上海地区猪源大肠杆菌血清型及其毒力因子分布调查

对上海地区分离的199株猪源大肠杆菌分离株进行血清型鉴定和毒力因子检测,使用大肠杆菌标准抗O因子血清进行玻片凝集试验;采用普通和多重PCR方法,对分离株进行STa、STb、LT、Stx2e等4种肠毒素和K88、K99、987P、F18、F41等5种黏附素检测。结果:199株猪源大肠杆菌分离株有110株鉴定出血清型,占检测菌株的55.28%,覆盖了30种血清型,优势血清型为O8、O45、O64、O78、O138、O157、O163、O7+O8,占定型菌株的60%。共有32株大肠杆菌检测到毒力因子,阳性率为16.08%,检测出9种毒力因子。其中有21株单独检测到肠毒素,占65.63%,STa和STb+LT阳性的菌株分别为14和4;6株单独检测到黏附素,3株菌株K99阳性;5株同时检测到肠毒素和黏附素。在32株毒力基因阳性的大肠杆菌中,有15株血清型定型,覆盖7种血清型,其中O138、O7+O8与肠毒素STa相关,O8、O45与STb+LT相关。研究表明,上海地区猪源大肠杆菌分离株优势血清型至少覆盖了包括O8、O45、O64、O78、O138、O163等在内的8种血清型;大肠杆菌分离株以产肠毒素为主,主要毒力因子包括黏附素K99和肠毒素STa、STb+LT等。     

宁夏部分地区鸡大肠埃希菌的分离与血清型鉴定

为调查宁夏地区鸡大肠杆菌病的流行情况,采集宁夏4个地区的103个规模化鸡场疑似病料151份,通过病原菌分离鉴定及生化试验,鉴定出鸡大肠埃希菌109株,平均分离率为72.19%(109/151);动物试验证实,59株分离菌有致病性,占分离菌54.13%(59/109);对致病性菌株进行血清型鉴定,确定血清型种类13种38株,占致病性菌株64.41%(38/59);优势血清型分别为O15、O18、O78、O88,占定型菌株63.16%(24/38)。     

泰安地区鸡源致病性大肠杆菌流行病学调查与耐药性分析

正本调查对鸡场采集的疑似鸡大肠杆菌病例的相关病料进行大肠杆菌的分离鉴定,共分离鉴定出14株鸡源大肠杆菌,以微量平板凝集试验鉴定分离菌株的O抗原血清型,分别属于O78、O2和O1等3个血清型,其中O78(6/14)、O2(6/14)为主要流行血清型,占分离菌株总数的85.71%。选取了20种常用治疗肠杆菌病的抗生素对14株鸡源大肠杆菌分离株进行药敏试验,对14株大肠杆菌     

禽源和猪源大肠杆菌1型菌毛主要亚单位抗原多样性分子基础的初步研究

从禽源大肠杆菌037(O78)、166(O78)、120(O18)分离株和猪源大肠杆菌107/86分离株分别提取基因组DNA,并以此为聚合酶链反应(polymerasechainreaction,PCR)的模板,扩增上述分离株的1型菌毛主要亚单位结构基因pilA,得到了大小约570bp的扩增产物,将此扩增产物克隆于T载体,通过内切酶酶切分析得到4个阳性重组质粒;对上述4个大肠杆菌分离株的pilA基因进行序列测定,通过其编码的主要菌毛亚单位FimA蛋白氨基酸的序列比较发现:3个禽源株间FimA的同源性为94.3%至99.0%;禽源株和猪源株间FimA的同源性为89.6%至91.1%。在pilA开放性阅读框所编码的FimA182个氨基酸序列中,禽源大肠杆菌O78血清型的2个分离株037株和166株间只有2个氨基酸不同,其同源性为99.0%。     

皖北地区仔猪黄痢大肠杆菌血清型的分离鉴定与免疫保护试验

为探明皖北地区导致仔猪黄痢的大肠杆菌的主要血清型,从皖北地区仔猪黄痢病料中,分离到37株致病性大肠杆菌菌株,并用O抗原单价血清、ELISA试验和菌毛抗原进行了血清型鉴定。结果表明,引发该地区仔猪黄痢的优势血清型为O8、O9、O141,这3种血清型占全部分离菌株的63.6%,其余血清型为O45、O147、O20、O101、O157、O119,占36.4%;对O8、O9、O141分离菌株进行了菌毛抗原的鉴定,菌毛抗原主要有K88、K99、F41。采用分离的优势菌株制成灭活疫苗,免疫妊娠猪,进行疫苗免疫效果比较试验。结果显示,自制分离株灭活苗组、K88-LTB基因工程苗组和对照组仔猪黄痢的发病率分别为11.5%、18.3%和28.4%,死亡率为8.6%、12.0%和18.1%,表明自制分离株灭活苗有较好的保护效果,为下一步有针对性地防制仔猪黄痢提供科学依据。     

鸡源致病性大肠杆菌血清型鉴定及耐药性分析

为了了解山东、河北和河南等省部分地区养殖场鸡源致病性大肠杆菌的血清型及耐药性,试验采用细菌分离和生化鉴定方法对鸡大肠杆菌进行分离和鉴定,通过动物试验确定分离大肠杆菌的致病性,应用血清学方法和药敏试验对致病性大肠杆菌的血清型及耐药性进行研究。结果表明:分离鉴定的119株大肠杆菌中致病性菌株有110株,其中92株致病性菌株能够定型,分别属于15个血清型,有5株出现交叉凝集,O14、O35、O1、O2、O78、O88为优势血清型;分离鉴定的致病性大肠杆菌对环丙沙星、头孢噻肟、头孢哌酮等最敏感,对林可霉素、头孢克肟、卡那霉素、庆大霉素、红霉素的耐药率达60%以上。说明分离的致病性大肠杆菌血清型存在复杂性,对大部分临床常用药物产生了不同程度的耐药。     

新疆北疆地区致犊牛腹泻大肠杆菌的分离鉴定及部分生物学特性

为研究确定新疆北疆地区规模化奶牛场犊牛腹泻病原性大肠杆菌的优势血清型、致病性及毒力因子特征。采用细菌学、免疫学及分子生物学的方法对从新疆呼图壁、石河子、奎屯3个主要奶牛生产基地14个规模化奶牛场10日龄内腹泻犊牛直肠棉拭子样品进行了大肠杆菌的分离与鉴定、O血清群、黏附素及肠毒素的测定。结果从302份样品中分离并经生化鉴定获得180株呈β溶血的大肠埃希菌,其中94株对小鼠有致病性;对其中53株代表菌株的O血清型、黏附素及肠毒素测定,结果为8株携带K99菌毛及STa毒素基因,6株携带F41茵毛基因,6株产生LT毒素;28株分离株分布于16个O血清型,其中O101,O6,O114,O78为优势血清型,占被测菌株的50％。结果表明,新疆北疆主要奶牛养殖区致犊牛腹泻大肠杆菌是以携带K99、F41和产ST的溶血性大肠杆菌为主,其研究结果为犊牛大肠杆菌性腹泻的免疫防治提供病原学依据。     

山东省部分地区禽源致病性大肠杆菌的血清流行病学调查与耐药性检测

为调查山东省禽源致病性大肠杆菌流行的血清型及耐药性,从山东部分地区的45家养禽场分离到致病性大肠杆菌96株,应用微量平板凝集试验进行了血清型鉴定,共鉴定出18种血清型,其中优势血清型6种,分别为O78、O2、O15、O18、O143、O88,占定型致病性菌株的64％。抗菌药物敏感性试验发现,96株大肠杆菌对20种药物有不同程度的耐药性。75％以上的菌株对氨苄青霉素、阿莫西林、土霉素等5种抗菌药耐药,50％以上的菌株对卡那霉素、多西环素、环丙沙星等7种抗菌药表现为耐药;所有分离株存在多重耐药现象,75％的受检菌对9种或9种以上的被测药物耐药。结果表明,O78、O2、O15、O18、O143、O886种血清型是山东省部分地区近年来禽源致病性大肠杆菌的优势血清型,且禽源致病性大肠杆菌的耐药现象严重,有必要加强耐药性检测,以指导兽医临床合理使用抗菌药物。     

猪水肿病大肠杆菌分离、鉴定及药敏试验

本实验从疑似猪水肿病的病例分离到5株大肠杆菌,O抗原鉴定结果表明所有菌株均为O139血清型;应用F18ab菌毛单克隆抗体对这5株大肠杆菌能否表达F18ab菌毛进行了鉴定,结果表明其中2个菌株能表达F18ab菌毛;利用聚合酶链式反应(PCR)对志贺氏菌样毒素Ⅱ型变异体(SLT-Ⅱe)操纵子基因保守区进行了扩增,结果发现在能表达F18ab菌毛2个菌株中可扩增一段特异性序列。以上数据表明这2株大肠杆菌为致仔猪水肿病大肠杆菌。药敏试验表明这两株菌株均对氟哌酸、妥布霉素、庆大霉素、利福平等抗生素高度敏感。     

河北省鸡病原性大肠杆菌的分离与鉴定

从河北省不同地区的发病鸡场分离培养出91株大肠杆菌，经致病力试验鉴定为病原性大肠杆菌，血清学鉴定，除有23株未能定型外，其余68株共鉴定出13个血清型，其中以O78、O2、O111、和O35等5个血清型检出率较高，分别占定型菌株的33．80％、26．50％、11．80％、5．88％和5．88％，而O78，O2和O111仅3个血清型却拥有49个分离株，占定型菌株的72．10％。     

Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis

A comprehensive study of 223 Escherichia coli isolates from pigs with colibacillosis included determination of O serogroups, detection of heat-labile enterotoxin, heat-stable enterotoxin (STa and STb), and identification of K88, K99, 987-P, F-41, and type 1 fimbriae. The incidence of the various E coli types among isolates of pigs of different ages was also determined. Escherichia coli bearing K88 fimbriae accounted for 48% of all isolates studied, were most often of serogroup O157, O149, or O8, and usually produced labile toxin alone or in combination with STa or STb. These E coli were commonly isolated from pigs in each age group studied (0 to 5 days, 6 to 10 days, 11 to 24 days, and greater than 24 days). Escherichia coli bearing 987-P accounted for 30% of the isolates, were most often of serogroup O141 or O20, and usually produced STa. Escherichia coli bearing K99 accounted for 13% of the isolates, usually were of serogroup O101 or O8, and almost always produced STa. Escherichia coli bearing 987-P or K99 were most often isolated from pigs less than 6 days of age. Fimbriae F-41, when identified, were usually on E coli of serotype O101:K99. Although infrequently found, type 1 fimbriae were on E coli of most of the serogroups identified in this study.     

Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea

The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.     

Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.     

Isolation,Identification and Drug Resistance Analysis of Interstinal Pathogenic Escherichia coli and Salmonella Isolated from Diarrhea Piglets

In order to find out the serotype, resistant phenotype and genotype of Escherichia coli (E. coli) and Salmonella in piglets, this study collected 128 samples of diarrhea piglets from seven large-scale pig farms in five cities in Guizhou province, and the E. coli and Salmonella were isolated and identified. The pathogenicity of the strain was identified by animal test. The drug resistance of the main pathogen was tested by drug susceptibility paper. The resistance gene of each pathogen was detected by PCR. The drug resistance and genotype correlation of the bacterial were analyzed. The results showed that 78 strains of pathogenic E. coli and 21 strains of Salmonella were isolated and identified in this study. The serotypes of pathogenic E. coli were predominantly O138 and O87. Salmonella Typhimurium and Salmonella Enteritidis were predominant serotypes. The susceptibility test showed that the resistant strains of 78 strains of E. coli were more than 80% resistant to β-lactams and more than 40% for other antibacterials. The resistance rate of 21 strains of Salmonella to aminoglycosides was more than 50% and more than 20% to other types of antibacterials; 12 and 10 kinds of drug resistance-related genes of E. coli and Salmonella were detected, respectively; The coincidence rate of resistant genotype and phenotype of two kinds of bacteria were above 60%, and both were multiple drug resistance. This study provided a theoretical basis for comprehensive prevention and control of piglets diarrhea.     

腹泻仔猪肠道致病性大肠埃希氏菌和沙门氏菌的分离鉴定与耐药性研究

为探明仔猪细菌性腹泻肠道致病性大肠埃希氏菌和沙门氏菌流行的血清型、耐药表型及耐药基因型,本试验采集了贵州省5个地（州）市7个规模化养猪场的128份腹泻仔猪肠道样本,并对采集的样本进行了大肠埃希氏菌和沙门氏菌分离与鉴定,通过动物试验鉴定菌株的致病性,利用血清学方法鉴定其血清型,并通过药敏纸片法对主要致病菌进行耐药性研究,采用PCR技术检测各致病菌株耐药相关基因,分析细菌耐药表型和耐药基因型相关性。结果显示,本研究共分离鉴定到78株致病性大肠埃希氏菌与21株沙门氏菌,致病性大肠埃希氏菌血清型以O138、O87为主,沙门氏菌血清型以鼠伤寒沙门氏菌、肠炎沙门氏菌居多;药敏试验结果表明,本试验分离到的78株致病性大肠埃希氏菌对β-内酰胺类药物耐药率达80%以上,对其他种类的抗菌药耐药率均超过40%,分离鉴定的21株沙门氏菌对氨基糖苷类药物耐药率达50%以上,对其他种类的抗菌药耐药率均达20%以上;本试验分离鉴定的致病性大肠埃希氏菌共检出12种耐药相关基因,沙门氏菌共检出10种耐药相关基因,两种细菌耐药基因型与耐药表型符合率均达60%以上,且均为多重耐药。本研究为仔猪腹泻的综合防控提供了理论依据。     

Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan

The virulence factors of avian pathogenic Escherichia coli (APEC) isolated in Japan were investigated. Serogroups O, serotypes K1 and K5, and genes cva C, iss, iutA, papA, tsh, and usp, which have been thought to be related to virulence, were examined for their association with E. coli strains isolated from diseased and healthy chickens. The frequently recognized serogroups O1, O2, and O78 were found in 56 of 125 (44.8%) strains of diseased chickens (APEC) versus 13 of 100 (13.0%) strains of healthy chickens (commensal E. coli), a significant difference at risk ratio < 0.01. Although iss, iutA, and tsh were widely distributed in the APEC irrespective of O serogroup, papA, usp, and the K1 serotype were detected in serogroup O2 of APEC. The kfiD gene related to the K5 capsule and VT, LT, and ST genes related to exotoxins were not detected in any strains examined.     

Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in galicia (north-western Spain)

A total of 289 Escherichia coli colonies isolated from 78 diarrhoeic calves were studied for production of heat-labile (LT) and heat-stable (STa) enterotoxins, verotoxin (VT), cytotoxic necrotizing factor (CNF) and K99 antigen, and they were serotyped. Production of STa was detected in a single strain possessing both K99 and F41 antigens; the serotype was 09:K (A) 35. LT-producing strains were not detected. From 16 (20.5%) calves, 51 VT-producing colonies of E. coli were isolated. Production of the necrotic factor was detected in 33 E. coli colonies isolated from 14 (17.9%) calves. Serotype was a useful marker for production of VT and CNF. Among the 51 VT-producing colonies, 24 were untypable and the remainder belonged to serotypes O2:K?, O103:K--, O104:K?, O128:K?, O153:K-- and O157:K--:H7. Four of the 33 CNF-producing colonies were untypable and the majority of the remaining colonies belonged to serotypes O15:K14, O78:(K80), O123:K-- and O139:K--. Both VT and CNF were lethal for mice, but only CNF showed necrotizing reaction in rabbit skin. Our results indicate that VT-producing and CNF-producing E. coli strains are frequently isolated from diarrhoeic calves and that according to the serotypes exhibited, some of them might be considered potential pathogens for humans. The role of VT-producing and CNF-producing strains in calf diarhoea remains to be established.