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48h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。     

新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究

为了了解牛源大肠杆菌(E.coli)O157∶H7在新疆地区的污染状况以及遗传多样性,探究不同地区分离菌株的遗传关系,为控制牛源E.coli O157∶H7的传播提供试验依据。将采集的样品在EC肉汤中进行增菌(37℃、180 r/min),接着将增菌液划线接种到SMAC平板上,37℃培养箱中过夜培养18 h左右。挑取SMAC平板上白色或无色单菌落接种MUG培养基,37℃培养18 h左右,将无荧光样品接种到SMAC平板上,37℃培养18 h左右,隔天挑取白色或无色单菌落进行PCR鉴定,具有rfbE和fliC基因条带的即为阳性菌株。将阳性菌株进行肠杆菌基因间重复共有序列扩增(ERIC-PCR)指纹图谱聚类分析,分析菌株之间的同源性关系。ERIC-PCR结果显示,相似性100%的菌株有3组。从伊犁地区分离到的菌株差异性最大,具有6种分型;其次是乌鲁木齐,具有4种分型。菌株来源多样性最多的在D簇,由此可见通过ERIC-PCR分型,可以进行溯源观察。ERIC-PCR能够区分特定采样点或物种的分离物,它能够证明从不同来源的菌株之间,存在着某些相似的ERIC特性,并聚集在同一个簇群中。该研究中筛选出的E.coli O157∶H7菌株具有广泛的遗传多样性,该方法对于检测不同物种间的细菌差异非常敏感。由此可见ERIC-PCR可以作为E.coli O157∶H7常规监测和鉴定的一个有效的工具。     

某定点肉牛屠宰场中非O157致病性STEC的分离鉴定

为了了解新疆伊犁地区肉牛屠宰过程中大肠杆菌的污染情况，检测非O157致病性产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli，STEC）的感染情况，本试验采集新疆伊犁地区某定点肉牛屠宰场中屠宰肉牛的粪样和屠宰后的胴体表面拭子，并对样品进行了大肠杆菌的分离鉴定、毒力基因（eae、stx1、stx2）的PCR检测、O157鉴定（rfbE）、ERIC-PCR基因分型和小鼠致病性试验。结果显示，在采集的45份样品中分离鉴定出42株大肠杆菌，分离率为93.3%。其中2株菌株同时编码了毒力基因stx1和stx2，检出率为4.8%，毒力基因eae未被检出。PCR鉴定均为非O157 STEC。ERIC-PCR基因分型检测发现，2株菌的基因型非常相似，同源关系密切。对小鼠进行腹腔注射攻毒，攻菌6 h后，小鼠开始出现死亡，立即解剖死亡小鼠发现，其肠道出血，肝脏、脾脏、肾脏明显出血肿大，解剖对照小鼠表现正常，表明菌株具有一定的致病性。综上所述，在肉牛屠宰过程中存在大肠杆菌污染，其中粪便中非O157 STEC菌株对胴体造成了污染，需要加强控制肉牛的屠宰加工关键环节的环境卫生。     

猪源大肠杆菌O157:H7广西分离株的鉴定

为了解大肠杆菌O157:H7是否在猪场存在,采集有拉稀症状猪的粪便,接种到新生霉素mEC增菌肉汤,增菌培养后转接到O157:H7显色培养基上,对可疑菌株用因子血清和PCR方法做进一步鉴定,实验结果证实得到5株大肠杆菌为O157:H7血清型。动物试验发现,5个分离菌株对小白鼠的致死率为33.3%—100%之间。调查结果证实广西猪群中存在强致病性的O157:H7血清型大肠杆菌。     

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。     

季节对新疆牛源E.coli O157∶H7分布的影响研究

试验旨在研究季节对牛源E.coli O157∶H7分布的影响。在新疆地区A、B、C 3个牛场中,分别于春、夏、秋、冬4个季节采集牛肛拭子(399份)、粪便(68份)、水(29份)和饲料(43份)样本,先用EC肉汤增菌,再用山梨醇麦康凯培养基(SMAC)与4-甲基伞形酮-β-D葡萄糖醛酸苷(MUG)选择性培养,然后进行PCR鉴定和毒力基因检测。结果显示,从3个牛场539份样本中共检出E.coli O157∶H7菌株5株(0.93%,5/539),其中春季样本检出2株(1.44%,2/139),秋季样本检出1株(0.56%,1/180),冬季样本检出2株(1.38%,2/145),未能从夏季样本中检出目标菌;B牛场的菌株检自肛拭子样本(0.69%,1/145),C牛场的菌株分离于饲料(20.00%,3/15)和肛拭子(0.66%,1/152)样本,水中未分离到目标菌。牛源E.coli O157∶H7的分布具有明显的季节性,B牛场只在秋季的肛拭子样本中检出了1株E.coli O157∶H7,C牛场分别在春季和冬季各检出2株,春、冬季E.coli O157∶H7分离率高于夏、秋季。因此,在新疆特殊的气候特点和饲养模式下,防控牛源E.coli O157∶H7的传播时,要十分注意寒冷季节圈舍内卫生的管理,尤其要严防饲料被粪便污染。     

某定点肉牛屠宰场中非O157致病性STEC的分离鉴定

为了了解新疆伊犁地区肉牛屠宰过程中大肠杆菌的污染情况,检测非O157致病性产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC)的感染情况,本试验采集新疆伊犁地区某定点肉牛屠宰场中屠宰肉牛的粪样和屠宰后的胴体表面拭子,并对样品进行了大肠杆菌的分离鉴定、毒力基因(eae、stx1、stx2)的PCR检测、O157鉴定(rfbE)、ERIC-PCR基因分型和小鼠致病性试验。结果显示,在采集的45份样品中分离鉴定出42株大肠杆菌,分离率为93.3%。其中2株菌株同时编码了毒力基因stx1和stx2,检出率为4.8%,毒力基因eae未被检出。PCR鉴定均为非O157STEC。ERIC-PCR基因分型检测发现,2株菌的基因型非常相似,同源关系密切。对小鼠进行腹腔注射攻毒,攻菌6h后,小鼠开始出现死亡,立即解剖死亡小鼠发现,其肠道出血,肝脏、脾脏、肾脏明显出血肿大,解剖对照小鼠表现正常,表明菌株具有一定的致病性。综上所述,在肉牛屠宰过程中存在大肠杆菌污染,其中粪便中非O157STEC菌株对胴体造成了污染,需要加强控制肉牛的屠宰加工关键环节的环境卫生。     

新疆牛源E.coli O157:H7的致病力及PFGE分子分型

对2012-2015年新疆8株牛源E.coli O157:H7进行毒性强弱测定和PFGE分子分型。结果显示,几乎所有菌株能使小鼠出现精神沉郁、食欲减退、腹泻等症状,且3株导致小鼠死亡。病理组织学检查发现,死亡小鼠的肝、脾、肾等组织器官均表现较明显的病理损伤。8株E.coli O157:H7 PFGE图谱相似性为74.3%～100%。经聚类分析得到A-C共3类基因簇,其中优势基因簇为B。E.coli O157:H7在两地区具有水平传播途径。     

免疫磁珠法分离大肠杆菌O_(157):H_7及毒力基因的PCR检测

为了了解新疆不同牛场中大肠杆菌O157:H7的流行情况,对新疆乌鲁木齐、伊犁地区、塔城地区多个牛场采集的粪样及水样样本的检测,样品经EC肉汤增菌、免疫磁珠富集后,用SMAC平板和MUG培养基进行初步筛选,再用rfb E和fli C基因对筛选后的疑似大肠杆菌O157:H7菌株做PCR检测,同时结合生化试验的方法进行符合性检验。结果从新疆3个地区不同牛场的208份样品分离得到5株肠出血性大肠杆菌O157:H7,检出率为2.4%。其中粪样检出5株,检出率为2.4%;水样未检出。生化试验结果表明,5株分离出的大肠杆菌O157:H7菌株与国标规定的O157:H7特性相同。表明,部分被检牛场存在大肠杆菌O157:H7威胁,应该引起重视。     

新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究

为了了解牛源大肠杆菌（E.coli）O157∶H7在新疆地区的污染状况以及遗传多样性,探究不同地区分离菌株的遗传关系,为控制牛源E.coli O157∶H7的传播提供试验依据。将采集的样品在EC肉汤中进行增菌（37 ℃、180 r/min）,接着将增菌液划线接种到SMAC平板上,37 ℃培养箱中过夜培养18 h左右。挑取SMAC平板上白色或无色单菌落接种MUG培养基,37 ℃培养18 h左右,将无荧光样品接种到SMAC平板上,37 ℃培养18 h左右,隔天挑取白色或无色单菌落进行PCR鉴定,具有rfbE和fliC基因条带的即为阳性菌株。将阳性菌株进行肠杆菌基因间重复共有序列扩增（ERIC-PCR）指纹图谱聚类分析,分析菌株之间的同源性关系。ERIC-PCR结果显示,相似性100%的菌株有3组。从伊犁地区分离到的菌株差异性最大,具有6种分型;其次是乌鲁木齐,具有4种分型。菌株来源多样性最多的在D簇,由此可见通过ERIC-PCR分型,可以进行溯源观察。ERIC-PCR能够区分特定采样点或物种的分离物,它能够证明从不同来源的菌株之间,存在着某些相似的ERIC特性,并聚集在同一个簇群中。该研究中筛选出的E.coli O157∶H7菌株具有广泛的遗传多样性,该方法对于检测不同物种间的细菌差异非常敏感。由此可见ERIC-PCR可以作为E.coli O157∶H7常规监测和鉴定的一个有效的工具。     

Feedstuffs as a vehicle of cattle exposure to Escherichia coli O157:H7 and Salmonella enterica

Feed has been reported as a vehicle for transmission of Salmonella enterica in cattle and several lines of evidence suggest that feed can be a vehicle for transmitting Escherichia coli O157:H7 as well. To show whether microbial contamination of feeds could contribute to the populations of S. enterica and E. coli O157:H7 on a farm, we compared isolates from feed samples to bovine fecal isolates from the same farm using pulsed-field gel electrophoresis (PFGE). Four of 2365 component feed samples (0.2%) and 1 of 226 feed mill samples (0.4%) were positive for E. coli O157:H7. Twenty of 2405 (0.8%) component feed samples and none of 226 feed mill samples were positive for Salmonella. PFGE profiles from E. coli O157:H7 isolated from a component feed sample closely resembled that from a fecal isolate collected later from the same farm, and a similar observation was made of a Salmonella Tyhpimurium isolate from component feed on another farm. There were indistinguishable PFGE profiles from component feed Salmonella Tyhpimurium DT104 isolates and fecal isolates from the same farm. These results provide evidence for a role of cattle feed in transmission of E. coli O157:H7; S. enterica; cattle-bacteria.     

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.     

Influence of Immunomagnetic Enrichment Method for Isolation and Identification of E.coli O157∶H7 from Bovine

In order to prove the effect of immunomagnetic beads adsorption technology on separation efficiency of bovine E.coli O157∶H7 in practice,a total of 18 fecal samples, 162 anal samples,10 fodder samples,17 water samples and 36 samples of carcass surface cotton were collected from three cattle farms in Wujiaqu,Yining and Changji. The samples were taken by EC bead enrichment and direct SMAC and MUG test,respectively. Then,the bacterial rfbE gene and flagellarfliC gene were detected by PCR. Finally,the suspected E.coli O157∶H7 bacteria were detected by biochemical test. 8 and 4 strains of E.coliO157∶H7 were isolated from 243 samples,respectively. There was no statistical difference between the two methods. This experiment showed that the immunomagnetic beads adsorption technique in practice was not statistically significant compared with the conventional method, but it did increase the number of isolated E.coli O157∶H7,which was consistent with previous research results.     

免疫磁珠富集法对牛源大肠杆菌O157∶H7分离鉴定的影响

为证明免疫磁珠吸附技术对牛源大肠杆菌 O157∶H7分离效率的影响,从新疆五家渠市、伊宁县、昌吉市3个牛场的采集18份粪样、162份肛拭子、10份饲料样、17份水样和36份胴体表面棉拭子样本,经EC肉汤增菌后,分别采用免疫磁珠富集后和直接进行SMAC和MUG试验进行选择性培养,然后对菌体rfbE基因和鞭毛fliC基因进行PCR检测,最后对疑似大肠杆菌 O157∶H7菌用生化试验进行符合性检测。结果2种方法分别从243份样品中分离到8株和4株大肠杆菌 O157∶H7,统计学分析该差异不显著。本试验结果表明,在实践中免疫磁珠吸附技术和普通方法相比虽然在统计学上差异不显著,但确实能够增加大肠杆菌O157∶H7的分离数量,这与以前的相关研究结果基本一致。     

Testing of a Chemiluminescence Enzyme Immunoassay for selective detection of E. Coli O157 from ground beef samples

The aim of this study was to evaluate a Chemiluminescence Enzyme Immunoassay (CLIA) developed for the detection of E. coli O157:H7, using different E. coli O157 serotypes. The sensitivity and specificity of the kit were determined from the tenfold dilutions of the 24-hour broth cultures of the test strains. According to the results obtained in this trial, the sensitivity of the kit is 10(3)-10(4) cells ml-1, and it is specific for E. coli O157. Twenty-five g ground raw beef samples were prepared and inoculated with E. coli O157:H7 at different CFU g-1. The samples were incubated in 225 ml of modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 4 h and the immunoassays were performed following the instructions of the manufacturer. According to the results obtained by the CLIA test 10(1)-10(2) E. coli O157 g-1 can be detected from the sample. So this kit seems to be suitable for screening the samples before selective cultivation of E. coli O157:H7.     

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.     

Isolation of a rare Escherichia coli O157:H7 strain from farm animals in Greece

A strain of Escherichia coli O157:H7 was isolated from goat faeces during a surveillance study on the prevalence of this serotype of E. coli in farm animals in Greece. Three hundred and fifty one faecal samples were collected from goat, sheep and cattle breeding farms in the area of Epirus, Northwestern Greece. The E. coli O157:H7 isolate was nonsorbitol-fermenter, produced only VT2 and showed a beta-glucuronidase positive activity, a rather unusual biochemical feature for the E. coli O157:H7 serotype. No other strain of E. coli O157:H7 was isolated from the faecal samples of the rest farm animals examined, thus the overall prevalence of animal carriage was found to be 0.2%. The findings also indicate that goats can be a reservoir of E. coli O157:H7 and goat milk, dairy products and meat may serve as a vehicle for the pathogen transmission to humans.     

Usefulness of a commercially available enzyme immunoassay for Shiga-like toxins I and II as a presumptive test for the detection of Escherichia coli O157:H7 in cattle feces.

The performance of a commercially available enzyme immunoassay (EIA) for determining the presence of Shiga toxin I and II in human diarrheal stool samples was evaluated for use as a presumptive test for the presence of Escherichia coli O157:H7 in nondiarrheal bovine fecal samples collected from 10 Kansas cow-calf ranches. The prevalence of E. coli O157:H7 in 2,297 samples, as determined by selective bacterial culture, was 1.6%. The sample prevalence of non-E. coli O157:H7 Shiga toxin-producing bacteria, as detected by the Shiga toxin EIA, was 5.8%. Only 2 of 136 samples that tested positive with the Shiga toxin EIA were positive for E. coli O157:H7 by culture. Compared with bacterial culture, the sensitivity of the Shiga toxin EIA was 5.5% and the specificity was 94.1%. Agreement between the 2 tests, as measured by the kappa statistic, was poor (kappa = -0.002). Although the Shiga toxin EIA was not a good presumptive test for the determination of E. coli O157:H7 in bovine fecal samples because of its low sensitivity (5.5%), it might be a useful test for the detection of Shiga toxin producing non-E. coli O157:H7 organisms in bovine feces.