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。     

西藏林芝地区仔猪白痢血清型及毒力基因检测

为探究西藏林芝地区仔猪白痢大肠杆菌优势血清型和毒力基因间的相关性,采用玻片凝集试验测定与仔猪白痢有关的血清型,PCR方法调查11种毒力基因。结果显示,在分离的81株藏猪源大肠杆菌里有72株检测到毒力基因,其中黏附素K88和CS31A分别检测到1(1.39%)和34(47.22%)株;肠毒素STa、STb和EAST1分别检测到10(13.89%),16(22.22%)和38(52.78%)株;毒力岛eaeA、irp2和ETT2分别检测到12(16.57%),44(61.11%)和56(77.78%)株。72株中有59株鉴定出9种血清型,其中有15株定型为O8(20.99%),11株定型为O64(16.05%),10株定型为O138(13.58%)。研究表明:O8、O64、O138为优势血清型,且O8与STa+STb相关,O107与肠毒素STa相关,O64与毒力岛eaeA+ETT2有关,毒力基因以EAST1+CS31A+irp2+ETT2组合检出率最高为18(25%)株。     

仔猪腹泻致病性大肠杆菌分型鉴定及耐药性分析

为了解贵州省规模化养猪场腹泻仔猪致病性大肠杆菌流行情况及耐药性变化,本研究运用凝集试验、PCR和药敏纸片琼脂扩散法等方法对分离的78株致病性大肠杆菌进行血清型、毒力基因及耐药性分析。结果显示,78株致病性大肠杆菌以O138、O87血清型为主,占定型菌株的60.8%;其中62株致病性大肠杆菌检出毒力基因,检出率为79.5%,可分为8种毒力基因类型,分属肠致病性大肠杆菌（EPEC）、肠产毒性大肠杆菌（ETEC）和肠聚集性大肠杆菌（EAEC）,毒力基因eaeA、elt和escV检出率较高,分别为38.5%、28.2%和21.8%;分离到的致病性大肠杆菌对β-内酰胺类药物高度耐药,均为多重耐药株,耐药种类可达8种以上。结果表明,当前贵州省规模化养猪场腹泻仔猪致病性大肠杆菌的毒力基因检出率较高且基因型复杂,耐药性严重。本试验结果可为规模化养猪场防控仔猪腹泻提供基础资料及理论依据。     

一规模化猪场断奶仔猪腹泻大肠杆菌毒力因子的检测

从疑似断奶仔猪腹泻的仔猪中分离、鉴定出15株病原性大肠杆菌。经O血清型鉴定,11株为0131、4株未定型,所有O131血清型菌株呈β溶血。多重PCR检测毒素基因(STa、STb、LT、SLT-2e)和大肠杆菌粘附素单抗(F4、F5、F6、F41、F18)检测菌毛,O131菌株的毒素为STa、STb、SLT-2e,表达F18粘附素,未定型菌株的毒素为STa,共表达F6和F18粘附素。对15株大肠杆菌用16种抗生素进行药敏试验,结果表明:分离株对阿米卡星、痢特灵、新霉素敏感,而对多种抗生素产生了不同程度的耐药性。运用本场大肠杆菌分离株灭活苗免疫猪群,取得良好效果。     

皖北地区仔猪源大肠杆菌毒力因子及HPI毒力岛的检测

我国皖北地区哺乳仔猪腹泻频发,临床直肠棉拭子培养物有34例符合大肠杆菌特征,并进行产肠毒素大肠杆菌(ETEC)毒力因子(STa、STb、LT、SLT-2e)和HPI毒力岛的PCR快速检测。结果,仅有10例表达STa和STb(29.41%),其中3例表达STa基因(8.82%),2例表达STb基因(5.88%),2例表达STa和STb基因,1例表达STa和irp2基因(2.94%),2例表达STa和STb及irp2基因,揭示HPI+大肠杆菌与ETEC混合感染共有3例(8.82%);未检测到LT和STL-2e毒力因子。有10例表达irp2基因(29.41%),其中7例独立表达irp2基因(20.59%)。STa、STb和Irp2的PCR产物测序结果分别与GenBank检索的目标序列比对,其同源性均达到100%。结果初步揭示了皖北地区致哺乳仔猪腹泻ETEC毒力因子及HPI毒力岛的分布情况。     

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。     

猪大肠埃希菌分离鉴定及毒力因子PCR检测

为了解陕西省宁强县部分地区引起仔猪黄白痢的致病性大肠埃希菌的主要血清型及其主要毒力因子,用细菌分离培养方法,从宁强县5个猪场采集的60份有疑似黄白痢症状仔猪的肛门拭子中分离出20个大肠埃希菌菌株。经菌落形态观察、革兰染色镜检、培养特性、生化试验、血清型鉴定,确定分离菌株均为致病性大肠埃希菌。血清分型表明,20个分离菌株中有18株能确定血清型,分布在7个血清型中,其中优势血清型为O8(8株),占总数的40%。药物敏感性试验表明,分离菌株对临床常用药物具有抗药性。毒力因子的PCR检测结果表明,分离菌株均不携带STa、STb、LT和SLT-2e四个毒力基因。     

仔猪腹泻致病性大肠杆菌分型鉴定及耐药性分析

为了解贵州省规模化养猪场腹泻仔猪致病性大肠杆菌流行情况及耐药性变化,本研究运用凝集试验、PCR和药敏纸片琼脂扩散法等方法对分离的78株致病性大肠杆菌进行血清型、毒力基因及耐药性分析。结果显示,78株致病性大肠杆菌以O138、O87血清型为主,占定型菌株的60.8%;其中62株致病性大肠杆菌检出毒力基因,检出率为79.5%,可分为8种毒力基因类型,分属肠致病性大肠杆菌(EPEC)、肠产毒性大肠杆菌(ETEC)和肠聚集性大肠杆菌(EAEC),毒力基因eaeA、elt和escV检出率较高,分别为38.5%、28.2%和21.8%;分离到的致病性大肠杆菌对β-内酰胺类药物高度耐药,均为多重耐药株,耐药种类可达8种以上。结果表明,当前贵州省规模化养猪场腹泻仔猪致病性大肠杆菌的毒力基因检出率较高且基因型复杂,耐药性严重。本试验结果可为规模化养猪场防控仔猪腹泻提供基础资料及理论依据。     

貉致病性大肠杆菌分离鉴定及HPI、LEE毒力岛检测

为了解致病性大肠杆菌引起貉腹泻的机理,采用了貉致病性大肠杆菌分离鉴定、检测毒力岛基因HPI(irp2、fyuA)与LEE(ler、eaeA),小鼠腹腔注射检测菌株毒力的方法。结果表明:从腹泻貉体内分离出血清型分别为O_(29)、O_(38)、O_(78)和O_(107)的大肠杆菌,4个血清型中有2个血清型(O_(38)、O_(78))携带有irp2和fyuA,4个血清型均未检测到ler和eaeA;4个血清型的大肠杆菌均可使小鼠不同程度发病,其中以携带irp2和fyuA的菌株毒力较强,可见该菌对貉的健康存在危害。     

广东屠猪肉样品中大肠杆菌耐药性与毒力特征的分析

为分析广东地区屠猪肉中大肠杆菌(E.coli)药物敏感菌株的血清型、毒力基因和系统进化背景,本研究从屠猪肉样品中分离出112株E.coli,采用玻片凝集法鉴定血清型,琼脂稀释法测定10种抗菌药的敏感性,PCR方法检测7种毒力相关基因,多重PCR方法进行系统进化背景判定。结果显示,112株E.coli中,定型菌株95株,分别属于15种血清型,其中O65、O131、O8和O158为优势血清型。几乎所有菌株对氟苯尼考、多西环素和四环素高度耐药,而对头孢曲松高度敏感,其中多重耐药菌株多数耐5种以上药物,常见的多重耐药表型是氟苯尼考/氯霉素/多西环素/四环素/氨苄西林。PCR鉴定结果表明,含有毒力基因的菌株中38%至少具有两个毒力基因,其中EAST1+Stx2e和hlyF+Stx2e比较常见。比较常见的毒力基因为Stx2e和EAST1,STb基因仅在一株菌中检测到。多重PCR鉴定结果显示,屠猪肉样品中E.coli主要分布为共生型的A组和B1组。本研究为大肠杆菌病的控制和合理使用抗生素提供实验依据。     

Detection of Serotypes and Virulence Factors of Escherichia coli from Piglets in Beijing

To investigate the cause of piglets diarrhea, and the distribution of the serotype and virulence factors of swine Escherichia coli in Beijing, 400 diarrhea samples were collected. TSA serum agar culture method was used to isolate Escherichia coli, serotype identification test and virulence factor genes test were used to verify the presence of O-antigen. 64 strains of E.coli were isolated from 400 diarrhea samples, among which 42 strains of E.coli were classified as 8 serotypes:O101(18.7%),O64(12.5%),O8(10.9%),O20(10.9%),O45(4.7%),O149(4.7%),O2(1.6%) and O89(1.6%), and the major virulence factors were STa, Stx2e, astA and eaeA. There were 8 mainly serotypes that caused piglets diarrhea in Beijing area, among which O101 serotype accounted for the highest proportion. The major virulence factors were astA and eaeA, accounted for more than 50% of all strains, STa and Stx2e accounted for more than 30% of all strains. These data would provide effective data to support the prevention and control of piglet diarrhea in Beijing.     

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.     

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.     

Distribution of virulence genes in Escherichia coli strains isolated from diarrhoeic piglets in the Slovak Republic

Ninety-two Escherichia coli isolates from 14 to 28-day-old piglets that died because of diarrhoea were examined for genes for fimbriae (F4, F5, F6, F18 and F41), enterotoxins (STa, STb and LT), verotoxin (VT2e or Stx2e) and enteroaggregative heat-stable enterotoxin 1 (EAST1) by polymerase chain reaction. Twenty-two strains (24%) carried a gene for F4, whereas genes for F18, F6 and F5 + F41 were detected in 10.8, 3.3 and 1.1% of strains respectively. Genes for STb, LT, STa and Stx2e were detected in 40.2, 26.1, 14.1 and 1.1% of strains respectively. The astA gene was detected in 49 (53.3%) isolates, 35 of which also carried genes for enterotoxins and/or fimbriae. The major genotypes reached at (in decreasing order of prevalence) were F4/STb/LT/EAST1, F18/STa/STb/EAST1, STb/EAST1, F6/STa/STb/EAST1 and F18/STb/EAST1.     

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.     

Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina

Grazing-fed cattle were previously demonstrated to be reservoir of non-O157 Shigatoxigenic Escherichia coli (STEC) serotypes in Argentina. The acid-resistance of some STEC strains makes it reasonable to assume the presence in feedlot of particular STEC serotypes. Fifty-nine animals were sampled every 2 weeks during 6 months by rectal swabs. Twenty-seven of 59 animals (45.8%) were shown to be Stx2(+); 3/59 (5.1%) carried Stx1(+) and 7/59 (11.9%) were Stx1(+) Stx2(+). Among 44 STEC isolates, 31 isolates were associated to 10 O serogroups (O2, O15, O25, O103, O145, O146, O157, O171, O174, O175) and 13 were considered non-typable (NT). Six H antigens (H2, H7, H8, H19, H21, H25) were distributed in 21 isolates whereas 23 were non-mobile (H-). Seventeen of 44 strains (38.6%) were eaeA(+) and 14 (31.8%) harbored the 60MDa plasmid. The megaplasmid (Mp) and eaeA gene were simultaneously found in a limited number of serotypes belonging to the enterohaemorrhagic E. coli (EHEC). E. coli O157:H7 strains, isolated from four (6.8%) animals, corresponded to the Stx2(+), eaeA(+), Mp(+) pattern. Three O157:H7 strains belonged to phage type 4 and the other strain was atypical. Many serotypes isolated from grain-fed cattle (O2:H25, O15:H21, O25:H19, O145:H-, O146:H-, O146:H21, O157:H7, O175:H8) also differed from those isolated by us previously from grazing animals. The serotypes O15:H21, O25:H19 and O175:H8 had not been identified at present as belonging to STEC. This work provides new data for the understanding of the ecology of STEC in grain-fed cattle and confirms that cattle are an important reservoir of STEC.     

Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3?%) from diarrhoeic animals and 60 (85.7?%) from healthy calves. The virulence markers identified were: Stx1 (82.8?%), eae (24.3?%), F41 (11.4?%), F5 (10?%), STa (4.28?%) and Stx2 (4?%). In diarrhoeic animals, Stx1 (70?%) and F41 (30?%) were identified, while Stx1 (83.3?%), eae (28.3?%), F41 (8.3?%), F5 (11.6?%), STa (5?%) and Stx2 (1.6?%) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26?% of all calves and on 75?% of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.     

Rapid identification of virulence genes in enterotoxigenic Escherichia coli isolates associated with diarrhoea in Queensland piggeries

OBJECTIVE: To identify virulence genes in enterotoxigenic E coli (ETEC) isolates associated with diarrhoea in neonatal, 1 to 3 week-old and weaned pigs in southeast Queensland. DESIGN: Multiplex PCR and serotyping were applied to E coli isolates obtained over a 5-year period (1998-2002) from cases diagnosed at Toowoomba Veterinary Laboratory. PROCEDURE: A total of 126 isolates from 25 different Queensland piggeries were tested for haemolytic activity on 5% sheep blood agar and by multiplex PCR for the presence of five commonly recognised fimbrial (F4, F5, F6, F41 and F18) and three enterotoxin genes (STa, STb, LT). A subset of 62 representative isolates were serotyped by slide agglutination. For comparative purposes, multiplex PCR was also performed on the DNA of 31 ETEC isolates from 9 serotypes originating from piggeries in southern New South Wales. RESULTS: A total of 113 (89.7%) of the isolates from Queensland possessed ETEC virulence genes, including 14 of 15 isolates from neonatal pigs (93.3%), 18 of 23 isolates from 1 to 3 week old pigs (78.3%) and 81 of 88 isolates from weaned pigs (92.1%). F4:STa:STb:LT (serotype O149) was the most prevalent pathotype in neonatal and 1-3 week old pigs and F4:STa:STb:LT (serotype O149) and F18:STa:STb:LT (serotype O141) were most prevalent in weaned pigs. In comparison, isolates obtained from neonatal pigs from New South Wales belonged to a more diverse range of pathotypes and serotypes. CONCLUSION: Multiplex PCR was a rapid and specific method for detecting the presence of ETEC virulence genes in porcine E coli isolates. For isolates obtained from cases of suspected colibacillosis in Queensland, growth of a heavy pure culture of haemolytic E coli was a sensitive prognostic indicator of the presence of ETEC virulence genes in the isolate. ETEC pathotypes and serotypes remained stable in Queensland piggeries over the five-year study period and appear to have changed little over the last three decades.