广西猪水肿病志贺样毒素大肠杆菌的分离与鉴定

1材料与方法1.1材料样品采自桂林、玉林、南宁等地患水肿病仔猪器官组织样品。1.2培养基及参考菌株按常规方法制备麦康凯琼脂培养基、伊红-美蓝琼脂培养基和普通琼脂培养基;生化鉴定培养基购自杭州天和生物技术有限公司。参考菌株:DH5α为本实验室保藏菌株。1.3试剂Taq DNA聚合     

T-2毒素的毒性作用及其诱导细胞凋亡机制概述

T-2毒素是一种毒性作用很强的霉菌毒素,是以镰刀菌属为主要产毒菌株所产生的一种A类单端孢霉烯族毒素,在多种谷物中的污染水平较高,通过食物摄入后在人类和动物机体内产生一系列毒性作用,严重威胁人类和动物的健康。论文从T-2毒素的理化性质、产毒菌株、毒性作用及对细胞凋亡的作用机制进行了简述,重点介绍了T-2毒素在免疫系统、消化系统和肝脏毒性、神经和皮肤毒性、血液毒性、生殖毒性方面的研究进展,以及T-2毒素通过线粒体信号通路介导细胞凋亡机制的研究进展,为T-2毒素的深入研究提供参考。     

贵州猪、牛志贺样毒素大肠杆菌的检出与鉴定

以两对全盛的寡核苷酸引物,通过聚合酶链式反应,检测158份猪和牛腹泻粪便分离物,从8份猪源和3份牛源分离物中扩增出大肠杆菌志贺样毒素基因片段,其生化试验符合大肠杆菌特征,经Vero细胞检测均产生志贺样毒素,血清学试验证实分属不同的血清型,与志贺样毒素大肠杆菌血清型范围相符。     

川西北牦牛肉中产志贺毒素大肠杆菌的分离鉴定及stx2基因的序列分析

为了解中国川西北牦牛肉中产志贺毒素大肠杆菌(STEC)的携带情况及stx2的亚型和特征,试验将采集的204份川西北牦牛肉样品(各25g)增菌培养后,每份挑取5个可疑菌落,采用stx1、stx2双重PCR方法检测STEC,对分离株中的stx2分型并克隆测定stx2编码区序列。结果显示,在204份样品中分离出8株STEC,平均分离率为3.9%(8/204);存在4个不同的O血清型,分别为O38(4)、O50(1)、O74(2)、O150(1);在6株含有stx2的菌株中,其中有2株为stx2a型、4株为stx2c型。结果表明牦牛源分离株氨基酸序列与人源和牛源菌株同源性较高;由stx2A、B亚基的氨基酸序列系统进化树可知,牦牛源分离株与人源、牛源菌株聚为一支,表明它们之间遗传距离相对较近,牦牛源stx2各自分布在自己的小分支中,表明牦牛源STEC stx2与人源和牛源stx2相比,尽管亲缘关系较近,但仍存在一定程度的差异。     

副猪嗜血杆菌细胞致死膨胀毒素的细胞毒性研究

本研究旨在原核表达副猪嗜血杆菌细胞致死膨胀毒素(Cytolethal distending toxin,CDT),并作用于猪髋动脉内皮细胞(Pig iliac endothelial cells,PIEC),以研究其细胞毒性.根据本实验室完成的副猪嗜血杆菌SH 0165株(血清5型)全基因组序列,针对cdtA、cdtB和cdtC基因序列设计引物,扩增的基因片段大小分别约为681、834和531 bp.将靶基因克隆到原核表达载体pET28a中,再转化到E.coliBL21( DE3),IPTG诱导表达3h,SDSPAGE和Western blot检测证实表达产物以包涵体形式存在,大小分别约36、34和28 ku.通过体外重构毒素与PIEC细胞作用3h,继续培养72 h观察细胞形态学变化.结果表明,CdtABC全毒素致PIEC细胞膨胀、空泡形成、细胞凋亡等,而其他试验组变化不显著.结果提示,CDT毒素可能在细菌感染与致病中发挥着重要作用.     

伏马毒素B1的细胞毒性研究进展

伏马毒素主要污染玉米、小麦、高粱、水稻等粮食作物,通过食物链对人和畜禽健康造成巨大威胁。迄今为止,伏马毒素已被发现至少有15种,其中伏马毒素B1(FB1)的毒性最强,污染最广泛,也是目前研究最多的类型。论文就FB1对不同动物的肝细胞、肾细胞、肠道上皮细胞、免疫细胞、神经细胞、生殖细胞等的毒性作用进行综述,以期为FB1体内毒性评价及其毒性机制研究提供参考。     

猪源性水肿病大肠杆菌的分离鉴定及耐药性分析

仔猪水肿病又名猪胃肠水肿病.猪水肿病是由产类志贺毒素SLT-2e的大肠杆菌引起的断奶仔猪以神经症状和全身水肿,特别是胃大弯、肠系膜及脑部水肿为特征的一类传染病.该病主要发生在断奶后1～2周的仔猪,尤其在发育良好的仔猪群,造成很大的经济损失,是断奶仔猪危害较严重的疾病.本试验对仔猪水肿病大肠杆菌进行了体外抑菌试验,为研究仔猪水肿病的耐药性做了铺垫,并为进一步消除耐药性打下了基础.     

T-2毒素毒性作用研究进展

T-2是A类单端孢霉烯族化合物中毒性最强的一种,对动物机体具有较强的危害,可造成多个系统出现中毒效应。作者主要对T-2毒素对动物的广泛毒性作用进行论述,且对下一步的研究方向进行了展望。     

引致猪水肿病毒素的抗体检测

猪水肿病（ＥＤ）是断奶仔猪常见的一种传染病，与类志贺氏毒素Ⅱ变种（ＳＬＴ－Ⅱｖ）密切相关。本试验用多粘菌素Ｂ从基因工程菌大肠杆菌ＴＢ１中提取ＳＬＴ－Ⅱｖ，作中和试验检测来自江苏等地２６７份屠宰猪血清，发现９份血清有ＳＬＴ－Ⅱｖ的抗体，滴度由１∶４～１∶２５６不等，显示致猪水肿病毒素在猪群的实际存在     

牛源产志贺毒素大肠杆菌分离株的毒力基因分布和遗传进化分析

为了探讨牛源产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）分离株在毒力基因分布和遗传进化方面与人源EHEC O157菌株之间的关系,本试验选择收集来自江苏某奶牛场的STEC菌株18株以及人源、羊源、猪源、禽源STEC参考菌株9株,参照美国疾病预防控制中心PulseNet推荐的方法,运用XbaⅠ酶进行酶切并完成脉冲肠凝胶电泳（PFGE）分型和聚类分析;同时对部分STEC菌株进行毒力基因检测。结果表明,经毒力基因检测,不同来源的O157菌株毒力基因分布不尽相同,其中牛源STEC O157与参考株EHEC O157∶H7（EDL933W）的基因排谱最为相近;牛源STEC O18和O26的基因排谱与参考株EHEC O157∶H7（EDL933W）类似,但存在部分基因的缺失。对27株不同来源的STEC分离株进行PFGE,产生了22种不同的酶切图谱。总体来看,不同来源的STEC Dice相似性系数在72%~100%之间。牛源O157分离株与猪源及禽源O157菌株的相似度偏低,而与两株人源O157分离株的相似度偏高,Dice相似性系数在83%~95%之间,牛源O26（克隆群Ⅶ、Ⅷ）与人源O157的相似性系数 > 82%。显然,从牛群中分离到的部分STEC菌株与人源EHEC O157具有较近的遗传进化关系。     

Prevalence,O-genotype and Shiga toxin (Stx) 2 subtype of Stx-producing Escherichia coli strains isolated from Argentinean beef cattle

The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin (Stx) 2 gene-subtype of Stx-producing Escherichia coli (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. stx genes were detected in 90 (32%) out of the 283 rectal swabs by stx gene-specific PCR assay. The positive cases were 13 with stx1, 58 with stx2, and 19 with both stx1 and stx2. Among 90 stx gene-positive samples, 45 STEC strains were isolated, which included 3 stx1, 34 stx2, and eight stx1 and stx2 genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various stx2 gene-subtypes were identified in 42 STEC strains: 13 positive cases for stx2a, 11 for stx2c, 3 for stx2g, 10 for stx2a and stx2d, 4 for stx2a and stx2c, and 1 for stx2b, stx2c and stx2g. efaI gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that stx2a and stx2c were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.     

广东鸡致病性大肠杆菌对氟喹诺酮类药物的敏感性分析

以广东省21个地级市为单位,从鸡场病死鸡分离细菌,选取经生化和动物试验证明的鸡致病性大肠杆菌共115株,采用药敏纸片法测定其对氟喹诺酮类药物的敏感性.结果显示,72.2 %的试验菌对氟喹诺酮类药物产生抗药性;氟喹诺酮类药物之间交叉耐药严重,恩诺沙星与氟罗沙星交叉耐药率高达99%.     

猪源大肠杆菌质粒和染色体介导的喹诺酮类药的耐药机制

采用微量肉汤稀释法对31株猪源大肠杆菌进行6种喹诺酮类药物的敏感性测定,聚合酶链式反应检测质粒介导的喹诺酮类耐药（PMQR）基因qnr、qepA和aac（6′）-Ib-cr,并分析PMQR基因阳性菌株染色体gyrA、gyrB、parC、parE基因的喹诺酮耐药决定突变区（QRDRs）突变。结果显示,31株猪源大肠杆菌对兽医临床常用的氟喹诺酮类药物均呈现耐药。在31株猪源肠杆菌中共检测到2株携带qnrB10和4株携带qnrS1基因的大肠杆菌,未检测到qnrA、qepA和aac（6′）-Ib-cr。在PMQR阳性菌株gyrA基因的QRDRs中,低耐药菌株的gyrA基因出现83位S→W突变,高耐药菌株的gyrA基因同时出现83位S→L和87位D→N突变。而在parC基因的QRDRs中,大部分耐药菌株出现80位S→I突变,1株耐药菌株出现45位V→L突变。gyrB和parE基因的QRDRs未检测到突变。结果表明,本地区猪源大肠杆菌对兽医临床常用的氟喹诺酮类药物耐药严重,PMQR的出现和QRDRs的点突变可同时协同贡献对喹诺酮类耐药,而PMQR的出现加速了喹诺酮类耐药基因的快速传播。     

Molecular characteristics of ESBL-producing Escherichia coli isolated from chickens with colibacillosis

BackgroundAvian pathogenic Escherichia coli (APEC) causes colibacillosis, resulting in significant economic losses in the poultry industry.ObjectivesIn this study, the molecular characteristics of two extended-spectrum beta-lactamase (ESBL)-producing APEC isolates were compared with previously reported ESBL-producing E. coli isolates.MethodsThe molecular characteristics of E. coli isolates and the genetic environments of the ESBL genes were investigated using whole genome sequencing.ResultsThe two ESBL-producing APEC were classified into the phylogenetic groups C and B1 and ST410 and ST162, respectively. Moreover, the ESBL genes of the two isolates were harbored in different Inc plasmids. The EC1809182 strain, harboring the bla_CTX-M-55 gene on the plasmid, exhibited extensive homology to IncFIB (98.4%) and IncFIC(FII) (95.8%). The EC1809191 strain, harboring the bla_CTX-M-1 gene, was homologous to IncI1-I (Gamma) (99.3%). All chromosomes carried the multidrug transporter, mdf(A) gene. Mobile genetic elements, adjacent to CTX-M genes, facilitated the dissemination of genes in the two isolates, analogous to other ESBL-producing E. coli isolates.ConclusionsThis study clarifies the transmission dynamics of CTX-M genes and supports strengthened surveillance to prevent the transmission of the antimicrobial-resistant genes to humans via the food chain.     

Characterization of Escherichia coli strains isolated from pigs with edema disease.

Escherichia coli strains belonging to serogroups O 138 and O 139 isolated from pigs with edema disease, were characterized with respect to the presence of genes encoding Shiga-like toxin I, Shiga-like toxin II and Shiga-like toxin IIv (SLT I, SLT II and SLT IIv). Genes coding for the heat-stable and heat-labile enterotoxins (ST I and LT I) were also detected. Plasmid profiling, restriction enzyme digestion of total DNA, and ribotyping were performed for further characterization of the strains. The oligonucleotide probes applied in this study appeared to be useful tools for detecting genes coding cytotoxins and enterotoxins. DNA from 12 of 16 strains hybridized with two SLT II probes, and DNA from two SLT IIv encoding strains also hybridized with the ST I probe. DNA from one SLT IIv negative strain hybridized with the LT I probe. The results from plasmid profiling, restriction enzyme digestion, and ribotyping were compared with serogrouping in attempts to distinguish between the different E. coli edema disease isolates. Fourteen different plasmid profiles were identified, and as restriction patterns barely did, and ribotyping patterns did not, reveal any information useful for differentiation of the strains beyond serogroup level, plasmid profiling seemed to be the most suitable method for discrimination between the edema disease strains investigated here.     

Characterization of Escherichia coli isolated from healthy dogs

Five month old dogs from a Midwestern research kennel occasionally developed bloody diarrhea after shipment to other facilities. As previous diagnostic efforts failed to reveal any potential pathogens in feces from normal and diarrheic dogs, Escherichia coli was investigated for select virulence properties that may contribute to the occurrence of bloody diarrhea. Fecal swabs from 52 healthy dogs were examined for E. coli. Two hundred and sixty E. coli-like colonies were screened by PCR for the attaching and effacing (eae) gene, Shiga toxin (stx) genes, and the heat-stable enterotoxin type A (sta) gene. One hundred forty two of the 260 E. coli-like colonies (54.6%) from 43 dogs were eae or sta positive; and 60 of the eae and/or sta positive isolates were examined further. Among the 60 isolates, 23 (38.3%) possessed the eae gene, 32 (53.3%) possessed the sta gene, and five (8.3%) possessed both eae and sta genes (eae⁺/sta⁺). Of the 60 isolates, six sta⁺ and one eae⁺/sta⁺ isolates were hemolytic. When examined in the suckling mouse assay, five of six sta⁺ isolates and three of four eae⁺/sta⁺ isolates gave gut-to-remaining carcass ratios ≥0.083, indicating expression of heat-stable enterotoxin. These enterotoxin-producing isolates belonged to serogroups O42, O170, and O-negative.     

Shiga toxin 2eB‐transgenic lettuce vaccine is effective in protecting weaned piglets from edema disease caused by Shiga toxin‐producing Escherichia coli infection

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB‐transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB‐lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB‐lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB‐lettuce is a promising first oral vaccine candidate against ED.     

Drug resistance in strains of Escherichia coli isolated from the intestinal tract of pigs in Norway.

Faecal samples from 95 healthy pigs and samples of jejunal content from 85 piglets suffering from colienterotoxaemia were tested for the presence of drug resistant E. coli strains. Practically all pigs in both groups harboured E. coli strains resistant to one or more of the 6 antibiotics/chemotherapeutic agents tested (Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin, chloramphenicol). Almost 100% of healthy and approx. 90% of diseased pigs harboured strains resistant to Oxytetracycline, streptomycin and sulphaisodimidin. Pigs with strains resistant to neomycin, ampicillin and chloramphenicol were less frequently found. The predominant coliform flora consisted of E. coli strains” resistant to Oxytetracycline, streptomycin and sulphaisodimidin in 71% to 81% of diseased pigs and in 47% to 69% of the healthy pigs. In diseased pigs ¾ of the animals had a coliform flora dominated by neomycinresistant E. coli strains.Of the 721 resistant E. coli strains isolated from healthy pigs, 11% were single resistant while the corresponding figure for the 518 resistant strains isolated from diseased pigs was 6%. Thus 89% and 94% of strains showed simultaneous resistance to 2 or more antibiotics. E. coli strains resistant to 3 or more drugs were found in approx. 60% and 70% of the isolates from healthy and diseased animals, respectively. Oxytetracycline/streptomycin/sulphaisodimidin resistance was most commonly found, approx. 22% and 38% of the strains from healthy and diseased pigs, respectively, showing this resistance pattern.Transmission of drug resistance which was examined in E. coli strains originating from the diseased pigs was demonstrated in approx. 76% of the isolates. The incidence of drug resistance transfer in single, double, triple and quadruple resistant strains was 11%, 68%, 97% and 98%, respectively.     

猪和野生动物源大肠杆菌及沙门菌中磺胺类药物耐药基因的检测

对从四川省10个规模化猪场和16种野生动物的粪样中分离鉴定出的67株大肠杆菌、57株沙门菌进行了药敏试验。结果，猪源和野生动物源分离菌对磺胺类药物的耐药率分别为72．0％（72／100）和29．2％（7／24）。根据GenBank中登录的序列，设计了3对特异性引物，对磺胺类药物的耐药基因Sul1、Sul2、Sul3进行了三重PCR检测。在这124株细菌中，Sul1基因的检出率最高，为47．6％（59／124），Sul2基因的检出率为17．7％（22／124），Sul3基因的检出率为18．5％（23／124）。药敏试验结果与基因检测结果的符合率为89．9％。