本文選題：禽致病性大腸桿菌 + 分離鑒定��； 參考：《揚州大學(xué)》2016年博士論文

【摘要】：禽致病性大腸桿菌(avian pathogenic Escherichia coli, APEC)能引起禽類局部或全身性感染。全身感染即大腸桿菌型敗血癥,主要特征是大腸桿菌存在于血液中,其發(fā)展階段包括急性敗血癥、亞急性多發(fā)性漿膜炎和慢性肉芽腫性炎癥。局部感染性禽大腸桿菌病包括大腸桿菌型臍炎／卵黃囊感染、大腸桿菌型蜂窩織炎、腫頭綜合征、生殖道感染、大腸桿菌型輸卵管炎／腹膜炎和大腸桿菌型睪丸炎／附睪炎等。大量國內(nèi)外研究表明,因地理區(qū)域不同,大腸桿菌血清型呈現(xiàn)多樣性,但最常見的為O1、02、O35、O78。某些感染的暴發(fā)病例是由不常見的血清型造成的,如O111血清型分離株可造成產(chǎn)蛋母雞發(fā)生多臟器的漿膜炎、敗血癥。還有一些致病性大腸桿菌菌株血清型不明確或者不能分型。近年來,在江蘇、山東、安徽、河南等省規(guī)模化肉雞養(yǎng)殖場頻繁出現(xiàn)一種雛肉雞“黑腺胃病”(暫命名),發(fā)病區(qū)域分散,發(fā)病日齡早,主要集中在3-5日齡,有時在7-8日齡出現(xiàn),死淘率3%～12%。商品肉雞出雛時外觀健康,死亡前幾無可見臨診癥狀。剖檢病理變化以腺胃呈灰黑色為主(發(fā)黑程度隨病情輕重而呈淺灰色或灰色,嚴(yán)重時呈黑色)、病程稍長者,可見心包炎、肝周炎、氣囊炎。迄今,具有腺胃變黑變化的肉雛雞疾病尚未見報道。本研究旨在對臨床以腺胃發(fā)黑為特征的病死肉雛雞進行病原的分離鑒定,人工感染復(fù)制肉雛雞“黑腺胃病”,探明所分離的致病性大腸桿菌血清型、致病性、種系發(fā)生群、毒力基因型；選擇從同一病死個體腺胃分離的APEC 4d/9-1 O142菌株和APEC 4d/9-1 O142心血分離株進行毒力基因和模型動物的致病性比較,通過DNA芯片技術(shù)對這兩個分離株在SPF雞體內(nèi)和體外的基因表達(dá)譜進行分析,篩選APEC腺胃分離株潛在的毒力基因；選擇表達(dá)差異基因進行缺失株的構(gòu)建,評價基因缺失株與致病性的關(guān)系,最終為弄清APEC引發(fā)雛肉雞“黑腺胃病”的發(fā)病機制和防制措施提供依據(jù)。1.致雛肉雞“黑腺胃病”致病性大腸桿菌的分離鑒定本試驗對江蘇省某大型肉雞養(yǎng)殖場雛肉雞“黑腺胃病”病例進行了病原的分離鑒定,同時對該養(yǎng)殖場不同日齡死亡雞胚、飼料、出雛器和雞舍空氣進行大腸桿菌的分離鑒定。共分離獲得大腸桿菌96株,除5株未能定型外,共鑒定出88個分離株的O血清型,優(yōu)勢血清型分別為：O142、O45、O86和078,其中0142共有46個分離株,占本次定型菌株的52.27%(46/88),且0142血清型為首次在致死雞中分離到。用分離到的優(yōu)勢血清型0142大腸桿菌分離株進行了人工感染,結(jié)果該病原人工感染易感肉雞能復(fù)制出與臨診幾乎一致的疾病。選擇來自病死雞中同一個體的腺胃和心血分離株各10個,以107菌落形成單位(colony forming units, CFU)分別氣囊感染1日齡健康易感肉雞和SPF雞,結(jié)果顯示所有腺胃分離株恒能引起2種雞6/6(100%)死亡,而心血分離株則可引起3/6-4/6(50-67%)的雞死亡。腺胃分離株接種后病死肉雞83%-100%(5-6羽)腺胃變黑,而心血分離株接種的病死肉雞以及所有病死的SPF雞的腺胃無此變化。毒力基因檢查結(jié)果顯示,絕大部分菌株都攜帶iutA,fimH,frzorf4, ompT和feoB基因(≥88),50%以上分離株都攜帶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基因檢出率均低于10%。分離株種系發(fā)生分型結(jié)果顯示,以B2為主,占受試分離株的58.33%,D型、A型和B1型分別占11.46%、19.79%和10.42%。藥敏試驗結(jié)果顯示,分離株呈多重耐藥性,尤其大部分0142血清型分離株均對頭孢噻呋鈉耐藥。重金屬檢測結(jié)果顯示所有病死雞的內(nèi)臟組織中汞均超標(biāo),而發(fā)病雞舍污染飼料和試驗對照雞內(nèi)臟均不超標(biāo)。試驗結(jié)果表明分離的分離株經(jīng)氣囊接種肉雞和SPF雞均為高致病株；死亡雞胚和病肉雞舍料槽中污染的飼料樣品中,可分離出與雛雞病例一致的0142致病性大腸桿菌,暗示飼料可能成為同群易感肉雞致病性大腸桿菌的來源；汞不是造成雛肉雞死亡和黑腺胃的原因,但可能加重黑腺胃變黑的程度。2.同一個體APEC腺胃分離株和心血分離株毒力基因及致病性比較為探明前期研究中從同一病死個體腺胃分離的APEC 4d/9-1 O142菌株和APEC 4d/9-10142心血分離株是否為相同菌株及評價兩者對動物的致病性。首先對兩株菌進行了半數(shù)致死量(LD50)測定及體內(nèi)定居與持續(xù)試驗。半數(shù)致死量結(jié)果顯示APEC 4d/9-1 O142腺胃分離株比APEC 4d/9-1 O142心血分離株的毒力高100倍；體內(nèi)定居與持續(xù)試驗結(jié)果顯示,腺胃分離株在感染雞肺內(nèi)的細(xì)菌載量比心血分離株高10倍(P0.01),在心血、肝臟、腎臟和腺胃則高100倍(P0.001),腺胃分離株可使人工感染病死的AA肉雞的腺胃變黑,但病死SPF雞腺胃不變黑。動物試驗結(jié)果表明：來自同一個體的4d/9-1 O142腺胃分離株的毒力確實顯著高于4d/9-1 O142心血分離株。為進一步闡明這兩個菌株之間的差異,本研究對二者的毒力基因、種系發(fā)生群、多位點序列分型和脈沖場凝膠電泳進行了檢測。毒力基因檢測結(jié)果顯示：在受檢的32個毒力基因中,鐵攝取基因、保護素基因的出現(xiàn)率相對較高,而粘附素、毒素基因的出現(xiàn)率相對較低,腺胃分離株僅比心血分離株多了1個鐵攝取相關(guān)的毒力基因feoB。兩個分離株均屬B2分子發(fā)生群；但多位點序列分型顯示腺胃分離株為ST131,而心血分離株為ST2704,表明盡管分離自同一個體,但2個分離株基因組水平確實存在差異；脈沖場電泳顯示兩者的相似系數(shù)為97.3%,說明二者存在著遺傳差異。3.APEC O142腺胃分離株和心血分離株在雞體內(nèi)外的表達(dá)差異基因的篩選O142是一個十分罕見的禽源性大腸桿菌的血清型,前期研究結(jié)果顯示來自同一個體的O142腺胃分離株和心血分離株都屬強毒株,但前者毒力比后者高100倍。已知毒力基因檢測結(jié)果顯示,除了前者少一個feoB基因外所有毒力基因完全相同,已有研究表明feoB基因與致病性大腸桿菌致病性無關(guān)。為此,本研究選擇APEC4d/9-1 O142腺胃分離株和心血分離株為研究對象,應(yīng)用商品化大腸桿菌基因芯片對選擇的致病性大腸桿菌分離株進行以SPF雞作動物模型的體內(nèi)外表達(dá)基因譜檢測,篩選部分潛在的新毒力基因。該芯片系統(tǒng)共包含10113條探針,因此,可以實現(xiàn)大規(guī)模潛在毒力基因的篩選。試驗結(jié)果顯示,APEC4d/9-1腺胃分離株和心血分離株在體內(nèi)比體外差異表達(dá)基因數(shù)量分別達(dá)到1502個和2260個。APEC 4d/9-1腺胃分離株體內(nèi)對體外表達(dá)差異分析結(jié)果顯示,在1502個差異表達(dá)基因中表達(dá)上調(diào)的基因為527個,其中包含195個特異性探針對應(yīng)的已知基因,68個假定基因；表達(dá)下調(diào)的基因為975個,其中包含217個特異性探針對應(yīng)的已知基因,83個假定基因；5倍以上差異基因共97個,其中表達(dá)上調(diào)基因和下調(diào)基因分別為41個和56個。表達(dá)差異最顯著的基因是narG, narU, narJ, narH, nark, narl, ompF, metF和nanA基因。4d/9-1心血分離株體內(nèi)對體外表達(dá)差異分析結(jié)果顯示在2260個差異表達(dá)基因中表達(dá)上調(diào)的基因為651個,其中包含146個特異性探針對應(yīng)的已知基因,78個假定基因；表達(dá)下調(diào)的基因為1609個,其中包含281個特異性探針對應(yīng)已知基因,75個假定基因；5倍以上表達(dá)差異基因共177個,其中包含上調(diào)表達(dá)差異基因為44個,下調(diào)表達(dá)差異基因為133個。上調(diào)表達(dá)差異最顯著的基因是narU, narG, narH, narJ, nark, ompF, metF,pta,fdnG, gpmM和nanA基因等。腺胃分離株與心血分離株體內(nèi)分別與各自的體外表達(dá)譜相比上調(diào)或下調(diào)基因數(shù)量較多,但與心血分離株的體內(nèi)表達(dá)譜相比,腺胃分離株在體內(nèi)差異表達(dá)基因數(shù)量較少,共159個差異表達(dá)基因,原因可能是兩者是從同一個體內(nèi)分離出、同一血清型O142,種系發(fā)生群同屬B2群,雖然ST分型及動物試驗結(jié)果顯示兩者為兩株菌,但很可能兩者的同源率較高。在159個差異表達(dá)基因中包含了上調(diào)表達(dá)基因126個,其中差異表達(dá)基因最顯著為yehX, nrfG, lamb, ompF, gadB, ynjE, ygaM, yeiC, gltD和metF; 4d/9-1腺胃分離株與4d/9-1心血分離株相比共有33個下調(diào)表達(dá)差異基因,差異表達(dá)基因最顯著為leuB, nhaR, sdaB, yghJ, ytfQ, ycdT, c2481, kpsM, C3689和ECsl505。對4d/9-1腺胃分離株與4d/9-1心血分離株分離株在雞體內(nèi)差異表達(dá)基因進行Gene ontology(GO)分析,結(jié)果顯示,GO注釋結(jié)果中cell part, binding, catalytic, transporter, cellular process, metabolic process功能所占的比例最大。暗示這些基因在致病性大腸桿菌感染雞體內(nèi)生長繁殖及發(fā)揮致病作用時有主要作用。用qRT-PCR方法,分別對感染雞體內(nèi)表達(dá)上調(diào)的yjhQ, C3292, nuoM, narH, metF, ompF基因和表達(dá)下調(diào)的。fimA,C0719,yjdB,yedV基因表達(dá)做了解析驗證。結(jié)果顯示,除了表達(dá)上調(diào)的nuoM和表達(dá)下調(diào)的yjdB基因與基因芯片結(jié)果存在一定差異外,其他驗證基因均與芯片結(jié)果一致。4.禽致病性大腸桿菌E491P株ompF, metF基因缺失株的構(gòu)建及其生物學(xué)特性研究對在基因芯片試驗中篩選出的APEC4d/9-1腺胃分離株(4d/9-1 proventricular isolate, E491P)體內(nèi)外呈顯著差異表達(dá)基因ompF和metF基因,利用λRed重組系統(tǒng)分別構(gòu)建ompF和metF基因缺失株E491P△ompF和E491P△metF,研究兩個缺失株的生物學(xué)特性。試驗結(jié)果顯示,缺失株E491P△ompF和EA91P△metF的生長速度、抗血清補體殺菌能力較野生株無顯著差異,但缺失株△ompF生長速度略低于野生株。1日齡雛雞LD50致病性試驗結(jié)果顯示,野生株E491P、缺失株E491P△ompF和E491P△metFLD50分別為1038、1050和1040,結(jié)果表明僅缺失株E491P△ompF致病性被致弱；35日齡SPF雞體內(nèi)動態(tài)分布試驗結(jié)果顯示,缺失株AompF在雞體內(nèi)定植能力顯著下降,而E491P△metF較野生株致病性沒有明顯差異,結(jié)果表明ompF基因與致病性大腸桿菌致病性相關(guān)。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.31

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