鸡源大肠杆菌中HPI和LEE毒力岛相关基因的检测

采用PCR方法检测辽宁锦州地区分离的150株鸡源大肠杆菌中耶尔森菌强毒力岛基因(HPI)和肠细胞脱落位点毒力岛(LEE),利用多重PCR方法检测HPI irp2和fyuA基因,以及LEE ler和eaeA基因.在分离的鸡源大肠杆菌中,HPI毒力岛基因检测结果为:18.7％的菌株irp2和fyuA基因扩增阳性,6.7％的菌株irp2基因阳性;LEE毒力岛基因检测结果为:15.3％的菌株ler和eaeA基因扩增阳性.结果表明,25.3％的鸡源大肠杆菌携带HPI,15.3％的鸡源大肠杆菌携带LEE.     

狐狸源大肠杆菌毒力岛基因HPI、LEE的检测

为检测从狐狸体内分离的大肠杆菌的毒力岛基因和毒力,对从病死狐狸的肝脏、肺脏等器官分离的细菌进行了生化鉴定、毒力试验和毒力岛HPI(irp2、fyuA)与LEE(ler、eaeA)检测。结果表明:在所测的6株菌中有4株检测出HPI毒力岛基因,6株菌均未检测出LEE毒力岛基因,各注射菌液组小鼠均不同程度发病,且含有HPI毒力岛基因的菌株对小鼠的毒力较强,可见分离到的菌株对狐狸的健康养殖存在较大的危害。     

腹泻绵羔羊源大肠杆菌耐药性及LEE毒力岛部分相关基因的检测

为了解宁夏地区腹泻绵羔羊源大肠杆菌的耐药和肠细胞脱落位点(locus enierocyte effacement, LEE)毒力岛相关基因携带情况，试验从宁夏地区4个规模化羊场采集腹泻绵羔羊肛拭子样本120份，采用常规微生物分离培养方法对大肠杆菌进行分离培养，并进行16S rRNA基因PCR鉴定；对鉴定为大肠杆菌的分离菌株运用K-B纸片扩散法进行药敏试验，对分离菌株进行LEE毒力岛基因ler、eaeA PCR检测。结果表明：共分离得到87株大肠杆菌，分离菌株对恩诺沙星、复方新诺明、红霉素、四环素的耐药率分别为44.83%、29.89%、28.74%、27.59%,大于26.00%;对头孢唑啉、阿莫西林、氨苄西林、头孢拉定的耐药率次之，处于10.00%～18.00%之间；对链霉素、头孢氨苄、哌拉西林等12种药物的耐药率小于10.00%。LEE毒力岛基因ler、eaeA的检出率分别为83.91%和29.89%,二者同时检出率为25.29%。说明宁夏地区腹泻绵羔羊源大肠杆菌耐药率较低，携带LEE毒力岛可能是其条件性致病的原因之一。     

肉牛溶血性大肠埃希菌分离鉴定和毒力与耐药基因检测及药敏试验

为了解肉牛溶血性大肠埃希菌毒力基因和耐药基因分布情况及药物敏感性,从河北省围场县采集的健康肉牛鼻腔棉拭子中分离鉴定溶血性大肠埃希菌,采用PCR检测大肠埃希菌的4个毒力基因和12个耐药基因,并采用K-B法进行药敏试验。结果表明,从116份健康的肉牛鼻腔中分离鉴定出23株溶血性大肠埃希菌,分离率为19.8%;毒力基因检测结果显示,6株菌同时携带LEE(Ler、eaeA)毒力基因,携带率26.1%,18株同时携带高致病性毒力基因HPIirp2、fyuA,携带率78.3%,6株同时携带LEE和HPI毒力基因,携带率为26.1%;耐药基因检测结果显示,blaTEM、aadA1耐药基因的携带率最高,为100%;药敏试验结果显示,23株溶血性大肠埃希菌对头孢氨苄、复方新诺明、磺胺间甲氧嘧啶耐药。结果表明,溶血性大肠埃希菌普遍存在于健康肉牛鼻腔中,HPI毒力基因和β-内酰胺类和耐链霉素类耐药基因携带率高。对河北省围场县肉牛溶血性大肠埃希菌毒力基因和耐药基因进行了研究,并进行药物敏感性分析,结果提示溶血性大肠埃希菌对肉牛养殖存在潜在威胁。     

断奶仔猪源小肠结肠炎耶尔森氏菌的分离鉴定及其致病性研究

为研究小肠结肠炎耶尔森氏菌(Y.enterocolitica)在断奶仔猪腹泻发生中可能的致病作用,本研究采集黑龙江地区58份断奶仔猪腹泻粪便样品,经细菌分离鉴定,共分离到18株Y.enterocolitica,命名为Y1~Y18;对其6种毒力基因(ail、yst A、yst B、vir F、yad A和rfb C)进行检测,结果显示,其中10株携带yst B基因,3株携带ail基因,其余5株分离株未检测到任何毒力基因。致病性试验结果显示,这些分离株的致病性存在很大差异,含有ail基因的致病性分离株Y7对小鼠和猪均具有致病性,而Y17分离株则无致病性;含有yst B基因的Y11分离株对小鼠具有致病性对猪无致病性,Y18分离株则对小鼠也无致病性;不含毒力基因的分离株Y9和Y16则可以引起小鼠和猪不同程度发病。各分离株除引起腹泻的程度、持续时间和粪便带菌天数不同以外,在肺、后肢关节液、肠系膜淋巴结和小肠内容物中的分离情况也存在差异。本研究结果表明菌株携带毒力基因的情况与其致病性并不具有明确的相关性,并且初步证明Y.enterocolitica在断奶仔猪腹泻发生过程中起到了一定的作用。     

东北地区仔猪腹泻大肠杆菌EAST1基因调查

大肠杆菌肠聚集性热稳定肠毒素Ⅰ(EAST1)是近年来备受关注的一种肠毒素。为进一步了解EAST1毒力基因在东北地区仔猪腹泻大肠杆菌中的存在情况,以及EAST1毒力基因与其他毒力基因的存在关系进行研究,试验于2010—2013年从东北地区38个规模化猪场共采集断奶仔猪腹泻粪便样品290份,经染色镜检和生化试验鉴定出362株大肠杆菌,应用PCR检测方法对STa、STb、LTa、LTb、paa、AIDA-I、afa E、irp2和EAST1等毒力基因进行检测。结果表明:有286株大肠杆菌携带毒力基因,其中89株(占31.12%)携带EAST1毒力基因,只含有EAST1毒力基因的有49株(占17.13%),同时携带其他毒力基因的有40株(占13.99%)。EAST1毒力基因可以协同F1菌毛、paa基因频繁交叉出现在同一致病菌中,但这些毒力基因编码的毒素之间是否存在协同致病关系尚需进一步研究。     

不同毒力基因型大肠杆菌的毒力测定

为确定不同毒力因子对大肠杆菌毒力的影响,选用7株不同毒力基因型(分别为Stx2e 、LEE 、LEE HPI 、Stx2e HPI 、F18 Stx2e 、F18 Stx2e LEE 和F18 Stx2e ST1 LT1 LEE )的猪源大肠杆菌为材料,以消化道为感染途径,测定了其对30日龄健康ICR小鼠的半数致死量(LD50)。结果表明,以上7株大肠杆菌的LD50分别为1.504×109,5.986×108,3.777×108,1.504×108,5.986×107,3.777×107cfu/mL和1.504×107cfu/mL;确定了不同毒力基因型的大肠杆菌之间存在毒力差异,其中S462234株(F18 Stx2e ST1 LT1 LEE )的毒力最强,S452623株(Stx2e )的毒力最弱,S462234株的毒力是S452623株的100倍。     

奶牛犊牛腹泻源大肠埃希氏菌耐药情况及LEE相关基因的检测

为了明确宁夏地区奶牛犊牛腹泻源性大肠埃希氏菌的分离率、耐药情况和LEE毒力岛eaeA和ler基因的携带情况,采集宁夏5市16个规模化奶牛场1～2月龄犊牛病理性腹泻样本157份,采用微生物学方法培养、VITEK 2 Compact生化鉴定和PCR鉴定,应用PCR方法进行分离株LEE相关基因检测。结果分离鉴定到大肠埃希氏菌62株,对四环素耐药率为100%,对阿莫西林、氟苯尼考、哌拉西林等9种药物的耐药率大于90.00%,对阿米卡星较为敏感,耐药率为11.29%;LEE毒力岛eaeA基因的检出率为11.29%(7/62),ler基因的检出率为79.03%(49/62),二者同时检出率为3.23%(2/62)。结果表明,LEE的存在,可能是某些奶牛场犊牛腹泻大肠埃希氏菌病的原因之一,建议临床治疗犊牛腹泻大肠埃希氏菌病可以首选阿米卡星。     

牦牛产志贺毒素大肠杆菌主要黏附因子相关基因的分子流行病学调查

为了解中国牦牛产志贺毒素的大肠杆菌(Shiga toxin-producing Escherichia coli, STEC)中主要黏附因子的流行情况,采用PCR方法对来自四川甘孜阿坝等地区健康牦牛的70株STEC的eae、saa、iha 3种与黏附相关的毒力基因进行检测,并对部分含有相关黏附因子的阳性分离株的毒力基因进行了克隆及序列分析。结果显示,牦牛STEC中saa、iha的阳性率分别为71.42%(50/70)和78.57%(55/70),无eae基因序列(0/70),saa、iha的测序结果与GenBank上序列的同源性分别为100%和93%~99%。健康牦牛分离的STEC无LEE毒力岛编码eae,其他的一些与黏附相关的主要毒力基因saa、iha的携带率较高。     

应用PCR方法快速检测兔腹泻肠致病性大肠杆菌LEE致病岛

近年来,在我国集约化规模化养兔业发展过程中,断奶后幼兔的腹泻症已成为发病死亡的重要原因之一.国内外研究表明,引起家兔腹泻的大肠杆菌主要为非产肠毒素的致病性大肠杆菌(EPEC)[1].目前,我国对EPEC的鉴别诊断主要依靠菌株的血清型鉴定,但其并不是EPEC诊断和流行病学调查的理想方法,检测LEE毒力岛(通过检测eaeA基因)应为鉴定EPEC的主要依据.现将应用PCR方法快速鉴别兔肠致病性大肠杆菌LEE致病岛的方法介绍如下.     

断奶仔猪源大肠杆菌F18菌毛的分子流行病学研究

利用F18菌毛a因子单克降抗体以及已建立的鉴定F18菌毛及其亚型的双重PCR法,对来自断奶仔猪水肿病和／或腹泻病例的60株VTEC、24株VTEC／ETEC以及24株ETEC的进行了F18菌毛检测,以了解F18ab^＋和F18ac^＋大肠杆菌在江苏省断奶仔猪群的分子流行病学。结果表明：通过F18菌毛a因子单克隆抗体,可检测出52株大肠杆菌为F18^＋,检出率为48．15％;而通过双重PCIL方法,共检测出63株大肠杆菌为F18^＋,检出率为58．33％,其中53株（49．07％）为F18ab^＋10株（92．6％）为F18ac^＋。另外还发现：在VTEC、VTEC／ETEC以及ETEC的菌株之间,这2种F18菌毛亚型的分子流行病学是不同的。在VTEC中,F18ab^＋,菌株37株（61．67％）,未发现F18ac^＋菌株;在VTEC／ETEC中,F18ab^＋菌株15株（62．50%）,F18ac^＋菌株8株（33．33％）;而在ETEC中F18ab^＋菌株只有1株（4．17％）,F18ac^＋菌株只有2株（8.33％）。以上数据表明：④PCR法检测F18菌毛优于单抗法;②F18菌毛是VTEC／ETEC、VTEC的重要致病因子,而在ETEC中则明显低于VTEC／ETEC和VTEC;⑧F18ab^＋菌株一般为SLT-IIe^＋,而F8ac^＋菌株一般为STI^＋。     

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。     

Prevalence of fimbial colonization factors F18ab and F18ac in Escherichia coli isolates from weaned piglets with edema and/or diarrhea in China

F18ab and F18ac are important fimbrial colonization factors of verotoxigenic Escherichia coli (VTEC) and/or enterotoxigenic E. coli (ETEC) in weaned piglets with edema disease and/or diarrhea. To further investigate their prevalence and correlation to pathogenic E. coli, a duplex PCR, using three primers derived from the nucleotide sequence of the F18 major fimbrial subunit gene (fedA), and a direct agglutination test, using a monoclonal antibody specific for the antigenic factor 'a' of F18, were performed. Among 60VTEC, 24VTEC/ETEC and 24 ETEC isolates tested from weaned piglets with edema disease and/or diarrhea in different pig farms in the Jiangsu Province of China, 52 isolates (48.15%) were positive in the direct agglutination test and 63 isolates (58.33%) were positive in the duplex PCR. Among 63 PCR-positive isolates, 53 isolates (49.07%) were F18ab-positive and 10 isolates (9.26%) were F18ac-positive. In addition, the F18ab gene was more frequently detected in VTEC (61.67%) or VTEC/ETEC (62.50%) than in ETEC (4.17% only), while the F18ac gene was more frequently detected in VTEC/ETEC (33.33%) than in ETEC (8.33%) or VTEC (0%). Furthermore, F18ab was more frequently associated with Shiga toxin 2e (Stx2e), whereas F18ac was more frequently associated with enterotoxin ST I. These results suggest that the duplex PCR performed in this experiment is a more reliable method for identification of F18+E. coli, and that F18 is a more important virulence factor of VTEC and VTEC/ETEC.     

Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.     

Occurrence of verotoxin-producing Escherichia coli (VTEC) in fecal swabs from slaughter cattle and sheep--an observation from a meat hygiene view

Fecal samples from 544 beef cattle and 140 sheep were investigated by PCR for verotoxin (VT)-producing Escherichia coli (VTEC) without and with an enrichment step. 6.1% (after enrichment 14%) of cattle samples and 10% (after enrichment 29.2%) of sheep samples were VT-PCR-positive. Moreover, a noticeable age-depending prevalence in cattle was found. Eleven VTEC strains isolated from fecal samples of 5 cattle and 6 sheep were taken for further characterization. None of the strains belonged to serogroup O157. But, as reported previously, we also found in this study strains with virulence genes that are associated with increased pathogenicity. The importance of slaughter hygiene and of bacteriological monitoring of carcass contamination has to be pointed out.     

Detection of the saa gene in verotoxin-producing Escherichia coli from ruminants.

A total of 163 verotoxin-producing Escherichia coli (VTEC) strains isolated from diarrheic and healthy cattle, sheep, and goats were analyzed for the presence of the saa gene by polymerase chain reaction. Seventeen (45.9%) and 5 (29.4%) of the VTEC isolated from healthy cattle and diarrheic calves, respectively, had the saa gene. None of the saa-positive strains carried the eae gene, but 20 of the 22 saa positive were ehxA positive. In contrast with cattle VTEC, none of the VTEC isolated from small ruminants were saa positive. These results show that the saa gene is commonly associated with bovine eae-negative VTEC strains but not with ovine or caprine VTEC strains.     

Morphologic changes in the intestine of swine after infection with verotoxin-producing Escherichia coli

Spontaneous morphologic lesions are described in 12 of 66 pigs submitted for necropsy. All 12 pigs were culture positive for Verotoxin-producing E. coli (VTEC). 10 of them were weaned pigs, one a suckling piglet and one a fattening hog. In 6 cases E. coli serovar 0139:H1 and in one case each the serovars 0139:H40; 0138:H-; 0125ac:H27 and 0154:H- were isolated. From the fecal samples of 2 animals E. coli ONT (O-group non typable):H- were cultured. Macroscopically there were cyanosis, edema of the eye lids, catarrhal enteritis and/or colitis as well as edema of the mesentery, swelling of the mesenteric lymph nodes and congestion of the lung to varying degrees. Histopathologic examination of 5 animals was carried out. In 3 animals atrophy and edema of the villi in the jejunum and ileum were discovered. In one animal an additional infection with corona virus was confirmed electron microscopically. Furthermore there was disseminated necrosis of lymphocytes in Peyer's patches of the small intestine and in secondary follicles of the mesenteric lymph nodes. In one of the animals a hemorrhagic-necrotising ileitis, occurred characterized by necrosis of villi and thrombosis of blood vessels in the mucosa. The highest number of VTEC with seven out of twelve animals was found in weaned pigs in association with the E. coli serovar 0139.     

Replicon typing of F18 fimbriae encoding plasmids of enterotoxigenic and verotoxigenic Escherichia coli strains from porcine postweaning diarrhoea and oedema disease

The presence of fimbrial adhesin F18 is frequently found in enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) strains responsible for diarrhoea and oedema disease of weaned pigs. The F18 adhesin occurs in two antigenic variants: F18ab is characteristic of VTEC while F18ac is more typical for ETEC. F18 encoding plasmids of 17 phenotypically characterized porcine E. coli isolates (10 ETEC, 6 VTEC and 1 ETEC/VTEC) were tested with a DNA probe for F18 fimbrial adhesin and with replicon probes for the RepFIa, RepFIb and for the RepFIc family of basic replicons. In all the cases, the F18 probe hybridized to only one plasmid band of size higher than 42MDa. All F18 plasmids were determined to be unireplicon plasmids belonging to the RepFIc replicon family of the F incompatibility complex. There was no difference between F18ac plasmids of ETEC and F18ab plasmids of VTEC strains in terms of replicon type or subtype. However, the size of F18ab plasmids of the VTEC strains varied between 42 and 98MDa, in contrast to F18ac plasmids of ETEC strains (constantly approximately 98MDa).     

Prevalence of a gene encoding adhesin involved in diffuse adherence among Escherichia coli isolates in pigs with postweaning diarrhea or edema disease.

A total of 604 Escherichia coli strains isolated from weaned pigs with diarrhea or edema disease on 653 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of 5 fimbriae (F4, F5, F6, F18, and F41), 3 heat-stable (STa, STb, and EAST1) and 1 heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e) genes. Forty-five (7.5%) of the 604 E. coli isolates carried the gene for AIDA. Of these 45 isolates, 5 (11.1%) carried EAST1 genes only, 1 (2.2%) carried genes for at least one of the fimbrial adhesins, 12 (26.7%) carried genes for at least one of the toxins, and 27 (60%) carried genes for at least one of the fimbrial adhesins and toxins. Fifty-one percent of strains that carried AIDA genes carried Stx2e genes, and 40% of strains that carried AIDA genes carried F18ab. The isolation rate of enterotoxigenic E. coli strain carrying genes for AIDA was 87%, and the isolation rate of Shiga toxin-producing E. coil strain carrying genes for AIDA was 49%. AIDA may represent an important virulence determinant in pigs with postweaning diarrhea or edema disease.     

Serotypes, virulence genes and intimin types of verotoxin-producing Escherichia coli and enteropathogenic E. coli isolated from healthy dairy goats in Spain

Faecal samples from 222 healthy dairy goats on 12 farms in Spain, as well as bulk tank milk samples of these farms, were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and enteropathogenic E. coli (EPEC). VTEC and EPEC were isolated in 47.7 and 7.7% of the animals, respectively. VTEC were isolated more frequently from adults and replacement animals than from goat kids. In contrast, EPEC were detected more frequently from goat kids than from replacement animals and adults. VTEC or EPEC strains were not detected in the bulk tank milk samples. Although a selective enrichment protocol was used, the serotype O157:H7 was not detected. The most frequent serotypes among the 106 VTEC strains isolated from goats were O5:H-, O76:H19, O126:H8, O146:H21, ONT:H- and ONT:H21. None VTEC strain was eae-positive. The absence of the eae gene in the VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, 16% of VTEC strains isolated from healthy goats belonged to serotypes associated with haemolytic uraemic syndrome in humans. The ehxA gene was detected in 84.9 and 52.9% of the VTEC and EPEC from goats, respectively. The beta1, theta/gamma2 and zeta were the most frequent intimin types among the 17 EPEC strains studied and the most prevalent serotypes of these strains were O156:H25 and O177:H11. Our data show that in Spain healthy goats are an important reservoir of VTEC and EPEC, and a potential source of infection for humans.