鸡源大肠埃希菌抗“O”血清型鉴定及药敏试验研究

大肠埃希菌耐药性的增加及大肠埃希菌血清型众多,加大了药物防治大肠杆菌引发病症的难度。为了减少耐药菌株的产生及养殖者的经济损失,笔者对2011年4月～2012年7月门诊分离的鸡源大肠埃希菌进行抗"O"血清型鉴定及药敏试验,并与2005年11月～2007年4月门诊病死鸡疑似大肠埃希氏菌病例的相关统计数据进行了对比。     

ENTERITIS IN FOALS INDUCED BY ROTAVIRUS AND ENTEROTOXIGENIC ESCHERICHIA COLI

Colostrum-deprived, colostrum-fed or suckling foals were orally inoculated with foal rotavirus and enterotoxigenic Escherichia coli derived from a calf. Neither agent given alone caused diarrhoea in foals aged 1 or 2 days, although with rotavirus, 2 of the 3 inoculated foals became depressed 3 days after inoculation and all 3 were excreting rotavirus in the faeces. Inoculation of both agents induced diarrhoea in colostrum-deprived, colostrum-fed or suckling foals aged up to 16 days. There was an apparent age-related resistance to diarrhoea which developed between 2 and 3 weeks of age. It was related to failure of rotavirus to establish apparent infection in older foals and was independent of preinoculation maternal antibody.     

犬抗细粒棘球绦虫疫苗候选蛋白EgM9的原核表达及纯化

把含有EgM9基因的质粒pET-41b转化至大肠杆菌BL-21中,IPTG诱导重组质粒的表达,亲和层析纯化表达产物,用SDS-PAGE和Western blot进行分析鉴定,显示分离纯化的EgM9和GST蛋白纯度较高,1L的培养物约可以得到5mg纯化蛋白,分子量为60kDa。EgM9免疫后的血清与成熟虫体蛋白有特异性反应。融合蛋白获得高效表达,并成功纯化,初步实验证明EgM9具有良好的免疫原性,可进一步用于疫苗的免疫实验。     

镰形扇头蜱肌钙蛋白Ⅰ基因的克隆及其在大肠杆菌中的表达

目的克隆镰形扇头蜱肌钙蛋白Ⅰ(TnI)的cDNA并进行表达及鉴定。方法用RT-PCR方法从镰形扇头蜱总RNA中克隆编码肌钙蛋白Ⅰ的cDNA片段,将该cDNA连接到pET-32a( )表达载体,并转入宿主菌BL21(DE3),经IPTG诱导表达,并对表达产物进行Western blot分析。结果获得镰形扇头蜱的eDNA,并在大肠杆菌中以包含体的形式高效表达,Westem blot分析表明,抗TnI重组蛋白兔血清可以识别蜱体内的TnI,并在22 kDa和36 kDa之间有两条带。结论蜱体内可能同时存在两个大小不等的TnI,为进一步的研究打下基础。     

ESCHERICHIA COLI AND ROTAVIRUS INFECTIONS IN FOUR-WEEKOLD GNOTOBIOTIC PIGLETS FED MILK OR DRY FOOD

A haemolytic enteropathogenic E. coli (WG) and pig rotavirus were isolated from a field case of postweaning diarrhoea in pigs. Four-week-old gnotobiotic piglets fed on milk diet were found to be extremely susceptible to infection with WG E. coli . Piglets were less susceptible to the infection immediately after the diet was changed from milk to dry food, and were almost completely resistant 4 days after the change to dry food. There was no difference in the clinical response to infection with WG E. coli when the piglets were fed either a high energy diet or low energy diet. Four-week-old piglets fed milk showed mild symptoms of diarrhoea when inoculated with pig rotavirus. Symptoms were more severe when piglets were inoculated immediately after the change from milk to dry food. Piglets inoculated 4 days after the change of diet showed no symptoms of diarrhoea at all. Under the conditions of these experiments the enteropathogenic E. col produced a more serious disease than did pig rotavirus. Infection of 4-week-old gnotobiotic piglets with both agents given sequentially produced a diarrhoeal disease that was more severe than that produced by each agent separately.     

大肠杆菌不耐热性肠毒素LT_B基因的克隆及其核苷酸序列分析

用内切酶ＳａｃⅠ和ＨｉｎｄⅢ双酶切大肠杆菌质粒ｐＥＷＤ２９９，回收５０５ｂｐ的ＬＴＢ基因片段，再将载体ｐＵＣ１８用ＳａｃⅠ和ＨｉｎｄⅢ双酶切，最后将ｐＵＣ１８ＤＮＡ与５０５ｂｐ的ＬＴＢＤＮＡ进行连接，转化至受体菌ＤＨ５α中，经ＳａｃⅠ／ＨｉｎｄⅢ、ＥｃｏＲⅠ／ＨｉｎｄⅢ酶切反应鉴定重组子，得到了理想重组子质粒ｐＸＬＴ１。再将ｐＸＬＴ１进行ＥｃｏＲⅠ酶切、大肠杆菌聚合酶ⅠＫｌｅｎｏｗ大片段补平、ＨｉｎｄⅢ酶切处理，然后与用ＨｉｃⅡ和ＨｉｎｄⅢ酶切的ｐＵＣ１８连接，转化至受体菌ＤＨ５α中，经ＸｂａⅠ／ＨｉｎｄⅢ酶切反应鉴定重组子，得到了理想重组子质粒ｐＸＬＴ１－１。将质粒ｐＸＬＴ１－１进行核苷酸序列分析，确定了插入的ＬＴＢ基因与载体的连接向位及其全部核苷酸序列     

病原性大肠杆菌人工感染鸡外膜蛋白抗体和脂多糖抗体的测定

以提纯的外膜蛋白（Ｏuter membuane puoteins,ＯＭＰs）作包被抗原,应用酶联免疫吸附试验（Ｅnzyme-linked immunosorbant assay,ＥＬＩＳＡ）,测定了禽病原性大肠杆菌Ｏ １８－５６６、Ｏ７８－１６６分离株人工感染鸡的ＯＭＰs抗体。上述分离株２次攻毒鸡的ＯＭＰs抗体高峰期在攻毒后的３～５周;同时,我们以间接血凝试验（Ｉndirict hemagglutination tist,ＩＨＴ）测定了其脂多糖（Ｌipopolysacchauide,ＬＰＳ）抗体,该抗     

鸭致病性大肠杆菌的分离鉴定及其生物学特性分析

鸭致病性大肠杆菌病是危害养鸭业的最为重要的细菌性传染病之一。本研究分离鉴定了65株鸭致病性大肠杆菌,并对其O血清型、耐药谱以及毒力相关基因进行了检测。结果表明：大肠杆菌O78,O2和O1为主要流行血清型,各占分离株的75％、11％和5％。对15种常规抗菌素或药物的药敏试验结果表明：100％的分离株对利福平和红霉素耐药;94％以上的分离株对头孢噻吩等10种抗菌素或药物耐受;70％以上的分离株对氯霉素、卡那霉素、庆大霉素、壮观霉素和头孢噻肟敏感。对14种可能的大肠杆菌毒力相关基因的检测结果表明：ompA、pfs和luxS三种基因高度保守,在分离菌株中的检出率为100％;iss、iuCD、tsh、fimC、cvaC和iroC基因的检出率达到72％以上,为重要的鸭致病性大肠杆菌毒力相关基因。     

鸡毒支原体抗原在大肠杆菌中的表达

利用表达载体λgt11构建鸡毒支原体（MG）S6株基因文库。根据载体中lacZ基因插入失活的原理,重组子中大约69％为重组噬菌体。应用MG纯化抗体筛选文库,得到179个阳性克隆,不足重组噬菌体的1％。随机选择一个阳性克隆,收集其蛋白进行Western免疫印迹,检测到－38kD蛋白,表明在大肠杆菌中能全盛具有免疫学活性的MG蛋白,为MG的特异性血清学诊断提供良好工具。     

我国部分地区禽病原性大肠杆菌的分离与鉴定

从江苏、广东、河南、新疆、四川、北京、黑龙江等１８个省、市、自治区临 有典型大肠杆菌病病变的病禽１０５１羽采集病料,分离培养和鉴定出５９５株大肠杆菌。经Ｏ血清型测定,除１４４株未能定型、１１株自凝外,共出４４０个分离株的Ｏ血清型。这些 ５０个血清型,但以Ｏ１８、Ｏ７８、Ｏ２、Ｏ８８、Ｏ１１、Ｏ２６、Ｏ４、为、Ｏ１２７６、Ｏ１３１等１０个血清型为主,这些分离株覆盖了６０个血清型,但以血Ｏ１８、Ｏ７８     

VACCINATION OF PIGLETS AGAINST ESCHERICHIA COLI ENTERITIS

An immunisation procedure involving intraperitoneal followed by oral administration of a killed Escherichia coli vaccine was used to reduce losses associated with colibacillosis in young pigs reared under commercial conditions. Mortality was reduced in both the preweaning and postweaning period. The mortality rate among vaccinated piglets was half that of unvaccinated controls and by 3 weeks after weaning the total weight of pigs produced was 33% higher in the vaccinated group than in the control group.     

大肠杆菌热敏肠毒素B亚基的原核表达及生物学活性分析

从大肠杆菌K88（LT＋,ST＋）菌株中扩增热敏肠毒素B亚基基因（ltb）,得到454 bp片段,克隆至pMD18-T载体后,与pET32a（＋）定向连接,并转化入大肠杆菌BL21中,用IPTG 30℃诱导表达重组LTB蛋白。SDS-PAGE显示,重组蛋白分子量约为34 kDa,超声波裂解后,表达产物主要以包涵体形式存在。经鉴定,纯化复性后的LTB蛋白保留部分与GM1结合的生物学活性。     

鸡大肠杆菌病研究Ⅰ．鸡E．Coli人工感染模型的建立及致病力评价

本文对分离自典型大肠杆菌病变和粪便的４５株Ｅ．Ｃｏｌｉ通过４日龄雏鸡人工感染试验建立了大肠杆菌病的人工感染模型，并对菌株的致病性进行评价。结果表明：４日龄雏鸡颈部皮下接种０．２ｍｌ菌液，接种后，前６５ｈ内雏鸡死亡率≥６０％的菌株为强致病力菌株；死亡率＜６０％，或出现典型大肠杆菌病变为中等致病力菌株；试验期不引起雏鸡死亡也无病变者为无致病力大肠杆菌。试验的１３株分离自粪便的苗株，２株为中等致病力，１１株为无致病力；３２株分离自典型病变的菌，９株为强致病力菌，１５株为中等致病力，８株为无致病力。     

乙型脑炎病毒基因组cDNA克隆在宿主菌中不稳定因子的初步研究

为了探求乙型脑炎病毒(Japanese encephalitis virus,JEV)基因组cDNA克隆在宿主菌中不稳定遗传的影响因子,本研究以猪源JEV HEN0701株基因组片段1~2913 bp为研究对象,分析基因组原核启动子和相关毒力基因在不稳定遗传中的作用。在5’端,分析片段中潜在的原核启动子序列,通过PCR扩增得到不同长度的cDNA片段;在3’端,通过内切酶酶切截断基因组prM蛋白或E蛋白编码区,获得3’末端不同的cDNA片段。将各片段克隆入pCR-BluntII-TOPO载体,转化大肠杆菌,于37℃条件下培养。结果显示,片段73~2913 bp、97~2913 bp、355~2913 bp及1~933 bp、1~1275 bp能够在转化菌中稳定遗传;含有其他片段39~2913 bp、54~2913 bp及1~1800 bp、1~1971 bp的重组细菌不能在37℃条件下生长;片段1~1600 bp虽然能够在转化菌中传代,但是出现不规律突变。以上结果表明:软件所预测各原核启动子片段中,基因组73~118 bp区域与cDNA克隆稳定性有关,且上游的54 bp至73 bp的序列对该区域启动子存在影响;在JEV基因组1275 bp至1600 bp之间可能存在毒性蛋白编码序列。