不同猪种肠毒素大肠杆菌（ETEC）F4受体的微卫星标记遗传效应分析

从猪13号染色体选取与E.coli F4受体基因连锁的4个微卫星座位研究中外猪种间的遗传差异性,并分析不同基因型与F4受体黏附表型的关系。结果表明,4个猪种在4个基因座均具有高度多态性,杂合度（H）在0.6117～0.7500之间,多态信息含量（PIC）达0.5749以上;同时中外猪种基因频率存在差异,微卫星座位连锁越紧密,差异性越大。微卫星S0222不同基因型间在沙子岭猪F4ab血清型黏附表型中差异显著;SW458座位AC基因型在沙子岭、大白两个品种中无黏附表型,可望作为对E.coliF4抗性基因的遗传标记。     

产志贺样毒素和肠毒素大肠杆菌分子流行病学

[目的]人和动物腹泻的主要病原菌为大肠杆菌,本文主要研究贵州省致腹泻大肠杆菌毒力因子的分布类型.[方法]采用PCR技术对各毒力因子的基因分布进行研究.[结果]共分离到333株大肠杆菌,其中产肠毒素大肠杆菌(ETEC)在腹泻的人、猪、牛群中占优势,分别为:人群73(n=112),猪群82(n=106),牛群18(n=115).在ETEC菌株中检测到热敏肠毒素(lt)和不耐热肠毒素(st)基因,还存在lt/st并存现象.从人、猪、牛群中还检测到产志贺样毒素大肠杆菌(STEC),其中源自猪的STEC的检出率最高.大部分STEC同时携带lt、st或lt和st同时并存.编码F18菌毛的主亚基由fedA基因编码.对所分离大肠杆菌F18菌毛进行的研究结果表明,fedA基因主要与肠毒素基因共存,与stx基因并存的类型较少,25份猪源STEC菌株中仅有4份检测到fedA基因.[结论]贵州省人群、猪群和牛群致腹泻病原菌中以带F18菌毛的ETEC为主,STEC主要分布在腹泻的猪群中.     

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.     

Characterization of an F18+ enterotoxigenic Escherichia coli strain from post weaning diarrhoea of swine, and of its conjugative virulence plasmid pTC

The enterotoxigenic Escherichia coli (ETEC) strain Ec2173, causing post weaning diarrhoea in swine, harbours six plasmids ranging from 13 to 200 kb in size. The heat stable toxin genes sta, stb and a tetracycline resistance gene were located on a self conjugative 120-kb plasmid, called pTC. In the cloned ColE1 type origin of replication of pTC a deletion was detected compared to other ColE1 replicons affecting the replication modulator gene rom. Epidemiological studies on ETEC isolates showed that pTC-like plasmids are widely distributed among porcine ETEC strains; thus representing an example of co-evolution of antibacterial resistance and virulence in pathogenic E. coli.     

产肠毒素大肠杆菌快速检测方法的建立和评价

目的利用环介导等温扩增(LAMP)技术,建立产肠毒素大肠杆菌(ETEC)的快速、便捷、敏感、特异的检测方法,并对该方法的特异性和敏感性进行评价,为实验动物检测和细菌性腹泻的诊断提供技术支持。方法根据GenBank公布的产肠毒素大肠杆菌的LT毒素基因序列(S60731.1)设计外引物和内引物进行LAMP扩增,对LAMP特异性和敏感性与PCR方法做比较。结果建立的LAMP方法检测最低浓度为100 pg/μL,灵敏度是PCR的10倍以上并具有较高的特异性,利用该方法对27份猴腹泻样品进行LAMP和PCR方法检测,发现PCR检出率为33.3%,LAMP(60 min内)结果与PCR相同,而LAMP(90 min内)检出率为92.6%,约是PCR检出率的3倍。结论建立了一种用于检测肠毒性大肠杆菌(ETEC)的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,适合于ETEC临床快速检测。     

猪大肠杆菌耐热肠毒素和热敏肠毒素基因的融合及表达

用聚合酶链反应扩增出猪源大肠杆菌编码ST前体（proST）和LT的B亚单位(LTB)成熟多肽的序列,再通过套式PCR将proST编码序列3′端和LTB编码序列5′端融合,并置于同一阅读框内,得到ST和LTB的融合基因,将此序列克隆到pGEMT质粒中,序列分析后,亚克隆到表达载体pQE30中,在大肠杆菌细胞中得到表达,表达的融合蛋白同时具有ST和LTB的抗原性,且无ST和LT的生物毒性。     

CTB/CS3融合蛋白与GM1结合能力和免疫原性的分析

免疫霍乱毒素B亚单位（CTB）或肠毒素大肠杆菌（ETEC）定居因子CS3可使人体对ETEC的侵染有保护作用.为探索研制ETEC双组分亚单位疫苗的可行性,利用大肠杆菌诱导表达系统表达了CTB与CS3的融合蛋白（CTB/CS3）.蛋白质印迹结果表明,诱导表达的29 ku蛋白具有CTB和CS3蛋白双重抗原性.经Ni-NTA亲和层析纯化获得重组蛋白CTB/CS3,复性的重组蛋白可以部分形成五聚体并保留了与神经节苷脂GM1的结合能力.动物实验表明,融合蛋白CTB/CS3具有CTB和CS3蛋白的双重免疫原性,同时,CTB的免疫载体作用提高了CS3的免疫强度.     

肠毒素大肠杆菌定居因子CS3和肠毒素融合抗原基因在减毒鼠伤寒沙门氏菌中的共表达

不耐热肠毒素（LT）和耐热肠毒素（ST）是产肠毒素大肠杆菌的主要致病因素,CS3为该菌的优势定居因子,是定居因子CFA/Ⅱ菌毛抗原的共有抗原组分。采用基因操作技术将编码CS3和融合肠毒素蛋白基因转化到减毒鼠伤寒沙门氏菌疫苗侯选株X4072中进行表达。用重组菌株口服免疫小鼠后,免疫动物能产生抗CS3、LT和ST的血清抗体。特别有意义的是,所产生的抗ST抗体能中和天然ST的生物活性。这一结果为研究载体疫苗防治肠毒素大肠杆菌腹泻疾病奠定了基础。     

Purification and characterization of a dimethylsulfoniopropionate cleaving enzyme from Desulfovibrio acrylicus

Abstract Enterotoxigenic Escherichia coli (STa⁺) strains were isolated from adult bovine with diarrhea. These strains did not express any known ETEC-specific adhesins. Although hemagglutination with rat and sheep erythrocytes was observed in the presence of D-mannose (MRHA), these strains also showed mannose-sensitive hemagglutination (MSHA) with guinea-pig erythrocytes. Electron microscopic studies revealed the presence of fimbria-like structures (provisionally called "F43ms") on bacterial cells grown at 37°C but not on cells grown at 18°C. However, it was observed by SDS-PAGE that the J-1 strain (F43ms⁺) produces a protein similar to F1 fimbriae, and this strain hybridized with a DNA probe for F1 fimbriae. Immunogold-labelling techniques indicated that a rabbit anti-serum is specific for F43ms fimbrial structures, but not for Type 1 fimbriae. The immunofluorescence test carried out with semipurified F43ms on bovine brush borders suggests that the fimbria-like structures are responsible for the adhesion to bovine epithelial cells.     

Factors influencing survival of enterotoxigenic Escherichia coli, Salmonella enterica (serovar Typhimurium) and Vibrio parahaemolyticus in marine environments

The presence and persistence of enterotoxigenic Escherichia coli (ETEC) is poorly investigated in marine habitats. Here we compared ETEC with the more studied fecal contaminant, Salmonella enterica serotype Typhimurium ( S. enterica ) and the marine bacteria Vibrio parahaemolyticus . All three species of bacteria were culturable on agar plates during 8 weeks of incubation in seawater. However, the culturability of ETEC was positively affected by low temperature whereas V. parahaemolyticus was negatively affected. High-nutrient conditions favored the growth of ETEC but not the other bacteria. When the bacteria were fed to blue mussels, V. parahaemolyticus inhibited the filtration activity and the ingestion was lower than that of the enterobacteria. On the other hand, the mussels were less efficient in eliminating V. parahaemolyticus and an in vitro study showed that the hemocytes of three different species of bivalves were not able to kill this strain of V. parahaemolyticus . The bactericidal capacity of bivalves was seemingly an efficient elimination pathway for S. enterica and ETEC. This study showed that ETEC in endemic areas should, to the same degree as S. enterica and V. parahaemolyticus , be taken in consideration when assessing the role of marine environments as a source of enteric infection.