【作者】 陈薇蓉；

【导师】 高崧； 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2008， 硕士

【摘要】 禽大肠杆菌病(avianc olibacillosis)是指部分或全部由禽病原性大肠杆菌(avian pathogenic Escherichia coli, APEC)所引起的局部或全身性感染的疾病,包括大肠杆菌性败血症、大肠杆菌肉芽肿(Hjarre氏病)、气囊病(慢性呼吸道病, CRD)、禽蜂窝织炎、肿头综合症、腹膜炎、输卵管炎、滑膜炎、全眼球炎及脐炎/卵黄囊感染。禽大肠杆菌病是2至12周龄鸡和火鸡的一种常见传染病,给养禽业造成了严重的经济损失。大肠杆菌血清型复杂多样,在禽类中最常见的血清型是O1、O2和O78,与其致病相关的毒力因子已被相继报道,如脂多糖复合物、荚膜、黏附素、温度敏感性血凝素、铁摄取系统、外膜蛋白、毒素、AGI-3基因组岛、侵袭素IbeA、Pst系统、大肠杆菌素等。人们在致力于弄清APEC已知毒力因子的同时,也将注意力转移到新的致病基因的分离与鉴定方面。我们在大量禽源大肠杆菌的致病性实验中,发现有相当比例的分离株并不具备已知的毒力因子,这说明这些分离株还存在其他新的毒力因子。寻找和发现新的大肠杆菌致病因子,探讨其致病机理,具有重要的研究意义。1.禽病原性大肠杆菌E058株chp基因突变株的构建及其致病性研究本研究以构建基因突变株的方法,初步鉴定了APEC可能毒力相关片断aes-31的功能,同时结合其生物学特性,为研究该病的致病机理和防制措施奠定了基础。采用抑制差减杂交技术对禽病原性大肠杆菌高致病菌株E058(血清型O2)与低致病菌株E526进行基因组差异片段克隆与分析后检出的32个特异性差异片段中选取aes-31进行进一步的研究,该片段在基因芯片杂交试验中也表现为E058株在体外培养的上调基因。推测aes-31片断很可能与APEC对禽的致病力相关。本研究运用基因重组的方法构建了aes-31的基因等位突变株。经blast搜索,片断aes-31包含两个开放阅读框,chp1和chp2 ,均编码保守假定蛋白。PCR扩增chp1和chp2基因并将其分别插入到pBSK载体的多克隆位点中,再运用基因重组的方法将博来霉素抗性基因和卡那霉素抗性基因分别插入到两个目的基因chp1和chp2中,构建出带博来霉素抗性基因标志的载体pBSK-chp1-Zeo和带卡那霉素抗性基因标志的pBSK-chp2-Kan,通过PCR分别扩增片段chp1-Zeo和chp2-Kan,并电转化入禽病原性大肠杆菌分离株E058中,根据同源重组原理,筛选出E058突变株E058 (Δchp1)和E058 (Δchp2);将片断chp1-Zeo电转化到E058突变株E058 (Δchp2)中,再筛选出E058双突变株E058 (Δchp1Δchp2)。生长曲线测定的结果显示在LB液体培养基中突变株在对数生长期的生长速度均低于亲本株E058。E058 , E058(Δchp1) , E058(Δchp2)和E058 (Δchp1Δchp2)株的半数致死量(LD50)分别为104.5 CFU , 104.8 CFU, 105.1 CFU和105.4 CFU。1日龄雏鸡攻毒实验结果表明E058 (Δchp1)、E058 (Δchp2)、E058 (Δchp1Δchp2)攻毒后血液和各脏器中的的细菌清除率均高于E058株,尤其是双突变株E058 (Δchp1Δchp2)细菌清除率显著高于亲本株E058。攻毒48 h后大肠杆菌病病变比较中可以看出,各个突变株引起大肠杆菌病变的程度有了一定下降。因此,可以初步认定特异性差异片断aes-31在禽大肠杆菌E058的致病和持续感染的过程中起到了一定的作用。2. 1993至2006年禽病原性大肠杆菌分离株的耐药性研究从我国江苏等20个省、市、自治区临诊上具有典型大肠杆菌病病变的病、死家禽中分离到大肠杆菌1351株中随机挑选出不同时期、不同血清型的的分离株203株,按NCCLs推荐的纸片扩散法测定了其对21种抗菌药物的敏感性。结果表明,分离株对强力霉素和四环素的耐药率最高,均为98%,其余依次为磺胺甲基异噁唑、复方新诺明、链霉素、氨苄西林,耐药率在74%-84%之间,对环丙沙星、诺氟沙星、氯霉素、庆大霉素、卡那霉素、丁胺卡那霉素、新霉素、呋喃妥因、呋喃唑酮的耐药率为11%-51%,壮观霉素、头孢哌酮、头孢噻呋的耐药率分别为6%、2%和1%,对多黏菌素B无一例耐药,对本来不敏感的青霉素和林可霉素依旧保持高度耐药。以3-4年为一个时间段,将分离株分为四个时期, 1993-1995年58株,1996-1999年42株,2000-2003年39株,2004-2006年64株。可以看出,禽病原性大肠杆菌对各种药物的耐药性普遍呈现上升的趋势,表现最为明显是喹诺酮类药物,其次是磺胺类。在2000-2003时间段的分离株对多种药物的耐药性达到高峰,之后有所回落。将21种抗生素按照美国国家临床实验室标准委员会方法手册规定的标准分为7个大类,本研究结果显示所有的分离株都对三个以上大类的抗生素有耐药性,均为多重耐药菌株。更多还原

【Abstract】 Colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Escherichia coli (APEC), including septicaemia, granuloma (Hjarre’s disease), air sac disease, chronic respiratory disease (CRD), avian cellulitis, swollen head syndrome, peritonitis, salpingitis, osteomyelitis/synovitis, panophthalmitis, and omphalitis/yolk sac infection. Colibacillosis in mammals is most often a primary enteric disease, whereas colibacillosis in poultry is typically a secondary localized or systemic disease occurring when host defence has been impaired or overwhelmed. Collectively, infections caused by Escherichia coli (E. coli) are responsible for significant economic losses to the poultry industry in many parts of the world. The pioneer serological surveys indicate that the majority of avian septicaemic E. coli strains belong to a limited number of O serogroups (O1, O2, and O78). Analysis of the involvement of these potential virulence factors using experimental models of infection just began during the past few years. Nevertheless, the actual roles of the known virulence factors are not fully elucidated and certain steps of the infection process have not been related to previously identified factors. Furthermore, the mechanisms by which avian pathogenic E. coli cause infection are largely unknown.It has been reported that APEC has many virulence factors. Researchers are working hard on making clear the virulence factors, meanwhile, they pay attention to isolation and identification of the new virulence factors. Because many isolated strains don’t have the virulence factors ever known, according to the pathogenic experiment of the APEC. The above-mentioned information indicates that the isolated strains have other virulence factors. It is significant to acquire new virulence factors of the APEC.1. Construction of chp genes isogenic mutants of APEC E058 and evaluation of their pathogenicity in chickensIn our previous study,some unique DNA fragments associated with APEC E058 are obtained using suppression subtractive hybridization (SSH) in which the genome of avian highly pathogenic E. coli strain E058 serves as a tester and the genome of avian low pathogenic E. coli strain E526 serves as a driver. Among these unique DNA fragments, aes-31 is selected for future study since it is found to be up-regulated in APEC E058 compared to APEC E526 in chicken sera stationary cultures when analyzed by DNA microarray. In this study, we constructed the aes-31 gene isogenic mutants as follows. The 1200 bp fragment of aes-31 consists of two opening reading frames (ORF),the 753-bp gene of chp1 encoding a conserved hypothetical protein in E. coli K-12 and the 441-bp gene of chp2, encoding another conserved hypothetical protein in E. coli K-12 characterized by blast searching in GenBank. Chp1 and chp2 genes were generated by PCR and cloned into pBSK vector respectively. Zeocin resistant gene and kanamycin resistant gene were then cloned into the target gene chp1 and chp2, respectively, the resultant vectors containing the recombinant genes were named pBSK-chp1-Zeo and pBSK-chp2-Kan respectively. Mutant derivatives of strain E058 were generated by allelic replacement with electroporation technique and named E058 (Δchp1)、E058 (Δchp2) and E058 (Δchp1Δchp2).The growth curve showed that the mutant strains are slowly grown than the parental strain APEC E058 in logarithmic phase when they grew in LB broth. The 50% lethal dose (LD50) of E058, E058 (Δchp1), E058 (Δchp2) and E058 (Δchp1Δchp2) in commercial 1-day-old chickens challenged experimently via intratrachea were 104.5 CFU , 104.8 CFU , 105.1 CFU和105.4 CFU , respectively. In the chicken challenge model, the mutants were observed to have higher clearence rate of challenged strain than that of the parental strain, especially the double mutant strain E058 (Δchp1Δchp2). These results showed that the unique DNA fragment aes-31 contributes to the the virulence and persistance of APEC E058 in chickens.2. Prevalence of antimicrobial resistance of APEC isolates originated from 1993 to 2006In this study, 203 APEC isolates originated from 1993 to 2006 were characterized for antimicrobial susceptibilities. 203 APEC isolates include 58 collected during 1993-1995, 42 obtained during 1996-1999, 39 isolated during 2000-2003 and 64 recoverd during 2004-2006. Among the 21 antimicrobial agents tested, resistance of 203 APEC isolates to spectinomycin, tetracycline was the most frequent , and the resistance rate of all tested isolates for each of the two agents was 98% (199/203). None of the 203 isolates have antimicrobial resistance to polymycin B. High resistance rates were also observed in sulfamethoxazole (84%) , trimethoprim-sulfamethoxazole (78%), streptomycin (76%) and ampicillin (74%) . The results showed that a trend of increasing and broadening their antimicrobial resistance in tested isolates was observed, the nearer the isolates were isolated, the stronger and wider antimicrobial resistance were presented. It was interesting that all tested isolates collected during 2002-2003 experienced the strongest and widest antimicrobial resistance among the 14 years, especially for quinolones and sulfanilamide group agents. Our findings also showed that all isolates were multi-resistant.更多还原