牛源大肠杆菌O157:H7的分离鉴定

为调查新疆部分地区E.coli O157：H7的感染情况和菌株致病性,从新疆阿克苏、伊犁、塔城3个地区的牛场采集新鲜粪样564份,对E.coli O157：H7进行分离与鉴定。利用E.coli营养肉汤（EC肉汤）对样品进行增菌后,用山梨醇麦康凯培养基（SMAC）平板选择性培养,再经过4-甲基伞形酮-β-D葡萄糖醛酸苷培养基（MUG）的筛选,对疑似菌株进行生化和PCR鉴定,并将分离鉴定到的菌株进行小鼠攻毒试验。结果显示,从伊犁地区采集的样品中共分离出2株E.coli O157：H7（Y166和Y226）,其检出率为0.88%;小鼠攻毒试验中,Y166和Y226试验组小鼠在48 h内全部死亡,具有一定致病性;从阿克苏、塔城所采样品中未分离到E.coli O157：H7。     

从长春地区牛肉和猪肉中检出产vero毒素大肠杆菌O157:H7

用分离培养法、生化反应鉴定、胶乳凝集试验和聚合酶链式反应（ＰＣＲ）对长春地区市售的４０份牛肉和３０份猪肉样品进行了调查,结果从２份牛肉（５％）和１份猪肉（３．３％）样品中检出了产ｖｅｒｏ毒素大肠杆菌Ｏ１５７：Ｈ７。     

Isolation and characterisation of Shiga toxin-producing Escherichia coli O157:H7 from calves in Argentina

Four hundred and twenty-two calves were examined for intestinal carriage of Shiga toxin-producing Escherichia coli O157:H7 using conventional plating. Two (0.5%) E. coli O157 were recovered. They were compared with 96 Argentine strains of different origin by pulsed-field gel electrophoresis, phage typing and PCR-RFLP of stx2 genes. One strain isolated from a calf, was closely related with 18 strains of clinical origin.     

Isolation of Verocytotoxin-producing Escherichia coli O157:H7 from cattle at slaughter in Italy.

Cattle arriving for slaughter at a large abattoir in northern Italy between April 1997 and January 1998 were examined for intestinal carriage of Verocytotoxin-producing Escherichia coli (VTEC) O157 using an immunomagnetic separation technique. Sixty sorbitol non-fermenting VTEC O157 strains were isolated from 59 (13.1%) of the 450 cattle examined. In particular, VTEC O157 was found in 37 (16.6%) of 223 feedlot cattle and in 22 (16.1%) of 137 dairy cull cows, but not in the 90 veal calves sampled. The isolation rate was higher during warm weather (17.5%), falling to an average of 2.9% during the winter months. VT-negative, O157 latex-agglutinating E. coli strains were isolated from 23 (5.1%) of the 450 animals. PCR analysis showed that all 60 VTEC O157 strains carried the VT2 gene and that 25 strains also carried the VT1 gene. In addition, four of the VT-negative, O157 latex-agglutinating E. coli strains carried the VT2 gene. Atypical biochemical features were observed in some VTEC O157: two strains (3.3%) showed beta-glucuronidase activity, and seven (11.7%) produced urease.     

牛源大肠杆菌O157：H7的分离及毒力基因鉴定

从2个牛场采集新鲜粪便,增菌后,免疫磁珠富集,涂布筛选性培养基,挑取可疑菌落用rfbE/fliC二重PCR和血清学方法鉴定。设计毒力基因stx1、stx2、eae、hlyA和tccp相应引物,针对O157：H7对分离株进行PCR鉴定。口服攻毒链霉素处理的BALB/c小鼠明确分离株致病性。结果显示,成功分离到7株出血性大肠杆菌O157：H7,并且有1株迟缓性发酵山梨醇麦康凯培养基。毒力基因检测显示,其中6株毒力因子表型为stx1-stx2＋eae＋hlyA＋tccp＋,另有1株表现型为stx1＋stx2＋eae＋hlyA＋tccp＋,各分离株tccp基因均为阳性,但携带的重复片段数量有差异。所采集样品中肠出血性大肠杆菌O157：H7的检出率高达12%。1×1010 CFU同剂量口服接种经PBS洗涤的5株O157：H7分离株全菌,小鼠存活率有差异分别为40%,50%,60%,20%,50%,各分离株在小鼠体内排菌时间也有差异分别为攻毒后7,9,13,13,15d。     

Escherichia coli O157:H7 in healthy dairy cows

Faecal samples were taken from 371 cows originating from 55 dairy farms and slaughtered at one slaughterhouse; tonsils were taken from 215 of these animals. Escherichia coli 0157:H7 was found in the faeces of only two animals and was not found in any tonsils. The farm supplying the first positive cow detected at the slaughterhouse was visited 3 months later and 160 animals (80 cows and 80 heifers) were tested by rectal swabs; E. coli 0157:H7 was not isolated.     

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.     

用PCR鉴定大肠杆菌O157:H7

根据大肠杆菌Ｏ１５７：Ｈ７的编码ｅａｅ蛋白的ｅａｅ Ａ基因和大肠杆菌编码Ｈ７抗原的ｆｌｉＣ基因的核苷酸序列,合成了２对窦核苷酸引物,建立了一个检测大肠杆菌Ｏ１５７：Ｈ７的ＰＣＲ方法,对１１株已知大肠杆菌Ｏ１５７：Ｈ７（ＮＭ：无运动性）株和其他不同属的４２株已知肠道致病菌的检测结果表明,该方法只从大肠杆菌Ｏ１５７：Ｈ７（ＮＭ）株的ＤＮＡ中产生预期的扩增产物,而从其他菌株的ＤＮＡ中未扩增出任何ＤＮＡ不     

Isolation of Escherichia coli O157:H7 from the gall bladder of inoculated and naturally-infected cattle

To determine if Escherichia coli O157:H7 is capable of residing in the gall bladder of cattle, inoculation studies were conducted with O157:H7 strain 86-24 in weaned Holstein calves. Strain 86-24 was isolated from the gall bladders of five calves 36 days after inoculation. Two other calves contained the inoculation strain in the distal colon but the organism was absent in their gall bladders. A second trial in which the calves were euthanized 15 days after inoculation found strain 86-24 in six of seven inoculated calves but only in colon and/or rumen samples. In a third trial that inoculated eight calves with a four-strain cocktail of O157:H7 strains, the gall bladders from all eight animals were positive 9 days after inoculation. The colon and rumen samples from these calves were also positive. E. coli O157:H7 isolates recovered from bile samples and subtyped by pulsed field gel electrophoresis found that three of the four inoculation strains were present in one or more of the calves. Thus, residence in the gall bladder is not restricted to a single strain. Additional evidence of the ability to localize in the gall bladder of cattle was provided by testing the bile from 150 gall bladders (five collection dates, 30 samples each) obtained at an abbatoir and the isolation of E. coli O157:H7 from four samples (2.7%). This study establishes that E. coli O157:H7 can reside transiently or permanently at a low level in the gall bladder of cattle.     

用PCR鉴定大肠杆菌O157∶H7

根据大肠杆菌Ｏ１５７∶Ｈ７的编码ｅａｅ蛋白的ｅａｅＡ基因和大肠杆菌编码Ｈ７抗原的ｆｌｉＣ基因的核甘酸序列,合成了２对寡核苷酸引物,建立了一个检测大肠杆菌Ｏ１５７∶Ｈ７的ＰＣＲ方法。对１１株已知大肠杆菌Ｏ１５７∶Ｈ７（ＮＭ;无运动性）株和其他不同属的４２株已知肠道致病菌的检测结果表明,该方法只从大肠杆菌Ｏ１５７∶Ｈ７（ＮＭ）株的ＤＮＡ中产生预期的扩增产物,而从其他菌株的ＤＮＡ中未扩增出任何ＤＮＡ产物。该方法从基因水平直接确定大肠杆菌的血清型,特异性强,克服了以往血清学方法有非特异性反应的缺陷,为检测和鉴定大肠杆菌Ｏ１５７∶Ｈ７（ＮＭ）提供了一个新方法。     

Experimental infection of calves with Escherichia coli O157:H7

Three 3-month-old Japanese Black calves were experimentally infected with Escherichia coli O157:H7 to define the magnitude (CFU/g) and duration of fecal shedding of the organism. In two of the three calves, fecal shedding of E. coli O157:H7 ceased in 5 and 9 weeks. The remaining calf continued shedding E. coli O157:H7 for more than 31 weeks, and the magnitude of the shedding ranged from 10(1) to 10(4) CFU/g of feces. The possibility is suggested that a percentage of animals naturally infected with E. coli O157:H7 on farms may become long-term shedders, transmitting the organism to other animals in the herd and to the proximate environment.     

Persistence of Escherichia coli O157:H7 in experimentally infected swine

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.     

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     

Verocytotoxin-producing Escherichia coli O157:H7 in the Swedish pig population

Verocytotoxin-producing Escherichia coli O157:H7 (VTEC O157:H7) was detected in two of 2446 individual faecal samples collected from pigs slaughtered at five Swedish slaughterhouses, indicating a prevalence of 0.08 per cent, with a 95 per cent confidence interval from 0 to 0.16 per cent Four Swedish VTEC O157:H7-positive farms which kept ruminants and pigs were studied by repeated faecal sampling; VTEC O157:H7 was isolated from the ruminants and pigs on all the farms and the same strains were present in the pigs and the ruminants. On one of the farms, the organism persisted in the pig population for 11 months. On all four farms, management practices which might have influenced the isolation rate in pigs were identified. A group of young VTEC O157:H7-positive pigs was moved from one of the VTEC O157:H7-positive farms to a fattening herd where there were no ruminants. The number of VTEC O157:H7-positive faecal samples decreased gradually and after nine weeks the pigs were all negative; at slaughter none of the pigs was VTEC O157:H7-positive.     

应用多重PCR方法检测出血性大肠杆菌O157:H7

选择O157：H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因(uidA)分别设计引物,在同一扩增体系中进行PCR反应。在优化好的多重PCR反应条件下,对菌株及其它肠道菌进行检测。试验结果为：O157：H7菌株在250,207,179bp处均出现特异条带。试验结果表明,选择3对引物的多重PCR方法可特异、快速而且灵敏地对大肠杆菌O157：H7进行检测。     

Prevalence of Shiga-toxigenic Escherichia coli O157:H7 in adult dairy cattle

OBJECTIVE: To describe shiga-toxigenic Escherichia coil O157:H7 (STEC O157:H7) fecal shedding prevalence, seasonal fecal shedding patterns, and site-specific prevalence from the oral cavity, skin, and feces of dairy cattle. DESIGN: Cross-sectional study. ANIMALS: Adult dairy cattle from 13 herds in Louisiana. PROCEDURE: Samples were cultured for STEC O157 by use of sensitive and specific techniques, including selective broth enrichment, immunomagnetic separation, monoclonal antibody-based O:H enzyme immunoassay serotyping, and polymerase chain reaction virulence gene characterization. Point estimates and 95% confidence intervals were calculated for fecal shedding prevalence as well as site-specific prevalence from the oral cavity, skin, and feces. Logistic regression was used to assess seasonal variation and differences at various stages of lactation with respect to fecal shedding of STEC O157 in cattle sampled longitudinally. RESULTS: Summer prevalence in herds in = 13) was 38.5%, with a cow-level prevalence of 6.5%. Among positive herds, prevalence ranged from 3% to 34.6%. Samples from 3 of 5 herds sampled quarterly over 1 year yielded positive results for STEC O157. In herds with STEC O157, an increase in cow-level prevalence was detected during spring (13.3%) and summer (10.5%), compared with values for fall and winter. Site-specific prevalences of STEC O157:H7 from oral cavity, skin, and fecal samples were 0%, 0.7%, and 25.2%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicated that STEC O157:H7 was commonly isolated from dairy cows in Louisiana, seasonally shed, and isolated from the skin surface but not the oral cavity of cows.