ETEC growth, reduced adherence of ETEC to intestinal epithelial cells J2 and damaged the morphology and integrity of ETEC cell. Oral administration of anti-ETEC IgY effectively ameliorated ETEC-induced clinical signs, reduced ETEC colonization and intestinal permeability, alleviated inflammatory response through reducing the production and expression of proinflammatory cytokines, improved intestinal morphology, and inhibited excessive activation of the mucosal immune response of challenged mice. The overall protective effects of high-dose and medium-dose anti-ETEC IgY against ETEC infection were more effective. These results suggest that anti-ETEC IgY may function as a promising novel prophylactic agent against enteric pathogens infection.

Tannins extract from Galla Chinensis can protect mice from infection by Enterotoxigenic Escherichia coli O101

Background Enterotoxigenic Escherichia coli (ETEC) is classically associated with acute secretory diarrhea, which induces 2 million people death in developing countries over a year, predominantly children in the first years of life. Previously, tannins (47.75%) were extracted from Galla Chinensis and prepared as Galla Chinensis oral solution (GOS) which showed significant antidiarrheal activity in a castor oil-induced diarrhea in mice. Whether the tannins extract were also effective in treatment of ETEC-induced diarrhea was determined in this study. Methods Mice were randomly divided into 6 groups (n = 22). The mice in the normal and untreated groups were given normal saline. Three GOS-treated groups were received different concentrations of GOS (5, 10 and 15%, respectively) at a dose of 10 mL/kg. Mice in the positive control group were fed with loperamide (10 mg/kg). The treatment with GOS started 3 days before infection with ETEC and continued for 4 consecutive days after infection. On day 3, mice were all infected with one dose of LD50 of ETEC, except those in the normal group. Survival of mice was observed daily and recorded throughout the study. On days 4 and 7, samples were collected from 6 mice in each group. Results GOS could increase the survival rate up to 75%, while in the untreated group it is 43.75%. The body weights of mice treated with 15% GOS were significantly increased on day 7 in comparison with the untreated group and the normal group. GOS-treatment recovered the small intestine coefficient enhanced by ETEC-infection. The diarrhea index of mice treated with GOS was significantly decreased. GOS increased the levels of IgG and sIgA in the terminal ileum and decreased the levels of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-6 and IL-8) in serum. GOS could increase the amount of intestinal probiotics, Lactobacilli and Bifidobacteria. GOS could alleviate colon lesions induced by ETEC-infection. GOS showed higher potency than loperamide. Conclusions GOS could be a promising drug candidate for treating ETEC infections.