Molecular Mechanism of the Inflammatory Response Induced by Enterotrxigenic E. coli K88 in Piglets

To investigate the molecular mechanism of the inflammatory response in the piglets infected with enterotoxigenic E. coli (ETEC) K88, piglets were infected with ETEC K88,the IL-8 content in serum of piglets were assayed by ELISA,and the mRNA relative expression levels of TLR2/4 and its signal transduction pathway related genes (MyD88,Tollip and Bcl3) in mesenteric lymph nodes were detected by quantitative Real-time PCR. The results showed that compared with control group,the content of IL-8 in serum and the expressions of TLR2/4 in lymph nodes were all extremely significantly or significantly increased at 6 and 24 h after infection (P<0.01;P<0.05),and the IL-8 content and TLR2/4 mRNA expression at 24 h after infection were all significantly lower than those at 6 h after infection (P<0.05).In addition,the expressions of MyD88,Tollip and Bcl3 in lymph nodes were all extremely significantly increased at 24 h after infection compared with control group (P<0.01), but there was no significant difference between experimental group and control group at 6 h after infection (P>0.05). In conclusion,ETEC K88 infected piglets might produce inflammatory cytokines IL-8 through the TLR2/4-MyD88 signaling pathway,which could promote the inflammatory reaction in piglets. This inflammatory response might be regulated by Tollip and Bcl3,which could weak the inflammatory intensity in piglets.     

饲粮添加迷迭香酸对产肠毒素大肠杆菌K88攻毒断奶仔猪结肠菌群组成、屏障功能及炎症反应的影响

本研究旨在探究饲粮添加迷迭香酸（RA）对产肠毒素大肠杆菌K88(ETEC K88)攻毒断奶仔猪结肠菌群组成、屏障功能及炎症反应的影响。选取18头体重为（6.91±1.02） kg的健康21日龄断奶仔猪,随机分为对照组（CON组）、攻毒组（K88组）和迷迭香酸组（RA组）,每组6头仔猪。CON组和K88组每天饲喂基础饲粮,RA组每天饲喂含RA的试验饲粮（在基础饲粮基础上添加500 mg/kg RA）。试验第19～20天,K88组、RA组仔猪每天灌喂4 mL浓度为109CFU/mL的ETEC K88重悬液攻毒,对照组仔猪则每天灌喂同等剂量的无菌生理盐水。所有仔猪在试验第22天屠宰取样。结果显示：1）与CON组相比,K88组断奶仔猪腹泻指数显著增加（P<0.05）;与K88组相比,RA组断奶仔猪腹泻率极显著降低（P<0.01）。2）与K88组相比,RA组断奶仔猪结肠隐窝深度和黏膜厚度极显著降低（P<0.01）。3）与CON组相比,K88组断奶仔猪结肠菌群Chao1和ACE指数显著降低（P<0.05）。4）在门水平上,与K88组相比,RA组断奶仔猪结肠菌群中拟杆菌门（B...     

仔猪腹泻源大肠杆菌贵州株K88菌毛研究

通过玻片凝集试验、抗Ｄ－甘露糖微量血凝试验（ＭＲＭＨ）、离体细胞粘附试验、菌体蛋白的聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）和免疫印迹、质粒ＤＮ民泳等。分析了仔猪腹泻源大砀杆菌贵州株（４０９－１１）所产Ｋ８８菌毛的血凝谱、对肠上皮细胞粘附特性、菌毛蛋白单位分子量,并对其Ｋ８８基因进行了初步定位。结果表明：其所产Ｋ８８菌毛的血凝谱能凝集豚鼠和允许红细胞;能介导细菌粘会于仔猪离体肠上皮细胞,这种粘附作用     

饲用凝结芽孢杆菌对感染产肠毒素大肠杆菌K88断奶仔猪生长性能和肠道健康的影响

本文旨在研究饲用凝结芽孢杆菌对感染产肠毒素大肠杆菌(ETEC)K88断奶仔猪生长性能和肠道菌群、形态及细胞因子表达的影响.试验选用32头25日龄断奶的健康杜×长×大商品公猪,按照初始体重一致原则分成4个组,每组8个重复,单笼饲养.ETEC K88感染前7 d,2个组饲喂基础饲粮(BD组),另外2个组饲喂在基础饲粮中...     

K88、K99、987P仔猪大肠杆菌菌种的提纯和保存

由产肠毒素性大肠杆菌感染而引起的仔猪急性下痢分布十分广泛,本病主要危害一月龄以内的新生仔猪,尤其是一周龄以内的仔猪发病率和死亡率最高,多以急性、水样腹泻为特性。随着大型集约化养殖业的发展,病原性大肠杆菌对畜牧业所造成的损害已日益明显。产肠毒素性大肠杆菌含K88、     

大肠杆菌K88纤毛抗原的提纯和抗血清的制备

以5％绵羊血改良Minca琼脂筛选和克隆表达良好的K88^＋菌株，改良Minca琼脂培养获得K88菌液。用加热法及高速匀浆法使K88纤毛从菌体上脱下。饱和硫酸铵沉淀粗提，用Sepharose CL-4B凝胶过滤纯化K88抗原。提纯的抗原经SDS—PAGE电泳测定其分子量约为25000，且仅此一条蛋白带；与K88抗血清进行琼脂扩散试验只出现一条沉淀线。用提纯的抗原经多次免疫家兔制备了K88抗血清。抗血清在玻板凝集试验中只凝集K88菌株，不凝集K99，987P或F41菌株；在琼扩试验中，只与K88粗提抗原出现一条沉淀线，且效价为1：64，而不与K99，987P抗原出现沉淀线；在免疫电泳试验中，与无细胞K88纤毛液只出现一条沉淀线。鉴定结果表明，所制备的K88血清特异性强、效价高，可作为单因子血清用于凝集试验、琼扩试验等血清学试验，对K88菌株进行鉴定，对K88纤毛抗原进行定性和定量检测。     

产肠毒素大肠杆菌K88ab/K88ad菌毛操纵子fae全基因的克隆、表达及生物学活性的初步研究

为研究K88ab/K88ad菌毛对细胞的黏附作用,本研究分别以产肠毒素E.coli (ETEC) K88ab C83901株和K88ad C83903株基因组DNA为模板,采用PCR技术扩增这两种K88菌毛操纵子fae基因(均约7.9 kb).将其分别克隆于表达质粒pBR322中构建pBR-K88ab和pBR-K88ad重组质粒,并将其分别转化至不含任何菌毛的E.coli SE5000株中.该重组菌能够分别与鼠抗K88菌毛阳性血清和抗K88菌毛单克隆抗体(MAb)产生凝集反应;在电镜下观察到重组菌表面大量表达K88菌毛.采用热抽提法提取其体外表达的K88ab和K88ad菌毛,SDS-PAGE电泳检测结果显示,菌毛蛋白的分子量约为26 ku.玻板凝集试验和western blot结果表明:重组表达的K88ab及K88ad菌毛与K88+参考株菌毛均能够被抗K88菌毛阳性血清和MAb识别.以猪小肠上皮细胞系IPEC-J2为模型进行黏附和黏附抑制试验,结果表明表达K88菌毛的重组菌及K88+参考株均能够黏附于IPEC-J2上皮细胞表面;而且阳性血清和MAb能够有效抑制重组菌或K88+参考株对猪小肠上皮细胞系的黏附结合.     

动物性食品源大肠杆菌O血清型鉴定及其K88菌毛基因检测

本研究对河北省冀东地区农贸市场和超市采集的生猪肉、生鸡蛋和生羊肉等分离得到的20株大肠杆菌进行大肠杆菌血清型鉴定;并检测不同血清型大肠杆菌的K88菌毛基因。采用常规方法进行大肠杆菌的O血清型鉴定,用PCR方法检测K88菌毛基因。分离鉴定的20株大肠杆菌有7种血清型,包括O38、O78、O88、O11、O107、O91、O9,其中O38、O78为优势血清型菌,均占分离菌株的25%(5/20)。在分离的动物性食品源大肠杆菌中有30%(6/20)的菌株K88菌毛基因扩增呈阳性。结果表明,O78、O38为冀东地区动物性食品源大肠杆菌常见血清型,30%(6/20)的菌株K88菌毛基因扩增阳性。     

规模化猪场仔猪黄白痢的综合防治措施研究

作者在对乐山，新津、邛崃市县几个规模化猪场进行致病性大肠杆菌菌株的分离鉴定基础上，研制了针对性很强的灭活疫苗和生物制剂，提出了一套完善的综合防制措施，经临床应用证明可以很好控制仔猪黄白痢的发生。     

产肠毒素大肠杆菌双重PCR检测方法的建立

为了快速检测和鉴定产肠毒素大肠杆菌菌毛(K88和K99)基因,本研究设计合成了针对K88、K99的2对特异性引物,对扩增条件进行优化,建立了检测K88和K99的双重PCR方法。该方法对K88、K99基因的扩增产物大小分别为237和314 bp;最终确定dNTP终浓度0.4 mmol/L,K88、K99的引物终浓度均为25 μmol/L,退火温度为52℃。试验结果表明,该方法具有良好的灵敏性和特异性。用所建立的双重PCR方法对实验室分离的23株大肠杆菌进行检测,结果显示,K88单重PCR阳性2株,K99单重PCR阳性3株,K88和K99双重PCR阳性5株。本研究建立的双重PCR检测方法为致幼畜腹泻产肠毒素大肠杆菌的快速准确检测提供了方法。     

Identification of infant and adult swine susceptible to enterotoxigenic Escherichia coli by detection of receptors for F4(K88)ac fimbriae in brush borders or feces.

We attempted to determine F4(K88)-adhesive and non-adhesive phenotypes of infant (neonatal <3 day old and weaned <4 week old pigs) and adult (>6 month old) swine by ELISA using immobilized F4(K88)ac fimbrial antigen or whole F4(K88) + E. coli cells (strains M1823 and 1476) and isolated small intestinal brush borders or easily-obtainable fecal samples from the same animals. Nineteen of 22 neonates (86%), 17 of 20 weaners (85%), and 26 of 39 adults (67%) were classified identically as F4(K88) receptor-positive or negative by the ELISA. The ELISA with feces from adult swine was found to be almost equally specific (87%) as that with feces from neonatal (90%) and weaned (91%) pigs. However, the sensitivity of the assay was low (38%), indicating that fecal samples from adults contained less receptor-material than necessary for comparable phenotyping. The receptor-positive brush borders from neonates and weaners reacted significantly better (P < 0.02, < 0.001 respectively) with purified F4(K88) antigen than did those from adults. There was good agreement between the average ELISA values for feces from infant and adult swine regardless the source of coating antigen applied. With this assay we can determine F4(K88) phenotypes of infant swine using easily-collected fecal samples rather than isolated brush borders. It was also concluded that tested feces is not an acceptable alternate source of the receptor-material to brush borders from F4(K88)-susceptible adult swine.     

华东地区部分猪源大肠杆菌K88黏附素的生物学特性

近年来，从华东地区患腹泻仔猪中分离到一些表达K88菌毛的大肠杆菌，这些菌株只与K88a因子单抗反应，而不与b、c、d因子单抗反应。通过K88常规血清交叉吸收试验、SDS-PAGE、Western印迹，表明这些菌株不仅与K88ac参考菌株C83907制备的c因子血清反应，而且与以分离株SEC586制备且经K88ab、K88ac、K88ad参考菌株吸收后的血清也反应。对分离株SEC586、SEC464的K88主要亚单位结构基因faeG的克隆、测序，发现该基因由846对核苷酸组成，编码菌毛主要亚单位的262个氨基酸及21个氨基酸的信号肽，比国外报道的K88ac FaeG亚单位（263个氨基酸）少了1个氨基酸，比K88ab、K88ad（265个氨基酸）少了3个氨基酸。SEC586、SEC464菌株的FaeG亚单位氨基酸序列的同源性为97．7％，它们与K88ac的同源性为94．7％和96．2％；与K88ab的同源性为90．1％和91．2％；与K88ad的同源性为87．0％和88，6％。结果表明，新分离的K88ac大肠杆菌黏附素主要亚单位已发生了部分变异。     

葛根芩连汤对感染大肠杆菌小鼠血常规、血液内毒素及炎性细胞因子含量的影响

旨在研究葛根芩连汤预防及治疗感染大肠杆菌K_(88)小鼠的血常规、血液内毒素及炎性细胞因子含量的影响。统计小鼠的死亡率,血常规检测小鼠血液中白细胞、红细胞及血小板的变化,以及采用显色基质鲎试剂法和ELISA方法检测感染小鼠血液里内毒素及炎性因子TNF-α、IL-1β、IL-6的含量变化。结果显示,预防试验中葛根芩连汤组小鼠死亡率为20%,治疗试验中葛根芩连汤组死亡率为30%,均极显著地低于模型组(P0.01),且与氟苯尼考组相比没有差异;葛根芩连汤组小鼠血液中白细胞指标均极显著地低于模型组(P0.01),红细胞及血小板指标均极显著地高于模型组(P0.01);葛根芩连汤组小鼠白细胞、中性粒细胞及血小板数目极显著的高于氟苯尼考组(P0.01),葛根芩连汤组小鼠内毒素及TNF-α、IL-1β、IL-6的含量均极显著地低于模型组(P0.01)。结果表明:葛根芩连汤对感染大肠杆菌K_(88)小鼠具有预防与治疗作用,而且在抑制早期炎性因子的释放,降低机体炎症反应水平方面优于氟苯尼考。     

一株不表达K88、K99、987P粘附因子的鸽源产毒素性大肠杆菌的分离鉴定

从病鸽尸体中分离出一株大肠杆菌,通过生化试验、血清学鉴定及动物致病性试验,表明该菌是一株具有很强致病力的不表达Ｋ８８、Ｋ９９、９８７Ｐ粘附素的鸽源产毒素性大肠杆菌。     

猪K88、K99、987P、F41埃希氏大肠杆菌高密度发酵培养技术的初步探讨

采用高密度发酵和普通深层通气两种方法培养含K88、K99、987P、F41菌毛抗原的四株猪埃希氏大肠杆菌,对其培养液进行活菌数、OD值、pH值、效价的测定。实验结果表明,运用高密度发酵方法培养,各菌活菌数可达4.1×1010~4.9×1010CFU/mL,效价为211~213;运用普通深层通气方法培养,各菌活菌数为0.51×1010~0.59×1010CFU/mL,效价为24~25,可选择高密度发酵方法替代普通深层通气方法培养,用于制备猪埃希氏大肠杆菌K88、K99、987P、F41四价菌毛提纯苗。