Risk Factors for Prevalence of Enterotoxigenic Escherichia coli (ETEC) in Diarrheic and Non-diarrheic Neonatal and Weaner Pigs, South Africa

Enterotoxigenic Escherichio coli （ETEC） causes neonatal and post-weaning diarrhea in pigs. In order to determine the risk factors, rectal/fecal swabs and visceral organs obtained from pig farms in two regions of South Africa were analyzed microbiologically against risk variables. Seventy-two percent of young pigs were found to be positive for ETEC toxin genes; estB （38.9%）, estB/STAP （25%）, and estB/LT （13.9%） were dominant. Risk factors for ETEC-diarrhea in pigs include： leaving sick piglets in a pen with healthy piglets [odds ratio （OR） = 33.52; P 〈 0.0001]; water spillage in pen （OR = 42.87; P 〈 0.0001）; hypothermic piglets （OR = 7.29; P 〈 0.0001）; runt piglets in pen with healthy littermates （OR = 3.65; P 〈 0.0001）; and prolonged use of antibiotics （OR = 3.05; P = 0.05）.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of a-linolenic acid and 12,13-epoxyoctadecenoic acid

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium(DSS)-induced colitis in mice. To explore the protective effects of L. plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli(ETEC), 28 weaned piglets were assigned into four groups, namely, CON group—orally given 10 mL/d phosphate buffer saline(PBS), LI47 group—orally given a mixture of 10 m L/d L. plantarum L47 and inulin, ECON group—orally given 10 mL/d PBS and challenged by ETEC, and ELI47group—orally given 10 mL/d L. plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L. plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased antiinflammatory factors. Transcriptome analysis revealed that L. plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA(PLA2G2A)(P < 0.05) as well as affected alphalinolenic acid(ALA) metabolism and linoleic acid metabolism. Moreover, L. plantarum L47 and inulin increased the levels of ALA(P < 0.05), lipoteichoic acid(LTA)(P < 0.05), and 12,13-epoxyoctadecenoic acid(12,13-EpOME)(P < 0.05) and the protein expression of Toll-like receptor 2(TLR2)(P = 0.05) in the ileal mucosa. In conclusion, L. plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.

Micro-encapsulated essential oils and organic acids combination improves intestinal barrier function,inflammatory responses and microbiota of weaned piglets challenged with enterotoxigenic Escherichia coli F4(K88^+)

This study evaluated the effects of micro-encapsulated(protected)organic acids(OA)and essential oils(EO)combination,P(OA+EO),and effects of a regular blend of free acids(FA)on the growth,immune responses,intestinal barrier and microbiota of weaned piglets challenged with enterotoxigenic Escherichia coli(ETEC)F4(K88^+).A total of 30 crossbred(Duroc×Landrace×Large White)weaned barrows(7.41±0.06 kg,28 d old)were assigned randomly to 5 treatments:1)non-challenged positive control(PC),2)ETEC F4(K88^+)-challenged negative control(NC),3)NC+kitasamycin at 50 mg/kg+olaquindox at 100 mg/kg+free acidifier(FA)at 5 g/kg,4)NC+kitasamycin at 50 mg/kg+olaquindox at 100 mg/kg+P(OA+EO)at 1 g/kg(P1),5)NC+kitasamycin at 50 mg/kg+olaquindox at 100 mg/kg+P(OA+EO)at 2 g/kg(P2).Each dietary treatment had 6 replicates of one piglet each and the study lasted for 3 wk.On d 7,pigs in NC,FA,P1 and P2 were orally dosed with 10 mL of ETEC F4(K88^+)culture(1×10^9 CFU/mL).From d 7 to 14 after the ETEC F4(K88^+)challenge,P1 increased gain-to-feed ratio(G:F)significantly(P<0.05)compared with NC and FA groups.From d 14 to 21,P2 increased the average daily gain of pigs(P<0.05)compared with NC and FA groups.Compared with NC,P2 reduced tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-10 concentrations(P<0.05)in sera collected at 4 h later after ETEC F4(K88^+)challenge.On d 21,P1 increased occludin and zonula occludens-1 protein expression in ileum compared with NC(P<0.05).After this 3-wk experiment,alpha diversity of gut microbiota was decreased by P2 compared with PC,and P1 increased the relative abundance of Lactobacillus in ileum,cecum and colon(P<0.05).In co nclusio n,dietary P(OA+EO)additive at 2 g/kg combined with antibiotics could improve piglet performance and attenuate inflammation,and P(OA+EO)additive at 1 g/kg combined with antibiotics improved intestinal barrier and increased beneficial microbiota composition after an F4(K88^+)challenge.

Intestinal challenge with enterotoxigenic Escherichia coli in pigs,and nutritional intervention to prevent postweaning diarrhea

Gut health of nursery pigs immediately after weaning is tightly associated with their growth performance and economic values. Postweaning diarrhea(PWD) is one of the major concerns related to gut health of nursery pigs which often is caused by infections of enterotoxigenic Escherichia coli(ETEC),mainly including F4(K88)^+ and F18^+ E. coli. The main virulence factors of ETEC are adhesins(fimbriae or pili) and enterotoxins. The common types of fimbriae on ETEC from PWD pigs are F18^+ and F4^+. Typically, PWD in pigs is associated with both F18^+ and F4^+ ETEC infections whereas pre-weaning diarrhea in pigs is associated with F4+ ETEC infection. Enterotoxins including heat-labile enterotoxins(LT) and heatstable peptide toxins(ST) are associated with causing diarrhea in pigs. At least 109 to 1010 ETEC are required to induce diarrhea in nursery pigs typically lasting 1 to 5 days after ETEC infection. Antibiotics used to be the most effective way to prevent PWD, however, with the increased bacterial resistance to antibiotics, alternatives to the use of antibiotics are urgently needed to prevent PWD. Immunopropylaxis and nutritional intervention of antimicrobial minerals(such as zinc oxide and copper sulfate), organic acids, functional feedstuffs(such as blood plasma and egg yolk antibodies), direct fed microbials, phytobiotics, and bacteriophage can potentially prevent PWD associated with ETEC. Some other feed additives such as nucleotides, feed enzymes, prebiotic oligosaccharides, and clay minerals can enhance intestinal health and thus indirectly help with preventing PWD. Numerous papers show that nutritional intervention using selected feed additives can effectively prevent PWD.

Combined effects of chitosan and microencapsulated Enterococcus faecalis CG1.0007 probiotic supplementation on performance and diarrhea incidences in enterotoxigenic Escherichia coli K88^+ challenged piglets

The aim of this study was to investigate the combined effects of chitosan oligosaccharide(COS) and a microencapsulated Enterococcus faecalis CG1.0007 probiotic(PRO) on growth performance and diarrhea incidences in enterotoxigenic Escherichia coli(ETEC) K88^+ challenged piglets in a 14-d study. Thirty piglets,7.19 ± 0.52 kg initial BW weaned at 21 ± 1 d.were allotted to 5 treatment groups(n = 6)consisting of a corn-soybean meal diet with no additive(negative control, NC), NC + 0.25% chlortetracycline(positive control, PC), NC + 400 mg/kg COS(COS), NC + 100 mg/kg PRO(PRO) and NC + a combination of COS and PRO(CPRO). Pigs were individually housed in cages, acclimated to treatments for a 7-d period and had ad libitum access to feed and water throughout the study, On d 8, pigs were weighed, blood samples were collected, and then orally challenged with 6 mL(1 ×10^(11) cfu/mL) of freshly grown ETEC inoculum. During post-challenge period, blood was sampled at 24 and 48 h to determine plasma urea nitrogen(PUN), and diarrhea incidences and fecal consistency scores were recorded from d 9 to 12. On d 14, all pigs were weighed and then euthanized to obtain intestinal tissue samples for histomorphometric measurements. Growth performance responses were similar among treatments during the pre-and post-challenge periods. There were no significant differences in PUN content, incidences of diarrhea, and fecal consistency scores among treatments. The intestinal histomorphology results did not differ significantly among treatments except for PC with increased(P = 0.0001) villus:crypt ratio compared with the NC. Under the conditions of the present study, it can be concluded that supplementation of piglet diets with 400 mg/kg COS, 100 mg/kg microencapsulated PRO or their combination did not significantly improve piglet growth performance both during the pre-and post-ETEC K88+ oral inoculation. Also, there were no significant reduction of incidences and severity of diarrhea after challenge compared with the control group.

Melatonin shapes bacterial clearance function of porcine macrophages during enterotoxigenic Escherichia coli infection

Due to the immature gastrointestinal immune system,weaning piglets are highly susceptible to pathogens,e.g.,enterotoxigenic Escherichia coli(ETEC).Generally,pathogens activate the immune cells(e.g.,macrophages)and shape intracellular metabolism(including amino acid metabolism);nevertheless,the metabolic cues of tryptophan(especially melatonin pathway)in directing porcine macrophage function during ETEC infection remain unclear.Therefore,this study aimed to investigate the changes in the serotonin pathway of porcine macrophages during ETEC infection and the effect of melatonin on porcine macrophage functions.Porcine macrophages(3D4/21 cells)were infected with ETEC,and the change of serotonin pathway was analysed by reverse transcription PCR and metabolomic analysis.The effect of melatonin on porcine macrophage function was also studied with proteomic analysis.In order to investigate the effect of melatonin on bacterial clearance function of porcine macrophages during ETEC infection,methods such as bacterial counting,reverse transcription PCR and western blotting were used to detect the corresponding indicators.The results showed that ETEC infection blocked melatonin production in porcine macrophages(P<0.05)which is largely associated with the heat-stable enterotoxin b(STb)of ETEC(P<0.05).Interestingly,melatonin altered porcine macrophage functions,including bacteriostatic and bactericidal activities based on proteomic analysis.In addition,melatonin pretreatment significantly reduced extracellular lactate dehydrogenase(LDH)activity(P<0.05),indicating that melatonin also attenuated ETEC-triggered macrophage death.Moreover,melatonin pretreatment resulted in the decrease of viable ETEC in 3D4/21 cells(P<0.05),suggesting that melatonin enhances bacterial clearance of porcine macrophages.These results suggest that melatonin is particularly important in shaping porcine macrophage function during ETEC infection.

Oleanolic acid improved intestinal immune function by activating and potentiating bile acids receptor signaling in E. coli-challenged piglets

Background Infection with pathogenic bacteria during nonantibiotic breeding is one of the main causes of animal intestinal diseases.Oleanolic acid(OA)is a pentacyclic triterpene that is ubiquitous in plants.Our previous work demonstrated the protective effect of OA on intestinal health,but the underlying molecular mechanisms remain unclear.This study investigated whether dietary supplementation with OA can prevent diarrhea and intestinal immune dysregulation caused by enterotoxigenic Escherichia coli(ETEC)in piglets.The key molecular role of bile acid receptor signaling in this process has also been explored.Results Our results demonstrated that OA supplementation alleviated the disturbance of bile acid metabolism in ETEC-infected piglets(P<0.05).OA supplementation stabilized the composition of the bile acid pool in piglets by regulating the enterohepatic circulation of bile acids and significantly increased the contents of UDCA and CDCA in the ileum and cecum(P<0.05).This may also explain why OA can maintain the stability of the intestinal microbiota structure in ETEC-challenged piglets.In addition,as a natural ligand of bile acid receptors,OA can reduce the severity of intestinal inflammation and enhance the strength of intestinal epithelial cell antimicrobial programs through the bile acid receptors TGR5 and FXR(P<0.05).Specifically,OA inhibited NF-κB-mediated intestinal inflammation by directly activating TGR5 and its downstream c AMP-PKA-CREB signaling pathway(P<0.05).Furthermore,OA enhanced CDCA-mediated MEK-ERK signaling in intestinal epithelial cells by upregulating the expression of FXR(P<0.05),thereby upregulating the expression of endogenous defense molecules in intestinal epithelial cells.Conclusions In conclusion,our findings suggest that OA-mediated regulation of bile acid metabolism plays an important role in the innate immune response,which provides a new diet-based intervention for intestinal diseases caused by pathogenic bacterial infections in 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 antibiotic exacerbated 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;0.5 mg/kg carbadox)and label-recommended dose antibiotic(REC;50 mg/kg carbadox)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 labelrecommended dose antibiotic may contribute to the promotion of disease resistance in weaned pigs.