肠出血性大肠杆菌O157:H7紧密素单克隆抗体的制备及鉴定

以基因重组技术构建工程菌株表达肠出血性大肠杆菌O157:H7紧密素的融合蛋白。用纯化的紧密素蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,对杂交瘤细胞及时筛选,阳性孔经4次有限稀释法克隆,成功获得2株能稳定传代并分泌抗紧密素单克隆抗体(McAb)的杂交瘤细胞株。2株单抗分别制备腹水,ELISA检测效价分别为5.2×104,2.5×104。Western blot检测表明,2株单抗与融合蛋白发生特异性反应;ELISA检测表明,2株单抗可特异性检出大肠杆菌O157:H7。本研究为建立大肠杆菌O157检测方法提供了物质基础。     

Interaction with avian cells and colonisation of specific pathogen free chicks by Shiga-toxin negative Escherichia coli O157:H7 (NCTC 12900)

The prevalence of Escherichia coli O157:H7 infection in birds is low but several deliberate inoculation studies show that poultry are readily and persistently infected by this organism indicating a possible threat to public health. The mechanisms of colonisation of poultry are not understood and the aim is to establish models to study the interaction of E. coli O157:H7, at the cellular and whole animal levels. A non-toxigenic E. coli O157:H7 (NCTC 12900) was used in adherence assays with an avian epithelial cell line (Div-1) and used to inoculate 1-day-old SPF chicks. In vitro, NCTC 12900 induced micro-colonies associated with cytoskeletal arrangements and pedestal formation with intimate bacterial attachment. In the 1-day-old SPF chick, a dose of 1 x 10(5) cfu resulted in rapid and extensive colonisation of the gastrointestinal tract and transient colonisation of the liver and spleen. The number of E. coli O157:H7 organisms attained approximately 10(8) cfu/ml caecal homogenate 24h after inoculation and approximately 10(7) cfu/ml caecal homogenate was still present at day 92. Faecal shedding persisted for 169 days, ceasing 9 days after the birds came into lay and 6% of eggs were contaminated on the eggshell. Histological analysis of tissue samples from birds dosed with 1x10(7) cfu gave evidence for E. coli O157:H7 NCTC 12900 induced micro-colonies on the caecal mucosa, although evidence for attaching effacing lesions was equivocal. These models may be suitable to study those factors of E. coli O157:H7 that mediate persistent colonisation in avian species.     

Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxin-producing Escherichia coli from India

The aims of this study were to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in pigs as a possible STEC reservoir in India as well as to characterize the STEC strains and to determine the antimicrobial resistance pattern of the strains. A total of 782 E. coli isolates from clinically healthy (n?=?473) and diarrhoeic piglets (309) belonging to major pig-producing states of India were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga toxin-producing gene(s) (stx1, stx2), intimin (eae), enterohemolysin (hlyA) and STEC autoagglutinating adhesin (Saa). Overall STEC were detected in 113 (14.4 %) piglets, and the prevalence of E. coli O157 and non-O157 STEC were 4 (0.5 %) and 109 (13.9 %), respectively. None of the O157 STEC isolates carried gene encoding for H7 antigen (fliCh7). The various combinations of virulence genes present in the strains studied were stx1 in 4.6 %, stx1 in combination with stx2 gene in 5.1 %, stx1 in combination with stx2 and ehxA in 0.6 %, stx1 in combination with stx2 and eae in 0.2 % and stx2 alone in 3.7 %. All STEC isolates were found negative for STEC autoagglutinating adhesin (Saa). The number of STEC isolates which showed resistance to antimicrobials such as ampicillin, tetracycline, streptomycin, lincomycin, nalidixic acid, sulfadiazine, penicillin, gentamicin, kanamycin and ceftriaxone were 100, 99, 98, 97, 95, 94, 92, 88, 85 and 85, respectively. Ninety-seven isolates showed resistance to more than 2 antimicrobials, and 8 resistance groups (R1 to R8) were observed. This study demonstrates that pigs in India harbour both O157 and non-O157 STEC, and this may pose serious public health problems in future.     

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.     

A homolog of the O157 urease-encoding O island 48 is present in porcine O149:H10 enterotoxigenic Escherichia coli

The relationship of the urease operon in the highly virulent O149 porcine enterotoxigenic Escherichia coli (ETEC) strain Ro8 to a genomic island (GI) homologous to O island (OI) 48 of O157 enterohemorrhagic E. coli (EHEC) strain EDL933 was investigated. Eighty-four of 84 O149:H10 strains were urease positive whereas 44 of 44 O149:H43 porcine ETEC strains were urease-negative. Seventeen of 17 O149:H10 strains that were tested possessed the OI-48 homolog whereas 24 of 24 O149:H43 strains lacked this OI. Transposon insertions in lipB or guaA genes in strain Ro8 eliminated urease activity while insertions in the caiF gene increased urease activity. When the O149 ure operon was cloned on a high copy number plasmid, urease expression was increased approximately 11-fold in Ro8 and 83-fold in O157 strain EDL933 compared with that in the wild type Ro8. The O149 urease activity was expressed despite the presence of the same premature stop codon in ureD that is present in ure+ O157:H7 strains that are urease-negative. The ure operon in Ro8 consists of 4 893 nucleotides with 99% identity with the ure operons in EHEC O157:H7 strains EDL933 and Sakai, and is part of a GI similar to GI-48 of strain EDL933. This OI, designated OI-48149 , is inserted in the serX tRNA gene in strain Ro8 and contains genes for urease, tellurite resistance, iha and an AIDA-I-like adhesin. The presence of a homolog of the O157:H7 OI-48 in highly virulent O149 porcine ETEC suggests that this OI may contribute to establishment of the bacteria in the intestine.     

Occurrence of Escherichia coli O157 in Finnish cattle

Bovine faecal samples were collected during June-December 1997 at 14 major abattoirs slaughtering cattle in Finland. Escherichia coli O157 was isolated from 19 of the 1448 samples (1.31%) after enrichment and immunomagnetic separation (IMS). The positive faecal isolates originated from 16 farms and eight abattoirs. The occurrence of E. coli O157 was highest in July (8/204; 3.92%) and September (6/244; 2.46%). No E. coli O157 was detected in November and December, nor from the faecal samples from the northernmost region where cattle density is low. All of the isolates carried the eae gene and showed the enterohaemolytic phenotype. All except one were motile and had the flagella antigen H7. Seventeen of the isolates were positive for stx(2) gene and one carried both the stx(1) and stx(2) genes. Of the 17 isolates with stx genes, 16 were verocytotoxin-positive in a reversed passive latex agglutination test after polymyxin extraction but only eight without extraction. The isolates belonged to 10 different pulsed-field gel electrophoresis (PFGE) patterns. The most common PFGE pattern (1.42) was detected in eight isolates (42.1%). Four PFGE patterns (1.1; 1.6; 1.12; 1.14) were identical with those isolated from humans in Finland, suggesting that at least some human E. coli O157 infections may be of bovine origin.     

Spatial distribution of Escherichia coli O157-positive farms in Scotland

Using a sample of 949 Scottish farms with finishing cattle, the spatial distribution of Escherichia coli O157-positive farms was investigated using disease mapping models. The overall prevalence of E. coli O157-positive farms was estimated as 22%. The regions used in this study were the 16 postcode areas of Scotland. For each region, the posterior relative risk (RR) was estimated as a model-based alternative to the saturated standardized morbidity ratio (SMR), i.e., the ratio between observed and expected cases in a region. Three Bayesian hierarchical models with generalized linear modeling of the area-specific risks were used to estimate the posterior relative risk of E. coli O157-positive farms in the postcode areas: a random-effects model incorporating only spatially uncorrelated heterogeneity; a model incorporating both spatially correlated and uncorrelated heterogeneity; and a pseudo-mixture model with unstructured correlation and a weighted mix of two variance components representing the spatial correlation and a jump structure. None of the models identified any areas with a significant increase or decrease in risk. The deviance information criteria slightly favored the simplest model (RR range: 0.92--1.09). However, this model appeared to smooth out more of the variation in the RR compared to the pseudo-mixture model, which gave a more informative pattern of the posterior relative risks (range: 0.81--1.22).     

Environmental sources and transmission of Escherichia coli O157 in feedlot cattle

A study was conducted in 2 feedlots in southern Alberta to identify environmental sources and management factors associated with the prevalence and transmission of Escherichia coli O157:H7. Escherichia coli O157:H7 was isolated in preslaughter pens of cattle from feces (0.8%), feedbunks (1.7%), water troughs (12%), and incoming water supplies (4.5%), but not from fresh total mixed rations. Fresh total mixed rations did not support the growth of E. coli O157:H7 and E. coli from bovine feces following experimental inoculation. Within a feedlot, the feces, water troughs, and feedbunks shared a few indistinguishable subtypes of E. coli O157:H7. A few subtypes were repeatedly isolated in the same feedlot, and the 2 feedlots shared a few indistinguishable subtypes. The prevalence of E. coli O157:H7 in water troughs of preslaughter cattle in 1 feedlot was associated with season, maximum climatic temperatures the week before sampling; total precipitation the week before sampling, and coliform and E. coli counts in the water trough.     

大肠杆菌O157 Z4182基因的克隆与表达

根据大肠杆菌0157z4182基因设计1对引物，并在其5'端分别加入NcoI、XhoI酶切位点，用聚合酶链反应（PCR）从本试验用的大肠杆菌O157及1株产志贺毒素大肠杆菌（STEC）08、1株肠道聚集粘附大肠杆菌（EAggEC)和1株产肠毒素性大肠杆菌（ETEC）菌株中也扩增出了803BP的DNA片段。以大肠杆菌0157菌株94H的DNA为模板，用上述引物做PCR，将其产物连接到载体质粒pET28a( )，然后转化到宿主菌大肠杆菌LE392，从该菌中提取重组质粒，再将其转化到表达宿主大肠杆菌BL21（DE3），用PCR，限制性内切酶位点分析及核苷酸序列测定法对克隆的重组质粒进行鉴定，表明Z4182基因定向克隆到了载体质粒Pet28a( )。将转化子培养并经IPTG诱导后，做SDS-PAGE电泳，表明该菌株可以表达Z4182基因。本试验为阐明大肠杆菌O157Z4182基因的分布及探讨该基因产物的功能和致病作用奠定了基础。     

Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins

A multiplex real-time PCR (R-PCR) assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan) to detect enterohemorrhagic Escherichia coli (EHEC) O157:H7 in pure cultures, feces, and tissues. Three sets of primers and fluorogenic probes were used for amplification and real-time detection of a 106-bp region of the eae gene encoding EHEC O157:H7-specific intimin, and 150-bp and 200-bp segments of genes stx1 and stx2 encoding Shiga toxins 1 and 2, respectively. Analysis of 67 bacterial strains demonstrated that the R-PCR assay successfully distinguished EHEC O157:H7 serotype from non-O157 serotypes and provided accurate profiling of genes encoding intimin and Shiga toxins. Bacterial strains lacking these genes were not detected with this assay. The detection range of the R-PCR assay for the three genes was linear over DNA concentrations corresponding from 10(3) to 10(8)CFU/ml of EHEC O157:H7. The R-PCR allowed construction of standard curves that facilitated quantification of EHEC O157:H7 in feces and intestinal tissues. Detection sensitivity of the R-PCR assay ranged from 10(4) to 10(8)CFU/g of feces or tissues without enrichment. Enrichment of feces in a non-selective broth for 4 and 16h resulted in the detection of levels (from 10(0) to 10(3)CFU/g of feces) considered sufficient for infection in humans. The R-PCR assay for eae(O157:H7), stx1, and stx2 proved to be a rapid test for detection of EHEC O157:H7 in complex biological matrices and could also potentially be used for quantification of EHEC O157:H7 in foods or fecal samples.     

A three-year study of enterohemorrhagic Escherichia coli O157 on a farm in Japan

A long-term study was performed on the prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 in bovine faeces. The present study was conducted on heifers raised on a farm showing a high isolation rate of EHEC O157 in previous years. The prevalence of EHEC O157 isolated from faecal samples was 10.6% (222/2104), 5.6% (181/3225), and 5.6% (153/2744) from 1998 to 2000, respectively. The numbers of EHEC O157-positive heifers for the same 3 years were 46.3% (185/400), 36.8% (147/399), and 31.7% (130/410), respectively. The seasonal prevalence of EHEC O157 varied according to the year. Most positive heifers excreted the EHEC O157 only once during the survey, though it was excreted 2 or 3 times by some heifers. The results obtained in the present study showed that the farm examined was heavily contaminated with EHEC O157. It is assumed that EHEC O157 does not remain in individual cattle long-term, but does exist long-term on farms due to repeated infection.     

A preliminary study of Salmonella, verocytotoxigenic Escherichia coli/Escherichia coli O157 and Campylobacter on four mixed farms

The aims of this study were to investigate the incidence of Salmonella, verocytotoxigenic Escherichia coli (VTEC)/Escherichia coli O157 and Campylobacter on four mixed farms and to characterize the isolates in terms of a range of virulence factors. Eighty-nine composite (five different samples from the same animal species combined) faecal [cattle (24), pigs (14), sheep (4), poultry (4), horses (7), deer (4), dogs (9), rodents (2) and wild birds (20)] samples, 16 composite soil samples plus 35 individual water samples were screened using culture-based, immunomagnetic separation and molecular methods. Salmonella was detected in bovine faeces, cattle and poultry house water. Salmonella serotypes/phage types included Dublin, Kiel and Typhimurium DT193, and most isolates were spvC, invA and rck positive. The pefA and rck genes were found exclusively in the non-Typhimurium strains, while Salmonella Dublin and Salmonella Kiel strains carried Salmonella genomic island I marker(s). VTEC/E. coli O157 were found in deer and dog faeces only. The E. coli O157 isolate was an enteroinvasive E. coli, while the VTEC isolate was untypable but carried the vt1, eaeA, hlyA, tir and eptD genes. This article reports the first confirmed carriage of E. coli O157 in Irish deer. Campylobacter species were not detected over the course of this study. It was concluded that [1] Salmonella, VTEC and Campylobacter have low (<5%) prevalence or are absent on the farms in this study; [2] water was an important source of bacterial pathogens; [3] both dogs and deer may act as a source of pathogenic E. coli and [4] key virulence and resistance determinants are widespread in farm Salmonella strains. This study highlights the need to control water as a source of pathogens and suggests that the domestic pets and deer should be considered in any farm risk assessment.     

Significance of Escherichia coli O157 in sheep at slaughter in Switzerland

The importance of latent zoonoses has increased in recent years in view of foodborne diseases: (i) the "healthy" animal repesents a reservoir for specific pathogens; () no pathological-anatomical changes in the carcass and its organs show the presence of these pathogens; and (iii) these pathogens may enter the food chain via hygienic weak points in the slaughtering process. To estimate the risks involved and to take appropriate measures, analysis of the slaughtering process should be complemented by collecting data relating to the carriage of the animals of latent zoonotic pathogens. From October 2004 to June 2005, fecal samples from 630 slaughtered sheep were enriched and then examied by IMS technique and by PCR to assess the prevalence of E. coli O157 (OE). Seven samples (1.1%), distributed throughout the whole examination period, were found to be positive. To assess the potential pathogenicity for humans, E. coli O157 strains were isolated by colony hybridization and further characterized. The isolated strains fermented Sorbitol, showed four different H tys (H7, H12, H38, H48), and were all negative for stx. One O157:H7 strain harbored the gene for intimin (eae) in combination with ehxA, and paa. In consequence, the potential health hazard from sheep meat related to O157 STEC seems current not to be of particular importance in Switzerland. Results emphasize the fact that E. coli O157 are not always STEC but may belong to other pathotypes as nontraditional EPEC.     

Occurrence and Characterization of Escherichia coli O157 Isolated from Cattle in Norway

Faecal samples from 504 imported beef cattle were screened to investigate the occurrence of Escherichia coli O157. The results were compared with those from a previous screening of Norwegian dairy cattle, and the occurrence was found to be higher in the imported beef cattle. The E. coli O157 isolates from the previous and present studies were characterized for the genes encoding for shigatoxin 1 (stx ₁), shigatoxin 2 (stx ₂), the intimin protein (eae) and the flagellar protein H7 (fliC) using PCR analysis, pulsed-field gel electrophoresis (PFGE) with the restriction enzyme XbaI, and bacteriophage lambda RFLP analysis using the PvuII restriction enzyme. The isolates from the dairy and beef cattle could be distinguished by the profiles of the toxin genes and by PFGE patterns. Whether the importation of animals in itself should be regarded as a risk factor for the occurrence of E. coli O157, or whether other management factors contribute to the differences in carrier rates compared to the previous study on domestic cattle, is discussed.     

Prevalence and characterization of Shiga toxin-producing Escherichia coli O157 and O26 in beef farms

Rectal content grab samples were collected from 2436 beef cattle reared on 406 beef farms in Japan between November 2007 and March 2008. STEC strains O157 and O26 were isolated from 110 (27.1%) and 7 (1.7%) farms, respectively. Farms that tested positive for STEC O157 were located in 35 out of all 47 Japanese prefectures. This indicates that STEC O157 strains are widespread on beef farms nationwide. Of the 2436 tested beef cattle, 218 (8.9%) and 10 (0.4%) had STEC strains O157 and O26 in the rectal content, respectively. The most common Shiga toxin genes detected in the isolated STEC O157 strains were: stx(2c) alone (32.1%), stx(2)/stx(2c) (27.2%), and stx(1)/stx(2) (21.8%). Almost all of the STEC O157 and STEC O26 strains expressed Shiga toxins (Stx). Most of the STEC O157 and STEC O26 strains possessed eaeA and EHEC-hlyA. These results strongly suggest that STEC strains O157 and O26 from beef cattle would be pathogenic to humans. Therefore, it is important to reduce STEC strains O157 and O26 in beef cattle in order to prevent foodborne disease caused by STEC. The presence of dogs and/or cats on a farm was significantly (P=0.02) associated with the prevalence of STEC O157. More research is needed to clarify the role of dogs and cats.     

出血性大肠杆菌噬菌体933W的分离鉴定和保存

采用双层琼脂平板法从EHEC 0157 EDL933基因组中诱导分离出了编码志贺毒素的噬菌体933W，并利用PCR技术对其进行了鉴定。对933W的不同保存方法进行比较，结果表明，将噬菌体933W置于SM溶液中加入CaCl2、lgCl2至终浓度10mmol／L，明胶浓度至0．1％（W／V）并于4℃冷藏是保存933W较好的方法。     

Variable and strain dependent colonisation of chickens by Escherichia coli O157

The prevalence of enterohaemorrhagic Escherichia coli (EHEC) O157 in poultry is considered minimal compared with other species, especially ruminants. However, deliberate inoculation studies have shown that poultry are readily and persistently infected by this organism but that the mechanism of colonisation is independent of intimin, a recognised factor in host-EHEC interactions in mammalian species, and may be dependent upon flagella. Few strains of EHEC O157 have been tested in poultry and here 1-day-old and 6-week-old chicks were inoculated with seven non-toxigenic E. coli O157 strains in separate experiments. Persistence was measured semi-quantitatively by bacteriological assessment of E. coli O157 cultured from cloacal swabs (shedding score). In the 1-day-old chick model that was monitored for 43 days, all seven strains established well after inoculation. In the 6-week-old chicken model, one strain established and gave consistently high shedding for the duration of the experiment (156 days). Whereas of the remaining six strains, two persisted for 113 days, two persisted for 43 days, one persisted for 22 days and one strain was never detected.