致雏肉鸡“黑腺胃

发布时间：2018-06-12 07:48

本文选题：禽致病性大肠杆菌 + 分离鉴定　；参考：《扬州大学》2016年博士论文

【摘要】：禽致病性大肠杆菌(avian pathogenic Escherichia coli, APEC)能引起禽类局部或全身性感染。全身感染即大肠杆菌型败血症,主要特征是大肠杆菌存在于血液中,其发展阶段包括急性败血症、亚急性多发性浆膜炎和慢性肉芽肿性炎症。局部感染性禽大肠杆菌病包括大肠杆菌型脐炎／卵黄囊感染、大肠杆菌型蜂窝织炎、肿头综合征、生殖道感染、大肠杆菌型输卵管炎／腹膜炎和大肠杆菌型睾丸炎／附睾炎等。大量国内外研究表明,因地理区域不同,大肠杆菌血清型呈现多样性,但最常见的为O1、02、O35、O78。某些感染的暴发病例是由不常见的血清型造成的,如O111血清型分离株可造成产蛋母鸡发生多脏器的浆膜炎、败血症。还有一些致病性大肠杆菌菌株血清型不明确或者不能分型。近年来,在江苏、山东、安徽、河南等省规模化肉鸡养殖场频繁出现一种雏肉鸡“黑腺胃病”(暂命名),发病区域分散,发病日龄早,主要集中在3-5日龄,有时在7-8日龄出现,死淘率3%～12%。商品肉鸡出雏时外观健康,死亡前几无可见临诊症状。剖检病理变化以腺胃呈灰黑色为主(发黑程度随病情轻重而呈浅灰色或灰色,严重时呈黑色)、病程稍长者,可见心包炎、肝周炎、气囊炎。迄今,具有腺胃变黑变化的肉雏鸡疾病尚未见报道。本研究旨在对临床以腺胃发黑为特征的病死肉雏鸡进行病原的分离鉴定,人工感染复制肉雏鸡“黑腺胃病”,探明所分离的致病性大肠杆菌血清型、致病性、种系发生群、毒力基因型；选择从同一病死个体腺胃分离的APEC 4d/9-1 O142菌株和APEC 4d/9-1 O142心血分离株进行毒力基因和模型动物的致病性比较,通过DNA芯片技术对这两个分离株在SPF鸡体内和体外的基因表达谱进行分析,筛选APEC腺胃分离株潜在的毒力基因；选择表达差异基因进行缺失株的构建,评价基因缺失株与致病性的关系,最终为弄清APEC引发雏肉鸡“黑腺胃病”的发病机制和防制措施提供依据。1.致雏肉鸡“黑腺胃病”致病性大肠杆菌的分离鉴定本试验对江苏省某大型肉鸡养殖场雏肉鸡“黑腺胃病”病例进行了病原的分离鉴定,同时对该养殖场不同日龄死亡鸡胚、饲料、出雏器和鸡舍空气进行大肠杆菌的分离鉴定。共分离获得大肠杆菌96株,除5株未能定型外,共鉴定出88个分离株的O血清型,优势血清型分别为：O142、O45、O86和078,其中0142共有46个分离株,占本次定型菌株的52.27%(46/88),且0142血清型为首次在致死鸡中分离到。用分离到的优势血清型0142大肠杆菌分离株进行了人工感染,结果该病原人工感染易感肉鸡能复制出与临诊几乎一致的疾病。选择来自病死鸡中同一个体的腺胃和心血分离株各10个,以107菌落形成单位(colony forming units, CFU)分别气囊感染1日龄健康易感肉鸡和SPF鸡,结果显示所有腺胃分离株恒能引起2种鸡6/6(100%)死亡,而心血分离株则可引起3/6-4/6(50-67%)的鸡死亡。腺胃分离株接种后病死肉鸡83%-100%(5-6羽)腺胃变黑,而心血分离株接种的病死肉鸡以及所有病死的SPF鸡的腺胃无此变化。毒力基因检查结果显示,绝大部分菌株都携带iutA,fimH,frzorf4, ompT和feoB基因(≥88),50%以上分离株都携带cvaC, iroN, iucC, iucD, sitA, traT, irp-2, tsh, astA和neuC,而afa, papA, papG allele I, sfa, sfaS,fela, cdtB,vat和ace26基因检出率均低于10%。分离株种系发生分型结果显示,以B2为主,占受试分离株的58.33%,D型、A型和B1型分别占11.46%、19.79%和10.42%。药敏试验结果显示,分离株呈多重耐药性,尤其大部分0142血清型分离株均对头孢噻呋钠耐药。重金属检测结果显示所有病死鸡的内脏组织中汞均超标,而发病鸡舍污染饲料和试验对照鸡内脏均不超标。试验结果表明分离的分离株经气囊接种肉鸡和SPF鸡均为高致病株；死亡鸡胚和病肉鸡舍料槽中污染的饲料样品中,可分离出与雏鸡病例一致的0142致病性大肠杆菌,暗示饲料可能成为同群易感肉鸡致病性大肠杆菌的来源；汞不是造成雏肉鸡死亡和黑腺胃的原因,但可能加重黑腺胃变黑的程度。2.同一个体APEC腺胃分离株和心血分离株毒力基因及致病性比较为探明前期研究中从同一病死个体腺胃分离的APEC 4d/9-1 O142菌株和APEC 4d/9-10142心血分离株是否为相同菌株及评价两者对动物的致病性。首先对两株菌进行了半数致死量(LD50)测定及体内定居与持续试验。半数致死量结果显示APEC 4d/9-1 O142腺胃分离株比APEC 4d/9-1 O142心血分离株的毒力高100倍；体内定居与持续试验结果显示,腺胃分离株在感染鸡肺内的细菌载量比心血分离株高10倍(P0.01),在心血、肝脏、肾脏和腺胃则高100倍(P0.001),腺胃分离株可使人工感染病死的AA肉鸡的腺胃变黑,但病死SPF鸡腺胃不变黑。动物试验结果表明：来自同一个体的4d/9-1 O142腺胃分离株的毒力确实显著高于4d/9-1 O142心血分离株。为进一步阐明这两个菌株之间的差异,本研究对二者的毒力基因、种系发生群、多位点序列分型和脉冲场凝胶电泳进行了检测。毒力基因检测结果显示：在受检的32个毒力基因中,铁摄取基因、保护素基因的出现率相对较高,而粘附素、毒素基因的出现率相对较低,腺胃分离株仅比心血分离株多了1个铁摄取相关的毒力基因feoB。两个分离株均属B2分子发生群；但多位点序列分型显示腺胃分离株为ST131,而心血分离株为ST2704,表明尽管分离自同一个体,但2个分离株基因组水平确实存在差异；脉冲场电泳显示两者的相似系数为97.3%,说明二者存在着遗传差异。3.APEC O142腺胃分离株和心血分离株在鸡体内外的表达差异基因的筛选O142是一个十分罕见的禽源性大肠杆菌的血清型,前期研究结果显示来自同一个体的O142腺胃分离株和心血分离株都属强毒株,但前者毒力比后者高100倍。已知毒力基因检测结果显示,除了前者少一个feoB基因外所有毒力基因完全相同,已有研究表明feoB基因与致病性大肠杆菌致病性无关。为此,本研究选择APEC4d/9-1 O142腺胃分离株和心血分离株为研究对象,应用商品化大肠杆菌基因芯片对选择的致病性大肠杆菌分离株进行以SPF鸡作动物模型的体内外表达基因谱检测,筛选部分潜在的新毒力基因。该芯片系统共包含10113条探针,因此,可以实现大规模潜在毒力基因的筛选。试验结果显示,APEC4d/9-1腺胃分离株和心血分离株在体内比体外差异表达基因数量分别达到1502个和2260个。APEC 4d/9-1腺胃分离株体内对体外表达差异分析结果显示,在1502个差异表达基因中表达上调的基因为527个,其中包含195个特异性探针对应的已知基因,68个假定基因；表达下调的基因为975个,其中包含217个特异性探针对应的已知基因,83个假定基因；5倍以上差异基因共97个,其中表达上调基因和下调基因分别为41个和56个。表达差异最显著的基因是narG, narU, narJ, narH, nark, narl, ompF, metF和nanA基因。4d/9-1心血分离株体内对体外表达差异分析结果显示在2260个差异表达基因中表达上调的基因为651个,其中包含146个特异性探针对应的已知基因,78个假定基因；表达下调的基因为1609个,其中包含281个特异性探针对应已知基因,75个假定基因；5倍以上表达差异基因共177个,其中包含上调表达差异基因为44个,下调表达差异基因为133个。上调表达差异最显著的基因是narU, narG, narH, narJ, nark, ompF, metF,pta,fdnG, gpmM和nanA基因等。腺胃分离株与心血分离株体内分别与各自的体外表达谱相比上调或下调基因数量较多,但与心血分离株的体内表达谱相比,腺胃分离株在体内差异表达基因数量较少,共159个差异表达基因,原因可能是两者是从同一个体内分离出、同一血清型O142,种系发生群同属B2群,虽然ST分型及动物试验结果显示两者为两株菌,但很可能两者的同源率较高。在159个差异表达基因中包含了上调表达基因126个,其中差异表达基因最显著为yehX, nrfG, lamb, ompF, gadB, ynjE, ygaM, yeiC, gltD和metF; 4d/9-1腺胃分离株与4d/9-1心血分离株相比共有33个下调表达差异基因,差异表达基因最显著为leuB, nhaR, sdaB, yghJ, ytfQ, ycdT, c2481, kpsM, C3689和ECsl505。对4d/9-1腺胃分离株与4d/9-1心血分离株分离株在鸡体内差异表达基因进行Gene ontology(GO)分析,结果显示,GO注释结果中cell part, binding, catalytic, transporter, cellular process, metabolic process功能所占的比例最大。暗示这些基因在致病性大肠杆菌感染鸡体内生长繁殖及发挥致病作用时有主要作用。用qRT-PCR方法,分别对感染鸡体内表达上调的yjhQ, C3292, nuoM, narH, metF, ompF基因和表达下调的。fimA,C0719,yjdB,yedV基因表达做了解析验证。结果显示,除了表达上调的nuoM和表达下调的yjdB基因与基因芯片结果存在一定差异外,其他验证基因均与芯片结果一致。4.禽致病性大肠杆菌E491P株ompF, metF基因缺失株的构建及其生物学特性研究对在基因芯片试验中筛选出的APEC4d/9-1腺胃分离株(4d/9-1 proventricular isolate, E491P)体内外呈显著差异表达基因ompF和metF基因,利用λRed重组系统分别构建ompF和metF基因缺失株E491P△ompF和E491P△metF,研究两个缺失株的生物学特性。试验结果显示,缺失株E491P△ompF和EA91P△metF的生长速度、抗血清补体杀菌能力较野生株无显著差异,但缺失株△ompF生长速度略低于野生株。1日龄雏鸡LD50致病性试验结果显示,野生株E491P、缺失株E491P△ompF和E491P△metFLD50分别为1038、1050和1040,结果表明仅缺失株E491P△ompF致病性被致弱；35日龄SPF鸡体内动态分布试验结果显示,缺失株AompF在鸡体内定植能力显著下降,而E491P△metF较野生株致病性没有明显差异,结果表明ompF基因与致病性大肠杆菌致病性相关。
[Abstract]:Avian pathogenic Escherichia coli (avian pathogenic Escherichia coli, APEC) can cause local or systemic infection in poultry. Systemic infection, E. coli septicemia, is mainly characterized by the presence of Escherichia coli in the blood. Its developmental stages include acute sepsis, subacute seroserotis and chronic granulomatous inflammation. Local infection Avian colibacillosis includes Escherichia coli / yolk sac infection, Escherichia coli cellulitis, swollen head syndrome, reproductive tract infection, coliform salpingitis / peritonitis and Escherichia coli orchitis / epididymitis. A large number of domestic and foreign studies have shown that the serotypes of Escherichia coli are diverse, but the most diverse, but the most important. Some of the most common cases of O1,02, O35, and O78. infection are caused by uncommon serotypes, such as O111 serotype isolates that cause serotis, septicemia, and some pathogenic Escherichia coli serotypes. In recent years, in Jiangsu, Shandong, Anhui, Henan and other provincial regulations A type of broiler chicken "black gland stomach disease" (temporary naming) appeared frequently. The onset area was scattered and the onset of the disease was early. It was mainly concentrated at 3-5 days of age, sometimes at 7-8 days of age. The appearance of the chicks was healthy at the age of 3% to 12%., and there were no visible symptoms before death. The pathological changes were mainly gray and black (hair and black). With the severity of the disease, it is light gray or gray, and it is black in serious time. The course of the disease is a little longer, and the course of the disease is pericarditis, pericarditis, and gasbag inflammation. So far, the disease of the chicken with the change of the glandular stomach has not been reported. This study aims to isolate and identify the pathogen of the chicks with the characteristics of the glandular gastric blackening. Meat chicks "black gland stomach disease", explore the isolated pathogenic Escherichia coli serotype, pathogenicity, phylogeny, virulence genotype; select the virulence of the APEC 4d/9-1 O142 strain and the APEC 4d/9-1 O142 isolated strain from the same dead individual gland stomach, and compare the virulence of the virulence gene and the model animal by the DNA chip technology. The gene expression profiles of two isolated strains in SPF chickens and in vitro were analyzed, and the potential virulence genes of the APEC glandular stomach isolated strains were screened, and the difference genes were selected to construct the missing strains, and the relationship between the gene deletion and pathogenicity was evaluated. Finally, the pathogenesis and prevention measures of APEC induced "black gland stomach disease" were provided. According to the isolation and identification of the pathogenic Escherichia coli of "black gland stomach disease" caused by.1. chicks, the isolation and identification of the "black gland stomach disease" of broiler chickens in a large broiler breeding farm in Jiangsu province were isolated and identified. At the same time, the isolation and identification of the dead chicken embryo, feed, chick and chicken air in the farm were separated and identified. 96 strains of Escherichia coli were isolated. In addition to 5 strains, the O serotypes of 88 isolated strains were identified. The dominant serotypes were O142, O45, O86 and 078, of which 0142 had 46 isolates, accounting for 52.27% (46/88) of the stereotyped strain, and 0142 serotypes were isolated for the first time in the fatal chicken. The bacterial isolates were infected artificially, and the result was that the infected broiler was susceptible to replicating the disease which was almost identical with the immediate diagnosis. 10 of the 107 colony forming units (colony forming units, CFU) were selected from the same individual from the sick and dead chickens and infected with 1 day old chickens and SPF chickens. The results showed that all the glandular gastric isolates could cause the death of 2 chickens 6/6 (100%), while the isolated strains of heart and blood could cause the death of 3/6-4/6 (50-67%). The glandular stomach became black after the inoculation of the adenostomy isolate and the dead broilers and all the dead SPF chickens. The results showed that most of the strains carried iutA, fimH, frzorf4, ompT and feoB genes (> 88), and more than 50% of the isolates carried cvaC, iroN, iucC, iucD, sitA, and traT. 58.33%, D, A and B1 accounted for 58.33%, D, A and B1 respectively. The results of 19.79% and 10.42%. showed that the isolates showed multiple resistance, and most of the 0142 serotype isolates were resistant to ceftif sodium. The experimental results showed that the isolated isolates were high pathogenic strains of chicken and SPF chickens inoculated with air bag, and 0142 pathogenic Escherichia coli were isolated from the dead chicken embryos and the contaminated feed samples of the sick broiler chute, suggesting that the feed may be the same group of susceptible broilers. The origin of pathogenic Escherichia coli; mercury is not the cause of the death of chicks and the black gland stomach, but it may aggravate the degree of black glands and stomach blackening.2. the virulence genes and pathogenicity of the same individual APEC glandular gastric isolated strain and the heart blood isolate, and the APEC 4d/9-1 O142 strain and APEC 4d/ isolated from the same dead individual gland stomach in the early study. 9-10142 whether the isolated strain was the same strain and evaluate the pathogenicity of the two animals. First, the median lethal dose (LD50) was measured in two strains and in vivo and in vivo. The results of half lethal dose showed that the virulence of the APEC 4d/9-1 O142 glandular isolated strains was 100 times higher than that of the APEC 4d/9-1 O142 isolated strains; the body was settled and continued in vivo. The test results showed that the bacterial load in the infected chicken lung was 10 times higher than that of the isolated strain, 100 times higher in the heart blood, the liver, the kidney and the gland stomach, and the glandular stomach of the AA broiler infected by the artificial infection could blacken the gland stomach of the dead AA broiler, but the death of the SPF chicken gland was black. The animal test results showed that the same individual was from the same individual. The virulence of the 4d/9-1 O142 glandular gastric isolate was significantly higher than that of the 4d/9-1 O142 isolated strain. In order to further clarify the difference between the two strains, this study examined the virulence genes, the genotyping group, the multipoint sequence classification and the pulse field gel electrophoresis of the two, and the test results showed that 32 virulence bases were tested. In the case of iron uptake genes, the occurrence rate of protectin genes was relatively high, and the occurrence rate of adhesin and toxin genes was relatively low. The adenostomy isolates were only 1 different strains of iron uptake related virulence gene feoB. two more than that of the heart blood isolates, but the multiple point sequence classification showed that the glandular stomach isolated strain was ST131, and the heart blood was the heart blood. The isolated strain was ST2704, indicating that the genome level of the 2 isolates did exist in spite of the isolation of the same individual, and the similarity coefficient of the two isolates was 97.3%, indicating that the two people had a genetic difference of.3.APEC O142 adeno gastric isolate and the separation of heart blood isolates in the chicken body. A rare serotype of avian Escherichia coli showed that the O142 glands and blood isolates from the same individual were all strong strains, but the virulence of the former was 100 times higher than that of the latter. The results of the known virulence gene show that the virulence genes of one feoB gene, except for the former, are exactly the same, and the research has shown that feoB The gene is not related to the pathogenicity of the pathogenic Escherichia coli. Therefore, this study selected the APEC4d/9-1 O142 glandular gastric isolated strain and the heart blood isolate as the research object, and used the commercialized Escherichia coli gene chip to detect the gene spectrum of the SPF chicken as the animal model of the selected pathogenic Escherichia coli. The new virulence gene, which contains 10113 probes, can be used to screen large potential virulence genes. The results show that the number of differentially expressed genes in the APEC4d/9-1 glandular gastric separation plant and the heart blood isolate in vitro is 1502 and the in vitro expression difference of 2260.APEC 4d/9-1 adenosine isolated strains in vitro The results showed that 527 genes were up-regulated in 1502 differentially expressed genes, including 195 specific probes corresponding to known genes, 68 presumed genes, and 975 down-regulated genes, including 217 specific probes corresponding to known genes, 83 presumed genes, and 97 more than 5 times more than 97 genes. The expression of up-regulated and down regulated genes were 41 and 56 respectively. The most significant gene expression was narG, narU, narJ, narH, nark, NARL, ompF, metF and nanA gene.4d/9-1 heart blood isolates, which showed that 651 genes were up-regulated in 2260 differentially expressed genes, including 146 special genes. The heterosexual probes correspond to the known genes, 78 presumed genes, and 1609 down regulated genes, including 281 specific probes that correspond to the known genes and 75 presumed genes, and 5 times more than 177 genes, including 44 differentially expressed genes, and 133 differentially expressed differentially expressed genes. The genes are narU, narG, narH, narJ, nark, ompF, metF, PTA, fdnG, gpmM and nanA genes. A total of 159 differentially expressed genes may be caused by the separation from the same body, the same serotype O142, and the same B2 group. Although the ST typing and animal test results show that the two are two strains, they are likely to have a higher homologous rate. In the 159 differentially expressed genes, there are 126 up-regulated genes, of which the difference is poor. The most significant expression genes are yehX, nrfG, lamb, ompF, gadB, ynjE, ygaM, yeiC, gltD and metF, and there are 33 down-regulated genes in the 4d/9-1 gland gastric isolate. Gene Ontology (GO) analysis of the differentially expressed genes of /9-1 isolated strains in chickens showed that the proportion of cell part, binding, catalytic, transporter, cellular process was the largest in the GO annotation results, suggesting that these genes were grown and propagated in the chickens infected with pathogenic Escherichia coli. The expression of yjhQ, C3292, nuoM, narH, metF, ompF gene and down regulated.FimA, C0719, yjdB, yedV gene expression in infected chickens were analyzed by qRT-PCR method. The results showed that the expression of up regulated nuoM and down regulated genes and gene chip results were found. In addition, the other verifying genes were in accordance with the results of the chip, and the.4. avian pathogenic Escherichia coli E491P strain ompF, the construction of the metF gene deletion strain and the study on the biological characteristics of the APEC4d/9-1 gland gastric isolate (4d/9-1 proventricular isolate, E491P) found in the gene chip test (4d/9-1 proventricular isolate, E491P) showed significant differentially expressed gene ompF in vivo and in vitro. The metF gene, using the lambda Red recombination system, constructed the ompF and metF deletion strains E491P Delta ompF and E491P Delta metF to study the biological characteristics of the two missing strains. The experimental results showed that the growth rate of the deletion strain E491P Delta ompF and EA91P Delta metF was no significant difference compared with the wild strain, but the growth rate of the missing strain delta growth rate was not significant. The results of LD50 pathogenicity test of.1 day old chicks showed that the wild strain E491P, E491P Delta ompF and E491P Delta metFLD50 were 10381050 and 1040 respectively. The results showed that only the E491P Delta ompF pathogenicity of the missing strain was weak, and the dynamic distribution test of 35 day old SPF chicken showed that the deletion of AompF in the chicken was significant. The decline of E491P metF was not significantly different from that of wild plants. The results showed that ompF gene was associated with pathogenicity of E. coli.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.31

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