肠出血性大肠杆菌O157:H7研究进展

本文综述了近年来肠出血性大肠杆菌O157:H7的病原学、流行病学、临床诊断及预防控制等方面的研究进展。表明牛是该菌的主要贮存宿主,主要通过食源性途径传播,以患者出现出血性结肠炎、阑尾炎、食管狭窄和结肠穿孔等严重胃肠道并发症为基本特征。目前对该菌的自然携带宿主及其引起的感染尚缺乏有效的治疗手段,因此对该菌的防控就显得尤为重要。     

肠出血性大肠杆菌O157:H7感染小鼠模型的研究

目前制约肠出血性大肠杆菌(EHEC)疫苗发展的瓶颈之一,是国内外尚没有理想的动物感染模型评价疫苗保护效果,像其他致病菌一样EHEC对不同动物易感性是不同的。有报道100~200个EHECO157活菌就可以导致人发病,但多数实验动物对EHEC O157则不易感。为了更好地评价疫苗的安全性和有效     

牛肉中大肠杆菌O_(157)的调查

应用分离培养、形态特征观察、API2 0E生化特性鉴定、玻片凝集试验、快速检测试剂盒等方法对上海某地区 5家超市及 3个菜市场的生牛肉进行大肠杆菌O1 57的分离与鉴定 ,并测定分离株对小鼠的毒力。结果从 1 0 0份样本中检测出 3株大肠杆菌O1 57,且对小鼠均有致病性 ,表明超市、菜市场生牛肉中存在大肠杆菌O1 57的污染     

肠出血性大肠杆菌O157：H7噬菌体的分离纯化

分离纯化了抑制肠出血性大肠杆菌（Enterohemorrhagic E coli,EHEC）0157：H7的噬菌体,并对其进行效价测定。将宿主菌EHEC0157：H7培养制成悬液后与污水样品37℃共培养16h,将培养物离心、过滤除菌后得到该菌的噬菌体原液。噬菌体原液采用双层琼脂平板法进行5次纯化,得到直径相同的噬菌斑。纯化后噬菌体的效价达到12×10^pfu。电镜观察表明,该噬菌体呈蝌蚪状。根据ICTV对病毒的分类标准,该噬菌体属长尾噬菌体科,为烈性噬菌体。     

大肠杆菌O157:H7抗体胶体金免疫层析检测试纸条的研制

为建立快速检测鼠感染大肠杆菌O157∶H7后的抗体,本研究采用颗粒为20nm的胶体金溶液标记纯化的羊抗鼠IgG,制备金标探针,喷涂于玻璃纤维膜上;将大肠杆菌O157∶H7抗原和抗大肠杆菌O157∶H7单克隆抗体F1分别包被于硝酸纤维素膜上作为检测线和质控线,组装成鼠血清O157∶H7抗体胶体金免疫层析检测试纸条。实验结果表明,该试纸条仅与O157∶H7阳性鼠血清发生特异性反应,用该试纸条与常规间接ELISA平行比较检测O157∶H7免疫鼠血清,抗体效价分别为1×106和1×105,两者的敏感度相当。该试纸条操作简便,10min可出结果,适用于调查与追溯鼠O157∶H7感染状况时的现场检测。     

Sodium chlorate supplementation reduces E. coli O157:H7 populations in cattle

Cattle are a natural reservoir of the food-borne pathogen Escherichia coli O157:H7. Therefore, strategies that reduce E. coli O157:H7 prior to slaughter will reduce human exposures to this virulent pathogen. When bacteria that can anaerobically respire on nitrate (e.g., E. coli) are exposed to chlorate, they die because the intracellular enzyme nitrate reductase converts nitrate to nitrite, but also co-metabolically reduces chlorate to cytotoxic chlorite. Because chlorate is bactericidal only against nitrate reductase-positive bacteria, it has been suggested that chlorate supplementation be used as a strategy to reduce E. coli O157:H7 populations in cattle prior to harvest. Cattle (n = 8) were fed a feedlot-style high-grain diet experimentally infected with three strains of E. coli O157:H7. Cattle were given access to drinking water supplemented with 2.5 mM KNO3 and 100 mM NaCl (controls; n = 4) or 2.5 mM KNO3 and 100 mM NaClO3 (chlorate-treated; n = 4). Sodium chlorate treatment for 24 h reduced the population of all E. coli O157:H7 strains approximately two logs (10(4) to 10(2)) in the rumen and three logs (10(6) to 10(3)) in the feces. Chlorate treatment reduced total coliforms and generic E. coli from 106 to 10(4) in the rumen and by two logs throughout the rest of the gastrointestinal tract (ileum, cecum, colon, and rectum). Chlorate treatment reduced E. coli O157:H7 counts throughout the intestinal tract but did not alter total culturable anaerobic bacterial counts or the ruminal fermentation pattern. Therefore, it appears that chlorate supplementation is a viable potential strategy to reduce E. coli O157:H7 populations in cattle prior to harvest.     

Effect of pooling bovine fecal samples on the sensitivity of detection of E. coli O157:H7

To assess the effect of pooling fecal samples on the sensitivity of detection of E. coli O157:H7, 12 calves, inoculated orally with 10(8)cfu per calf of nalidixic acid resistant E. coli O157:H7, were used to provide positive fecal samples. After inoculation, calves were sampled twice weekly. Negative fecal samples were from calves at a local dairy. Samples from inoculated calves were incubated without pooling or were mixed with known negative fecal samples in a 1:4 ratio or a 2:3 ratio (positive:negative) for detection of E. coli O157:H7. Samples were enriched 6h in Gram negative broth with vancomycin, cefixime, and cefsoludin, underwent immunomagnetic separation with Dynabeads, and were plated onto sorbitol MacConkey agar with cefixime, and tellurite (SMACct). Morphologically typical colonies were plated onto blood agar, incubated overnight at 37 degrees C and an indole test was performed on each colony. Indole positives colonies were plated on SMAC agar with 20 microg/ml nalidixic acid (SMACnal). Colonies that grew on SMACnal were confirmed by O157 agglutination. Sensitivity of detection in non-pooled samples was 77%. Samples pooled 1:4 and 2:3 with negative samples were 55 and 52% sensitive, respectively. Pooling decreased sensitivity of detection for E. coli O157:H7 in bovine fecal samples (P<0.01). A deterministic binomial probability model was developed to assess the probability of detecting pens of cattle shedding E. coli O157 using a pooling protocol or individual samples. Pooling decreased sensitivity of detection at low pen prevalence compared to individual samples but was similar at high prevalence.     

A systemic vaccine based on Escherichia coli O157:H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157:H7 in calves

Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine.     

Pre-harvest factors influencing the acid resistance of Escherichia coli and E. coli O157:H7

The effects of pH, acetate, propionate, or butyrate concentration, and diet on acid resistance of fecal Escherichia coli and E. coli O157:H7 were determined by in vitro and in vivo experiments. The pH tested was from 4.0 to 8.0, and the VFA concentrations tested were 0 to 100 mM. The E. coli O157:H7 used was strain 505B. In an in vivo study, cattle were fed a grain-based diet, then either not switched or switched to a grain-based diet with 3% added calcium carbonate or two fiber-based diets (soybean hulls or hay). Acid resistance was expressed as viability after acid-shock at pH 2.0 for 1 h and 4 h for fecal E. coli and E. coli O157:H7, respectively. Enumeration methods used were multitube fermentation, agar plate, and petri-film methods. The E. coli O157:H7 was not found in continuous culture inocula or in vivo samples. The viability of fecal E. coli decreased linearly (P < 0.01) as the culture pH increased, and viability of E. coli O157:H7 was highest (P < 0.01) when cultivated at pH 6.0. The viability of fecal E. coli and E. coli O157:H7 showed quadratic responses (P < 0.05) as acetate and butyrate concentrations increased at pH 7.2, with maximal acid resistance at 20 and 12 mM, respectively. As propionate concentration increased, the acid resistance was not different (P > 0.05) for fecal E. coli. Acid resistance of E. coli was induced by acetate and butyrate, even though the environmental pH was near neutral. Similar results were measured in the in vivo study, where viability after acid shock was more dependent on VFA concentration than on pH. Increasing the dietary calcium carbonate concentration also increased (P < 0.05) acid resistance of fecal E. coli. Results from these studies demonstrated that culture pH and VFA affect acid resistance of E. coli.     

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli in white veal calves

The aims of the study were to determine the prevalence of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) and other Shiga toxin-producing E. coli (STEC) in feces of white veal calves in an operation in Ontario, to evaluate exposure of the calves to EHEC O157, and to investigate the milk replacer diet and antimicrobial resistance as factors that might influence the prevalence of EHEC O157. Feces from three cohorts of 20-21 calves were collected weekly for 20 weeks and processed for isolation of EHEC O157:H7 and detection of STEC by an ELISA. Exposure to EHEC O157 was also investigated by measuring IgG and IgM antibodies to the O157 lipopolysaccharide (O157 Ab) in sera by ELISA. The prevalences of EHEC O157 were 0.17% of 1151 fecal samples and 3.2% of 62 calves, and for STEC were 68% of 1005 fecal samples and 100% of 62 calves. Seroconversion to active IgG and IgM O157 Ab responses in some calves was not associated with isolation of EHEC O157. The milk replacer contained low levels of antibodies to EHEC antigens and without antimicrobial drugs, it did not inhibit the growth of EHEC O157 in vitro. Two E. coli O157:H7 that were isolated were totally drug sensitive whereas 60 commensal E. coli isolates that were examined were highly resistant. Antibodies in milk replacer that might be protective in vivo, and susceptibility to antimicrobial agents in the milk replacer may contribute to the low prevalence of EHEC O157 in white veal calves.     

应用荧光PCR方法检测动物产品中大肠杆菌O157:H7

大肠杆菌0157：H7是肠出血大肠杆菌(Enterohemorrhagic Escherichia coli。EHEC)的一种主要血清型．可引起人出血性结肠炎、溶血性尿综合征及血检形成性血小板减少性紫癜等严重并发症。该菌主要通过食物传播．动物是其主要的宿主。自1982年首次在美国暴发的出血性肠炎中分离到病原菌以来．在北欧、日本、加拿大、美国等地发生过多次流行，     

大肠杆菌O157:H7实时定量PCR检测方法的建立

根据GenBank公布的大肠杆菌O157:H7的Flic(H7)基因序列进行同源性比较分析,选择保守序列设计一对特异性扩增引物,通过优化反应条件,建立一个用于大肠杆菌O157:H7快速定量检测的实时定量PCR方法.该方法的最低检测极限是103CFU/mL,敏感性比常规PCR提高10倍.方法重复性好、特异性强,重复性检测的变异系数均小于2%;只能检测大肠杆菌O157:H7,对非大肠杆菌O157:H7血清型细菌、猪链球菌2型、副猪嗜血杆菌无反应.利用此方法对模拟样本进行定量检测,其结果与平板细菌计数基本一致,表明此方法可作为大肠杆菌O157:H7快速诊断和疫情监测的一种快速、准确、简便的检测工具.     

福建省猪源E.coli O157∶H7和O157∶NM的药物敏感试验分析

O157:H7大肠杆菌是一种新发现的肠道致病菌,也是人兽共患病病原菌.     

闽西地区猪源大肠杆菌O157∶H7的分离与鉴定

大肠杆菌 ( E.coli O157)是一种新型的致病性大肠杆菌 ,能引起人类出血性腹泻和溶血性尿毒综合征 ,该菌无侵袭力 ,也不产生肠毒素 ,但能产生一种毒力甚强的细胞毒素 ,引起盲肠、阑尾和升结肠黏膜上皮细胞坏死、黏膜充血、水肿和出血 ,还可进入血液 ,并通过血脑屏障 ,损伤血管内皮细胞而引起血栓性微血管病。病变部位主要在肾脏时可导致溶血尿毒综合征 ,也可引起肠壁梗死、出血 ,以及中枢神经系统病变。本病最早于 1 982年在美国首次暴发 ,之后加拿大、英国和日本等发达国家 ,都发生过大肠杆菌 O157的暴发流行 ;尤其是 1 996年日本暴发近万…     

Study on Seasonal Impact to the Distribution of Bovine E.coli O157: H7 in Xinjiang

To study the impact of season to the distribution of bovine E.coli O157:H7,samples of anus swabs (399),feces (68),water (29) and feed (43) were collected in the spring, summer,autumn and winter from A,B and C farms of Xinjiang. After enrichment by EC broth, SMAC and MUG selective culture were then performed. Finally,PCR was used for identification and virulence gene detection of isolated strains. A total of 5 E.coli O157:H7 strains were isolated from 539 samples from three farms (0.93%,5/539), 2 of them were from spring (1.44%,2/139),1 from autumn(0.56%,1/180),2 from winter (1.38%,2/145) and no strains were isolated from summer samples. One strain were isolated from anus swab samples in farm B (0.69%,1/145) and one were isolated from anus swabs (0.66%,1/152) and three strains were isolated from feed samples in farm C (20.00%,3/15),and no target strains were isolated from water samples. The distribution of bovine E. coli O157:H7 had obvious seasonal characteristics.One E.coli O157:H7 strain of farm B was isolated from autumn and four of farm C were from isolated spring (2 strain) and winter (2 strain),and the isolation rate of E. coli O157:H7 in spring and winter were higher than that in summer and autumn. In conclusion,under the special climate characteristics and feeding mode in Xinjiang,to prevent and control the spreading of E. coli O157:H7 of cattle,we must pay great attention on hygiene management of pens at cold season, specially avoiding the feed contaminated by feces.     

Presence of Shiga toxin-producing E. coli O157:H7 in a survey of wild artiodactyls

This study was carried out to evaluate the role of wild artiodactyls as reservoirs of Escherichia coli O157:H7 for livestock and humans. Retroanal mucosal swabs samples from 206 red deer (Cervus elaphus), 20 roe deer (Capreolus capreolus), 6 fallow deer (Dama dama) and 11 mouflon (Ovis musimon), collected during the hunting season (autumn-winter) in South-western Spain, were screened. Samples were pre-enriched in modified buffered peptone water, concentrated by an immunomagnetic separation technique and cultured onto selective cefixime tellurite sorbitol MacConkey agar. Polymerase chain reaction (PCR) was used to detect the presence of genes coding O157 and H7 antigens and the virulence factors verocytotoxin, intimin and enterohaemolysin. Three E. coli O157:H7 isolates were obtained from red deer (1.5%). Two of them showed inability to ferment sorbitol and lack of beta-d-glucuronidase (GUD) activity, however, the other strain investigated was an atypical sorbitol-fermenting E. coli O157:H7 with GUD(+) activity. This is the first report pointing to red deer as a reservoir of E. coli O157:H7 in Spain.     

Antigen Epitope Peptides and Immunogenicity Analysis of E. coli O157: H7 Ivy Protein

To evaluate the feasibility of synthetic polypeptides O157:H7 Ivy146-157 as a vaccine antigen, a bioinformatics method was employed to analyze the secondary structures, hydrophilicity plot, flexibility plot, surface probability plot and antigenic index of the Ivy. A B-cell epitope peptide sequence was identified and synthesized. BALB/c mice were immunized with Ivy146-157, antibody titers and splenic lymphocytes proliferations in mice were tested. The results showed that the antibody titers were 1:6 400 after immunized mice three times. And the native Ivy protein of O157:H7 R14 strain was identified by polyclone antibody Ivy146-157. The MTT assay results indicated the splenic lyphocytes were increased after immunized polypeptides Ivy146-157, and had significant difference with the control group (P<0.05). These results indicated that polypeptides Ivy146-157 could induce a strong humoral and celluar immune respond, and it would provide a theory as a vaccine antigen for further researches.     

大肠杆菌O157:H7 rfbE基因和fliC基因的克隆与序列分析

根据已发表的E．coli O157：H7 EDL933株的rfbE基因和fliC基因，设计了两对引物，分别对两个O157：H7，菌株的rfbE基因和一个菌株的fliC基因进行PCR，并将PCR产物克隆于pMD18-T栽体质粒。测序结果表明，获得到了与理论相符的582bp rfbE基因片段和802bp fliC基因片段。一株菌的rfbE基因存在无意义突变（两个）。而另一株菌fliC基因有一个有意义突变（aac→gac）     

Escherichia coli O115 forms fewer attaching and effacing lesions in the ovine colon in the presence of E. coli O157:H7

Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.     

Escherichia coli O157:H7 in beef cattle: on farm contamination and pre-slaughter control methods

This paper addresses food safety in beef cattle production, with particular emphasis on factors that affect the prevalence of Escherichia coli O157:H7 in beef cattle and on control methods that have been investigated. Product recalls and foodborne diseases due to this organism continue to occur even though control measures have been under investigation for over 20 years. Most meatborne outbreaks are due to improper food handling practices and consumption of undercooked meat. However, the majority of pathogenic bacteria that can spread at slaughter by cross-contamination can be traced back to the farm rather than originating from the slaughter plant. This would ideally require the adoption of rigorous on-farm intervention strategies to mitigate risks at the farm level. On-farm strategies to control and reduce E. coli O157:H7 at the farm level will reduce the risk of carcass contamination at slaughter and processing facilities although they will not eliminate E. coli O157:H7. The most successful strategy for reducing the risk of contamination of beef and beef products will involve the implementation of both pre- and post-harvest measures.