大肠杆菌K99纤毛抗原的提纯和抗血清制备

在引起初生犊牛、羔羊和仔猪腹泻的肠毒素大肠杆菌中,K99是常见的粘附素,这些细菌表面纤毛能与小肠黏膜上皮细胞表面的特异性受体位点结合,使细菌吸附于肠黏膜表面并产生肠毒素,导致急性腹泻、脱水,最后衰竭死亡.     

猪源大肠杆菌fedA基因的克隆及鉴定

断奶仔猪腹泻（post—weaning diarrhea,PWD）和猪水肿病（porcine edema disease,ED）是导致仔猪死亡的重要传染性疾病。PWD和ED分别由肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）和志贺氏毒素大肠杆菌（verotoxigenic Escherichia coli,VTEC）引起。ETEC和VTEC等病原菌除了产生毒素外,还同时具有菌毛粘附因子。F18菌毛是病原菌主要的粘附因子,介导细菌对小肠黏膜上皮细胞表面受体的粘附,在PWD和ED的发生中起着决定性的作用,是引起疾病的主要毒力因子之一。     

新生仔猪腹泻的体外抑菌治疗的研究

新生仔猪腹泻是由肠毒素性大肠杆菌(ETEC)产生一种或数种菌毛粘附素粘附于小肠黏膜上皮细胞及结膜层上而引起的。在实验中给禁食初乳的新生仔猪感染K88、K99、987P菌毛型的ETEC而导致腹泻,然后口服复方中药免疫球蛋白生物制剂进行治疗。体外抑菌研究结果表明:在大肠杆菌中加入复方中药免疫球蛋白生物制剂后,在最初1～3小时内其抑菌作用最强,3小时以后,其作用急剧降低。到5小时,起抑菌作用消失。体外粘附试验结果表明:效价为1:2560复方中药免疫球蛋白生物制剂在体外可以有效的抑制大肠杆菌在小肠上皮细胞的粘附,而且ETEC在小肠不同部位的菌毛受体并无明显的特异性。     

产肠毒素性大肠杆菌菌毛的研究进展

产肠毒素性大肠杆菌(ETEC)常引起仔猪腹泻,在世界范围内造成了严重的经济损失。ETEC的致病作用与其具有粘附性菌毛和肠毒素密切相关,ETEC菌株的菌毛可与宿主黏膜上皮细胞表面的相应受体结合,并在局部组织定居、繁殖和产生毒素,损伤小肠黏膜,使小肠吸收分泌功能失常而致腹泻。本文针对ETEC菌毛的生物学特性、表达条件、免疫原性等方面作一概述。     

卵黄抗体对ETEC粘附仔猪小肠上皮细胞的体外抑制作用研究

采用热抽提法提取 4种肠毒素性大肠杆菌菌毛蛋白 :K88、K99、F41和 987p。分别制成单价或多价的菌毛蛋白白油佐剂抗原 ,对产蛋鸡进行胸部肌肉分点注射免疫 ,初免后 2周加强免疫 1次。收集高效价卵黄抗体。用所获得各卵黄抗体对体外分离的初生仔猪小肠上皮细胞进行体外粘附抑制试验。结果表明 ,各种菌毛卵黄抗体均能特异地显著抑制相应大肠杆菌对仔猪上皮细胞的粘附 ,而对其他血清型大肠杆菌对肠上皮细胞的粘附无抑制作用     

K88-K99-987P-F41菌毛疫苗预防仔猪黄痢的效果

仔猪大肠杆菌性腹泻是养猪场最常见的疾病之一。而肠毒源性大肠杆菌(Enterotoxigenic Escherichia coli，ETEC)是引起仔猪腹泻的主要病菌之一。其致病的前提是必须能在小肠黏膜定居、繁殖并产生肠毒素。ETEC表面能产生菌毛粘附因子使得这些菌株很容易吸附并定居于小肠黏     

三起仔猪黄白痢的诊断与治疗

仔猪黄白痢是由产肠毒素性大肠杆菌(ETEC)引起的临床常见的一种新生仔猪传染病,其发病机理是致病性大肠杆菌靠其表的粘附素在仔猪小肠粘膜上皮定植,产生肠毒素,激活腺苷酸环化酶,引起离子平衡失调,从而引发剧烈腹泻,导致机体脱水和代谢性酸碱平衡紊乱。该病的发生往往给养     

复方中药口服液抗腹泻作用机理研究

目的：研究复方中药口服液抗腹泻的作用机理。方法：以发病仔猪病料（小肠及内容物）、蓖麻油和番泻叶分别对小鼠灌胃,后灌服复方中药口服液,研究复方中药口服液对小鼠攻毒后的保护作用。结果：病料攻毒后小鼠的主要病理变化在肝、胆及小肠,复方中药口服液对病料和蓖麻油灌胃致小鼠腹泻具有明显的治疗作用。结论：复方中药口服液抗腹泻作用主要表现在对小肠的保护作用。     

产肠毒素大肠杆菌疫苗在断奶仔猪中的应用

产肠毒素大肠杆菌(ETEC)是引起新生仔猪和断奶仔猪腹泻的主要病原之一。通过免疫母猪,新生仔猪通常可以从初乳和乳汁得到有效保护,但在断奶阶段,母乳的保护消失。有关研究发现,引起断奶仔猪腹泻常见的ETEC菌株均有菌毛F4或F18。这些菌毛是重要的毒力因子,它们使细菌结合到宿主易感肠上皮细胞,在肠内定植繁殖,随后分泌肠毒素引起腹泻。文章针对由菌毛F4和/或F18产毒素大肠杆菌引起的腹泻,概括了在养猪生产中其相关免疫接种途径,包括口服弱毒苗和亚单位疫苗、胶囊疫苗和注射免疫接种等。     

大肠杆菌多价卵黄抗体的研制及其体外抑制作用

由肠毒素性大肠杆菌（Enterotoxigenic Escherichia coli,ETEC）引起的新生仔猪腹泻是一种常见病,传染快且致死率高。20世纪90年代以来人们应用卵黄抗体防治仔猪大肠杆菌病取得满意效果。本试验的目的是试图研制出高效价的多价卵黄抗体,并观察和分析该卵黄抗体在体外试验中,能否有效地抑制相应ETEC对仔猪小肠上皮细胞的粘附。     

ITGB5和MUC13基因在产肠毒素大肠杆菌抗性和易感大白仔猪间的差异表达分析

为分析仔猪腹泻抗性候选基因在大白仔猪抗性和易感个体间的差异表达情况及组织表达特异性,实验利用荧光定量PCR技术检测整合素β5(ITGB5)基因和黏蛋白13(MUC13)基因在仔猪小肠、脾脏、肺脏、胸腺、肝脏和淋巴6种组织中的表达水平以及在产肠毒素大肠杆菌(ETEC F4)抗性和易感仔猪个体间的差异表达情况。结果表明:大白仔猪ITGB5基因在6种组织中均有一定的表达,在抗性个体小肠组织中的表达量低于易感个体(P>0.05);MUC13基因在小肠中高度表达,在其他组织中表达量较低,且抗性个体的表达量显著高于易感个体(P<0.05)。由此可知,小肠组织中ITGB5基因的低表达和MUC13基因的高表达可能有助于降低致病性大肠杆菌黏附到小肠上皮细胞上,进而实现对致病性大肠杆菌的抗性。ITGB5基因和MUC13基因可能与仔猪腹泻抗性存在密切关系,都可以作为抗性候选基因用于标记辅助选择,应用于抗腹泻仔猪的选育。     

苏太仔猪FUT1基因M307位点多态性与F18大肠杆菌抗病相关性的体外鉴定

采用PCR-RFLP方法检测了江苏苏太断奶仔猪FUT1基因M307位点等位基因多态性分布,在所检的49头仔猪中,GG基因型个体16头,AG基因型19头,AA基因型14头。在此基础上,制备上述不同基因型个体仔猪小肠上皮细胞,分别与表达F18ab菌毛的野生型大肠杆菌、表达F18ac菌毛含fed操纵子全基因的重组大肠杆菌和V型系统表面分泌表达F18abFedF亚单位的重组大肠杆菌进行体外黏附试验和黏附抑制试验。研究结果表明:FUT1基因M307位点中GG型和AG型仔猪小肠上皮细胞均能黏附上述3种大肠杆菌,而AA型个体小肠上皮细胞则不能黏附。将上述3种大肠杆菌分别与抗F18ab菌毛高免血清、F18ac菌毛高免血清及抗F18abFedF亚单位单因子血清作用后,则失去黏附仔猪肠上皮细胞能力。上述结果对苏太猪从体外试验上证明了FUT1基因M307位点多态性与断奶仔猪腹泻和水肿病存在着直接的相关性。     

产志贺毒素样大肠杆菌F18ab菌毛操纵子基因的体外非诱导系统的表达和鉴定

以产志贺毒素样大肠杆菌（SLTEC）F18ab血清型标准菌株107/86基因组DNA为模板,利用PCR技术成功扩增出编码F18ab完整菌毛操纵子fed基因,克隆入表达载体pBR322,经限制性内切酶酶切分析,DNA琼脂糖电泳鉴定并结合序列测定分析,构建和筛选出含fed完整基因正确插入的pBR322-fed重组质粒,将上述重组质粒转化至不含任何菌毛结构的大肠杆菌SE5000,该表达重组菌能分别与兔抗F18ab亚单位蛋白FedF高免血清、鼠抗F18ab菌毛a单因子单克隆抗体、兔抗F18ab菌毛高免血清和抗F18ab菌毛IgG抗体产生明显的凝集反应。用热抽提法分别抽提和纯化SLTEC F18ab标准株107/86和重组菌SE5000（pBR322-fed）体外表达的F18ab菌毛,纯化菌毛经SDS-PAGE电泳和考马斯亮蓝染色获单一相对分子质量约为15 000蛋白条带。Western-blotting结果表明：兔抗F18ab菌毛高免血清能特异性识别SLTEC F18ab标准株107/86和重组菌SE5000（pBR322-fed）所提纯的单一主要结构蛋白。用重组菌SE5000（pBR322-fed）进行易感仔猪小肠上皮细胞体外黏附试验和黏附抑制试验,结果表明：重组菌SE5000（pBR322-fed）和SLTEC F18ab标准株107/86一样具有较强的黏附易感仔猪小肠上皮细胞的能力,而兔抗F18ab菌毛高免血清能有效地抑制上述重组菌SE5000（pBR322-fed）和SLTEC F18ab标准株107/86对易感仔猪小肠上皮细胞的黏附结合。     

Invasive ability of Escherichia coli O18 isolated from swine neonatal diarrhea

Neonatal diarrhea occurred at two swine breeding farms in Hokkaido. Ten piglets aged 2 to 4 days were examined. Grossly, significant changes were confined to the small intestine. The mucous membrane was muddy and thickened. The intraluminal contents from the jejunum to the colon were liquid and yellow. In the small intestine, numerous Gram-negative bacilli preferentially adhered to the apex of villi. The mucosa was erosive with villous atrophy. There were bacilli also in the lamina propria and in the cytoplasm of degenerated enterocytes. Nonhemolytic Escherichia coli strains, belonging to serogroup E. coli O18 and possessing K88 fimbriae, were isolated from the small intestine. They could not be classified into any of the diarrheagenic E. coli groups because of the absence of genes of LT, STh, STp, VT1, VT2, eae, invE, and ipaH. After inoculation of the isolates on HEp-2 cells, some bacilli were engulfed by cytoplasmic projections resembling membrane ruffles and subsequently were localized in cytoplasmic vacuoles or free in the cytoplasm. These findings support the view that the present E. coli O18 is a new invasive strain enteropathogenic to piglets.     

Pharmacokinetics of amoxicillin after oral administration in recently weaned piglets with experimentally induced Escherichia coli subtype O149:F4 diarrhea

OBJECTIVE: To measure the effect of Escherichia coli subtype O149:F4-induced diarrhea on the pharmacokinetics of orally administered amoxicillin in affected piglets relative to that of uninfected piglets. ANIMALS: 22 healthy 4-week-old recently weaned Danish crossbred piglets. PROCEDURE: 12 piglets were orally inoculated through gastric intubation with 10(9) CFUs of an E. coli O149:F4 strain and responded by developing diarrhea 12 to 16 hours later. Piglets were dosed with amoxicillin trihydrate solution (20 mg/kg) by gastric intubation. A control group of 10 age-matched piglets without signs of diarrhea was dosed similarly. Blood samples were obtained before amoxicillin administration and at 0.5, 1, 1.5, 2, 3, 6, 12, and 24 hours after amoxicillin administration. The plasma concentration of amoxicillin was analyzed by high-performance liquid chromatography. RESULTS: A significant 39% decrease in the area under the plasma concentration versus time curve of amoxicillin was observed in piglets with diarrhea relative to that of control piglets. The maximum plasma concentration (Cmax) was significantly (52%) lower in piglets with diarrhea, compared with control piglets, while the elimination rate constant, time to reach Cmax, and elimination half-life were unchanged. CONCLUSIONS AND CLINICAL RELEVANCE: Escherichia coli-induced diarrhea may decrease systemic bioavailability of amoxicillin. Escherichia coli bacteria attach to the intestinal epithelial cells. Because it is assumed that the concentration of the antimicrobial at the site of infection reflects the systemic concentration, higher doses of amoxicillin in the treatment of piglets with E. coli O149:F4-induced diarrhea may be appropriate.     

中兽药防治大肠杆菌性仔猪腹泻研究

仔猪腹泻是严重的仔猪疾病之一,常用抗生素进行治疗,但细菌耐药性导致药物的治疗效果下降。目前,有很多研究报道中草药防治仔猪腹泻取得了良好的疗效。本文从中药抗动物腹泻的作用机理、仔猪大肠杆菌性腹泻的中兽医辨证、中药抗仔猪大肠杆菌性腹泻的作用机理三个方面展开论述中药防治仔猪腹泻的研究进展,为防治仔猪腹泻的中兽药开发提供理论基础。     

Thermostable factor(s) in soya producing a net excess of secretion in the ligated gut test in pigs

The ligated gut test (LGT) is the standard method for the examination of Escherichia coli strains for enterotoxin production in pigs. As solid pig feed has been associated with diarrhea, soya products (the main protein source for piglets) were investigated with the same test as E. coli strains. After injection of different soya products into ligated segments of the small intestine fluid accumulation was observed, indicating a net excess of secretion. The factor in soya products responsible for this effect was found to be thermostable, as its effect was unaltered after heating at 120 degrees C during an hour. No indications of a possible allergic phenomenon accounting for the fluid accumulation were found. From the results of this study it is concluded that soyabean products can produce results in the LGT similar to those produced by enterotoxigenic E. coli strains.     

对FC株猪源性肠毒素型大肠杆菌致病因子的研究

FC菌株是一株从腹泻仔猪粪便中分离的肠毒素型大肠杆菌(Enterotoxigenic E.coli,ETEC)。在MRHA反应中,本菌能凝集人O型、豚鼠、马、绵羊、牛、鸡和兔的红细胞,对人O型和豚鼠红细胞有很高的血凝性,血抗K88和K99血清不能抑制其对豚鼠和绵羊红细胞的血凝。在体外小肠上皮细胞吸附试验中,本菌对仔猪小肠上皮细胞具有强烈的吸附作用;透射电镜和扫描电镜观察证实了FC株菌除表面具有一种纤毛样结构外,还能定居在仔猪小肠段。血清学试验结果表明,本菌的O抗原属于O101。K88和987P两种抗血清均不能凝集本菌,而K99和F41抗血清均可凝集。对纯化的FC株菌粘着素抗原作等电聚焦和聚丙烯酰胺凝胶电泳分析,结果表明,该菌的粘着素是由等电点分别为4.61和9.78,分子量分别为29500和17500的两种蛋白质抗原所组成。此外,用乳鼠胃内投服试验和兔肠结扎试验证明,该菌只产生热稳定肠毒素。总之,本菌是一株能产生ST的K99,F41的肠毒素型大肠杆菌。     

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.     

仔猪产肠毒素大肠杆菌F4受体研究进展

猪肠毒素大肠杆菌F4(ETEC F4)是引起1～2周龄仔猪黄、白痢最普遍、危害最大的大肠杆菌。ETEC F4能否致病,决定于猪的小肠上皮细胞有无ETEC F4受体。本文综述了ETEC F4的黏附模式,F4特异受体在猪小肠中的分布,受体的生化特性,受体编码基因的定位、克隆现状及存在的问题,并对今后F4受体的研究方向及应用前景进行了展望。