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）.

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

Background:This study aimed to investigate the effects of oral administration of Enterococcus faecium NCIMB10415(E.faecium)on intestinal development,immunological parameters and gut microbiota of neonatal piglets challenged with enterotoxigenic Escherichia coli K88(ETEC).A total of 961-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 3×10~9 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.Results:Before 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 m RNA abundance of claudin-1 in the ileum,whereas increased the percentage of lymphocytes,concentrations of IL-1βin the plasma and TNF-αin the ileal mucosa,as well as increased the m RNA 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.Conclusions:These 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.

The Truncated Gene cfaD′ Positively Regulates CFA/Ⅰ Expression of Enterotoxigenic Escherichia coli

The gene cluster cfaABCED’ of enterotoxigenic Escherichia coli, encoding the fimbriae which is called colonization factor antigen located on a plasmid. It is positively regulated by cfaR, a member of the AraC family, and the cfaD’ gene region, which is located downstream of cfaE and is homologous to cfaR, had been described as a truncated cryptic gene. In the present study we observed that the CFA/ fimbriae subunit, cfaB, was expressed in lower amount by the cfaABCED’ clone pNTP513 in host E. coli HB101. The expression of CFA/ diminished by deletion of cfaD’ gene region from pNTP513, and was restored by acquisition of cfaD’ in trans. Furthermore, CFA/ expression by cfaD’ deletion mutant, the cfaABCE clone, was remarkably increased by the presence of cfaD’ in trans in a topoisomerase A deficient strain of E. coli DM800. These data suggest that cfaD’ region is a functional region of gene, that regulates the CFA/ expression with cfaR by unknown mechanism.

基于微生物组和宿主转录组整合分析香砂六君子汤对ETEC诱导断奶腹泻仔猪回肠损伤的调控机制

本研究旨在探讨香砂六君子汤对产肠毒素大肠杆菌(entexotoxigenic Escherichia coli,ETEC)诱导的断奶仔猪腹泻的干预作用及其机制。将24头21日龄断奶仔猪随机分为空白组(CON)、模型组(MOD)和香砂六君子汤组(XS),连续14 d给XS组灌服香砂六君子汤(1 mL·kg^(-1),1 g·mL^(-1)),其余组灌服等量无菌水,第15天给MOD组、XS组仔猪按1 mL·kg^(-1)连续3 d灌服1011 CFU·mL^(-1) ETEC菌液,XS组继续灌服香砂六君子汤。分别记录各组仔猪腹泻评分,HE法检测回肠组织病理学变化,实时荧光定量PCR检测回肠组织IL-1β、IL-6、IL-8 mRNA水平,转录组学分析回肠组织RNA差异表达情况,微生物宏基因组学分析回肠菌群变化情况,Western blot检测回肠组织p-p38/p38、p-ERK/ERK、p-JNK/JNK蛋白水平。结果表明,灌服ETEC后,MOD组腹泻评分极显著高于CON组(P<0.01),回肠组织结构破坏,V/C值极显著下降(P<0.01),且IL-1β、IL-6和IL-8 mRNA表达量极显著升高(P<0.01),肠道菌群门水平上变形菌门极显著上调(P<0.01),厚壁菌门极显著下调(P<0.01),属水平上乳酸菌属下调,志贺菌属上调,回肠组织p-p38/p38和p-JNK/JNK比值显著或极显著升高(P<0.05或P<0.01)。与MOD组相比,XS组腹泻评分极显著下降(P<0.01),回肠组织结构完整,V/C值极显著升高(P<0.01),回肠组织IL-1β、IL-6和IL-8 mRNA表达量显著或极显著降低(P<0.05或P<0.01),肠道菌群门水平上变形菌门极显著下调(P<0.01),厚壁菌门极显著上调(P<0.01),属水平上乳酸菌属上调,志贺菌属下调,回肠组织p-p38/p38和p-ERK/ERK比值极显著降低(P<0.01)。肠道组织基因转录组学筛选出与炎症免疫相关的差异显著的10个基因,分别为TNFAIP8L2、TRIM67、CXCL2、EGF、NOX1、CCL28、FABP2、FABP6、IL1RAP和CEBPB。微生物组学筛选出各组可能的标志物种分别为CON组的Lactobacillaceae,MOD组的Shigella,XS组的Deinococcus和Eubacterium。综上所述,香砂六君子汤可有效缓解ETEC诱导的断奶仔猪腹泻,提高肠道菌群中有益菌的丰度,逆转ETEC诱发的菌群结构的改变,并可通过抑制MAPK信号通路的激活,从而缓解ETEC诱导的肠道炎性损伤。

赤灵芝多糖对ETEC所致肠炎的影响及机制研究

目的研究赤灵芝多糖(ganoderma lucidum polysaccharides,GLP)对产肠毒素型大肠杆菌(enterotoxigenic Escherichia coli,ETEC)诱导小鼠肠炎的作用效果及机制。方法选用24只6~8周龄雄性SPF级昆明小鼠,利用ETEC菌液腹腔注射诱导建立小鼠肠炎模型。采用随机数字表法分为为正常组、ETEC感染组、ETEC感染+高剂量GLP组,ETEC感染+低剂量GLP组,每组6只。采用HE染色观察小鼠小肠组织病理变化,采用免疫印迹法检测各组小鼠小肠组织中C反应蛋白(C-reactive protein,CRP)、肿瘤坏死因子(tumour necrosis factor,TNF)-α和白细胞介素(interleukin,IL)-6蛋白的表达情况。检测各组小鼠小肠组织中Toll-样受体4(Toll-like receptor4,TLR4)、核因子κB p65亚基(nuclear factor-κB p65,NF-κB p65)、髓样分化因子88(myeloid differentiation factor88,MyD88)mRNA的表达水平,探查GLP通过TLR4核因子κB(nuclear factor-κB,NF-κB)信号通路对ETEC诱导的小鼠肠道炎症的调控作用。结果与正常组比较,ETEC感染组小鼠出现精神萎靡,食欲减弱,小肠绒毛破损,组织中可发现大量的炎性浸润,疾病活动指数(disease activity index,DAI)呈上升趋势(P<0.05),小鼠小肠组织中TLR4、NF-κB p65、MyD88 mRNA的相对表达量和CRP、TNF-α、IL-6的蛋白表达水平增高(P<0.01)。与ETEC感染组比较,ETEC感染+不同剂量组中小鼠精神状态和食欲有所改善,小鼠小肠组织中TLR4、NF-κBp65、MyD88的mRNA相对表达量均降低(P<0.01)。ETEC感染+高剂量组中小鼠小肠组织CRP、TNF-α、IL-6蛋白表达水平降低(P<0.01),ETEC感染+低剂量组降低程度差异无统计学意义(P>0.05)。与ETEC感染+低剂量组比较,ETEC感染+高剂量组小鼠小肠组织TLR4、MyD88 mRNA的相对表达量和IL-6蛋白表达水平均降低(P<0.05)。结论GLP可有效缓解ETEC诱导的小鼠肠道的炎症,且可能通过调控TLR4/NF-κB信号通路发挥抗炎作用,其作用效果可能与浓度呈正比。

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.

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

【目的】揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系。【方法】O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测。【结果】通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以O149、O107、O139、O93和O91为主,共133株,占定型菌株的57.1%;拥有estⅠ、estⅡ、elt、stx2e和eaeA基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,O149血清型与estⅠ或estⅡ+elt密切相关,在F18菌株中,O107血清型与estⅠ或estⅡ、O139血清型与stx2e紧密相关。依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%。通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以O149、O107、O93和O98等血清型为主,O149:K88菌株主要与estⅡ或estⅡ+elt肠毒素相关,O107:F18菌株主要与estⅡ相关,O93和O98血清型菌株主要与estⅡ肠毒素相关;STEC菌株以O139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以O107:F18和O116:F18血清型为主,主要与estⅠ+stx2e或estⅡ+stx2e密切相关,ETEC/AEEC菌株以O91和O107血清型为主,全部拥有肠毒素estⅠ和紧密素基因。【结论】我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂。

双歧杆菌纯化黏附素对ETEC和EPEC黏附肠上皮细胞的竞争抑制作用

目的 观察双歧杆菌分泌型黏附素对产毒性大肠杆菌 (ETEC)和致病性大肠杆菌 (ETEC)黏附肠上皮Lovo细胞的竞争抑制作用。方法 采用黏附试验计数肠上皮Lovo细胞黏附的细菌数并比较其结果。结果 除黏附素浓度为 1μg/ml和 5 μg/ml时不能明显抑制ETEC和EPEC对Lovo细胞的黏附外 ,10 μg/ml、2 0 μg/ml、30 μg/ml浓度组均能明显抑制ETEC和EPEC对Lovo细胞的黏附 ,且随浓度的逐渐增加 ,这种抑制作用也逐渐增强。

ETEC肠毒素基因多重PCR检测方法的建立

肠毒素性大肠杆菌的致病性与其具有粘附性的菌毛和产肠毒素的能力密切相关。由于菌毛的血清型多而复杂 ,肠毒素只有不耐热肠毒素 (LT)和耐热肠毒素 (ST)两种 ,因此成为研究的对象。用三对扩增产物分别为 1 1 0bp、2 37bp、368bp的引物建立了检测LT和STⅠ、STⅡ毒素基因的多重PCR方法。扩增产物分别用HindⅢ、HincⅡ、Sau3AⅠ限制性内切酶酶切 ,均得与预期一致的 2个片段。对各个参考株的检测结果为 1 0 0 %符合。结果表明该多重PCR方法具有很好的特异性和敏感性。

猪源ETEC的黏附素基因克隆与原核表达

[目的]克服传统方法制备黏附素抗原的缺点。[方法]应用PCR对猪源性产肠毒素性大肠杆菌(ETEC)的黏附素F4、F5和F6的质粒中扩增出faeG、fanC和fasG基因片段,克隆测序并与pET 32a(+)构建重组表达载体。将重组表达载体转化E.coli表达菌株BL21(DE3),并分析其免疫原性。[结果]重组表达载体经IPTG诱导获得高效表达。血凝抑制试验表明,鼠抗血清能抑制标准的ETEC强毒株凝集红细胞,抑制效价在1∶128以上。这说明克隆表达的猪源ETEC黏附素蛋白具有良好的免疫原性。[结论]该研究可为进一步开发抗体制剂奠定基础。

2个猪品种的ATP2B1基因表达与ETEC F4粘附性关联分析

以ATP2B1基因作为仔猪腹泻相关的候选基因,在2个品种63头猪的小肠组织中,采用实时荧光定量PCR技术对肠毒素大肠杆菌(ETEC)F4受体不同黏附表型个体进行表达量的比较分析,以验证其基因功能和作为仔猪腹泻相关候选基因的可能性。结果表明:ATP2B1基因在不同黏附表型间的表达量有显著的差异,可望作为F4ab受体的相关侯选基因。

基因探针技术与平板免疫溶血试验检测ETEC—LT的比较

本文报告从腹泻患者检出237株大肠艾希氏菌,用PIHT法与LT—DNA探针技术进行ETEC—LT检测比较。结果LT—DNA探针技术检出率(13.5％)明显高于PIHT法(4.7%),未发现PIHT法检出阳性而LT—DNA探针阴性者。认为LT—DNA探针特异性强,敏感度高,标本又可直接点膜检测,不需作系统培养鉴定,适用流行病学调查大批量的标本检测等优点。

表达猪乳铁蛋白肽重组屎肠球菌对断奶仔猪生长性能的影响及其抗ETEC感染效果研究

本试验以表达猪乳铁蛋白肽的重组屎肠球菌(pNZ8112-PLFcin/Ef)饲喂断奶仔猪,研究其对仔猪生长性能的影响和抗产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)感染的效果。选取28日龄体重相近的健康断奶仔猪36头,随机分为3组(重组屎肠球菌组、空载体组和培养基组),每组3个重复,每个重复4头仔猪。重组屎肠球菌组和空载体组分别饲喂添加pNZ8112-PLFcin/Ef(6×1012 CFU/kg)和pNZ8112/Ef(6×1012 CFU/kg)的基础日粮,而培养基组饲喂含有相同体积的GM17液体培养基的基础日粮。试验期26d。结果显示,与培养基组相比,重组屎肠球菌组断奶仔猪的平均日增重极显著提高(P<0.01);料重比显著降低(P<0.05);腹泻率明显降低,肠道菌群的均匀度和多样性指数均下降。为进一步探究表达猪乳铁蛋白肽的重组屎肠球菌对断奶仔猪抵抗ETEC感染的保护作用,在连续饲喂21d后,每个重复中随机挑选1头体况相近的断奶仔猪灌服ETEC。结果发现,与培养基组相比,攻菌后重组屎肠球菌组断奶仔猪血清白细胞介素-2(IL-2)、免疫球蛋白G(IgG)含量、肠黏液中分泌型免疫球蛋白A(sIgA)水平均显著升高(P<0.05);脾脏指数显著提高(P<0.05),但胸腺指数、肠段长度及重量则均无显著差异(P>0.05)。综上所述,表达猪乳铁蛋白肽的重组屎肠球菌能够起到促进断奶仔猪生长及抗ETEC感染的保护作用。

噬菌体防治ETEC感染小鼠效果的初步研究

为研究噬菌体防治产肠毒素大肠杆菌(ETEC)感染小鼠的效果,本研究通过双层平板法测定噬菌体DS2的效价,并利用双层琼脂空斑试验分别检测噬菌体DS2对致仔猪腹泻大肠杆菌K88、K99及987p的体外裂解作用。结果显示,噬菌体DS2的效价为6.9×10^(9)pfu/mL,且均能裂解上述3种ETEC。进一步经小鼠试验评估噬菌体在体内防治ETEC感染的效果:将噬菌体分别以2×10^(8)pfu/只(高剂量组)、2×10^(7)pfu/只(低剂量组)经腹腔注射接种小鼠,1 h后再经腹腔注射0.2 mL剂量均为3×10^(8)cfu/只的K88、K99及987p混合菌液。分别于感染后12 h、24 h、48 h无菌采集各组小鼠的肝脏,采用平板计数法检测各组小鼠肝脏载菌量;利用双层平板法检测各组小鼠肝脏中的噬菌体含量。感染后24 h,利用ELISA试剂盒检测各组小鼠血清中细胞因子IL-1β含量。感染后48 h,利用内毒素检测鲎试剂盒测定各组小鼠血浆内毒素的含量。小鼠肝脏载菌量和噬菌体含量检测结果显示:与接种LB肉汤的对照组相比,高、低剂量组小鼠的肝脏载菌量在感染后不同时间有升有降,但二者之间或者与对照组之间均无显著差异(P>0.05);随着感染时间的延长,高、低剂量组小鼠肝脏噬菌体的含量均在下降,且在感染后低剂量组小鼠肝脏噬菌体的含量极显著高于(感染后24 h)(P<0.01)或者高于(感染后48 h)高剂量组。IL-1β及内毒素检测结果显示,与对照组相比,高、低剂量组小鼠血清中IL-1β的含量显著下降(P<0.05)或者下降,但高、低剂量组小鼠血浆内毒素的含量均升高或显著升高(P<0.05)。将60只小鼠按照上述实验分组及处理,观察并记录感染后72 h内各组小鼠的发病率和死亡率,并统计感染后不同时间每组小鼠的平均体况得分。结果显示,对照组小鼠在感染接近24 h后体况计分降至25分以下,高、低剂量组小鼠在感染24 h后体况计分下降至70分及90分以上。高、低剂量组及对照组小鼠的存活率分别为65%、95%及15%。以上结果表明,DS2噬菌体可有效防治体内外ETEC的感染,且低剂量DS2噬菌体的疗效总体上要优于高剂量。本研究为防治仔猪大肠杆菌病提供了新思路。

共表达ETECK99、K88菌毛蛋白重组干酪乳杆菌的构建

将PCR扩增的K99和K88-LTB基因片段串联插入到干酪乳杆菌细胞表面表达载体pLA中,构建了重组表达载体pLA-K99-K88-LTB,将其电转化干酪乳杆菌CICC 6 105,获得了表达融合蛋白pLA-K99-K88-LTB的重组干酪乳杆菌表达系统。重组干酪乳杆菌在MRS培养基中表达后,经SDS-PAGE检测,约有90 KDa的融合蛋白得到表达,蛋白大小与理论值相符合。Western-blot分析表明表达的蛋白可被鼠源K99,K88抗血清所识别。间接免疫荧光技术和流式细胞术的结果表明,融合蛋白成功地利用多聚谷氨酸跨膜蛋白pgsA基因展示在干酪乳杆菌菌体表面。

共表达ETEC 987P、K88重组乳杆菌构建及其免疫原性分析

构建表达产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)987P和K88蛋白的重组干酪乳杆菌,并对重组干酪乳杆菌免疫原性进行分析。通过PCR构建重组质粒pLA-987P-K88并电转化至干酪乳杆菌6105后进行表达,通过Western blot、间接免疫荧光和流式细胞术检测目的蛋白的表达情况。将重组干酪乳杆菌灌胃免疫BALB/c小鼠,三次免疫后用ELISA检测小鼠血清中IgG抗体和肠冲洗液中sIgA抗体水平,采用CCK-8检测淋巴细胞的增殖情况,用C83916(987P)株和C83902(K88)株进行攻毒保护性试验检测其保护率。结果显示,重组干酪乳杆菌成功表达987P-K88融合蛋白,小鼠免疫重组菌后血清中IgG抗体水平和肠冲洗液sIgA水平显著升高,细胞免疫水平明显增长,攻毒保护性试验结果显示,重组菌能同时对C83916(987P)株和C83902(K88)株具有良好的保护作用。

Macleaya cordata extract alleviated oxidative stress and altered innate immune response in mice challenged with enterotoxigenic Escherichia coli

This study examines the effects of dietary Macleaya cordata extract(MCE) on bacterial burden and resistance to enterotoxigenic Escherichia coli(ETEC) in ICR mice. ICR mice were randomly distributed into one of the following groups:(i) basal diet;(ii)basal diet with 200 mg kg–1 MCE;(iii) basal diet challenged with ETEC;and(iv) basal diet with 200 mg kg–1 MCE and challenged with ETEC. Following a 7-day period of pre-treatment, CTRL-ETEC and MCE-ETEC mice were subjected to oral infection using 5×108 E. coli SEC 470. The results showed dietary 200 mg kg–1 MCE markedly reduced intestinal ETEC burden(P<0.05) and the disease-associated mortality was significantly alleviated in the MCE treated group(P<0.05). In addition,dietary MCE markedly alleviated ETEC-induced oxidative stress, evidenced by the lowered methane dicarboxylic aldehyde(MDA) abundance and enhanced activities of catalase and glutathione peroxidase(P<0.05). Furthermore, MCE mice exhibited higher immune activity, which might have further mediated ETEC infection. These results indicate MCE plays a preventative role with respect to ETEC infection. Future research should aim to develop MCE as a therapeutic approach to the promotion of intestinal health and a safeguard against ETEC infection.

Polymorphisms of three gene-derived STS on pig chromosome 13q41 are associated with susceptibility to enterotoxigenic Escherichia coli F4ab/ac in pigs

Neonatal diarrhea caused by enterotoxigenic Escherichia coli(ETEC)F4 is a common and serious disease,resulting in significant economical loss in the pig industry.The locus encoding ETEC F4 receptor has been mapped to pig chromosome(SSC)13q41,and one of the most significantly linked markers is S0075.In this study,we selected three genes including SLC12A8,MYLK and KPNA1 from a chromosomal region flanking S0075 on SSC13 to develop pig specific sequence tagged sites(STS). Seven single nucleotide polymorphisms were identified in the three pig STS using DNA of four full-sib susceptible and resistant animals in a White Duroc×Erhualian intercross.All grandparents,parents and 755 offspring in the intercross were genotyped for three polymorphisms,including SLC12A8 g.159A>G,MYLK g.1673A>G and KPNA1 g.306A>G.Family-based transmission disequilibrium test(TDT) revealed that all polymorphisms and the corresponding haplotypes are significantly associated with ETEC F4ab/ac(especially F4ac)brush border adhesion phenotypes,indicating that these polymor- phism are in linkage disequlibrium with causal mutation(s)of the gene encoding ETEC F4ab/ac receptor. Our results strengthen the evidence for the involvement of SSC13q41 in high acquiring risk of ETEC F4ab/ac infection,and provide novel polymorphic markers for fine mapping of the ETEC F4ab/ac receptor locus.

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.

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.