【摘要】：近年來,由禽致病性大腸桿菌(avian pathogenic Escherichia coli,APEC)引起的禽大腸桿菌病給全球養(yǎng)禽業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失。因此,快速準(zhǔn)確的診斷是治療和控制該病的重要前提。本研究自江蘇省徐州市周邊養(yǎng)鴨廠病死櫻桃谷鴨肝臟分離到7株APEC菌株,研究其種系發(fā)生型、耐藥性、毒力基因分布,并建立多重PCR檢測方法,為該地區(qū)APEC的臨床防控和進(jìn)一步研究提供支持。主要研究結(jié)果如下:1.對2015年采集自江蘇省徐州市周邊養(yǎng)鴨場臨床疑似大腸桿菌病的病死櫻桃谷鴨肝臟進(jìn)行病原菌的分離,根據(jù)形態(tài)特征、培養(yǎng)特性、生化試驗(yàn)、16S rRNA分子鑒定可確定分離到7株大腸桿菌。對分離菌株的種系發(fā)生型進(jìn)行鑒定可確定7株大腸桿菌均屬B2型。取編號為E17和E4的兩株大腸桿菌進(jìn)行動(dòng)物致病性試驗(yàn),表明分離到的兩株病原菌皆有致病性。為了解分離株對藥物敏感性的差異,采用kirby-Bauer紙片法,用29種藥敏紙片對分離到的7株鴨源大腸桿菌分離株進(jìn)行藥敏實(shí)驗(yàn),結(jié)果表明7株大腸桿菌都具有多重耐藥性,最少的對5種藥物耐藥,最多的對21種藥物耐藥。亞胺培南、呋喃妥因和呋喃唑酮可作為高敏藥物對本地區(qū)的鴨源大腸桿菌病防治選藥提供一定的指導(dǎo)作用。對7株分離菌株的12個(gè)毒力基因進(jìn)行檢測,結(jié)果顯示除菌株E14、E17、EB未檢出stx基因外,其余的4株都含有全部的12種毒力基因。2.根據(jù)Genbank中已發(fā)表的APEC iss、cvaC、iucD、irp-2、iroN、tsh基因序列,用Primer 5.0軟件設(shè)計(jì)6對特異引物,建立鴨源APEC的多重PCR檢測方法。檢測多重PCR方法的特異性和靈敏性,結(jié)果表明該方法具有特異性,擴(kuò)增最小檢出DNA核酸量為100 pg/μL,最小檢出菌落數(shù)為105 CFU。利用該多重PCR檢測25株APEC(均屬于B2型)和79株非致病大腸桿菌(31.65%屬于A群,50.63%屬于B1群,1.27%屬于B2群,16.46%屬于D群)驗(yàn)證多重PCR方法的可行性。結(jié)果為,在分離菌株含有上述6個(gè)毒力基因中3個(gè)或3個(gè)以上時(shí),即有92.0%的概率認(rèn)定該分離株具致病性,誤差率為8.0%。建立的多重PCR方法能夠簡便、快速地檢測APEC。本研究將為江蘇徐州地區(qū)APEC的進(jìn)一步研究提供一定支持。
[Abstract]:In recent years, avian Escherichia coli caused by avian pathogenic Escherichia coli (avian pathogenic Escherichia coli,APEC) has caused serious economic losses to poultry industry worldwide. Therefore, rapid and accurate diagnosis is an important prerequisite for the treatment and control of the disease. In this study, 7 strains of APEC were isolated from the liver of dead Cherry Valley Duck from a duck farm in Xuzhou City, Jiangsu Province. The genotypes, drug resistance, virulence gene distribution and multiplex PCR detection were studied. To provide support for clinical prevention and control and further research of APEC in this area. The main results are as follows: 1. The pathogenic bacteria were isolated from the dead Cherry Valley duck liver collected from the duck farm around Xuzhou City, Jiangsu Province in 2015. According to the morphological characteristics and the culture characteristics, the pathogenic bacteria were isolated from the liver of the dead Cherry Valley duck, which was suspected of E. coli disease. Biochemical test, 16s rRNA molecular identification can confirm that 7 strains of Escherichia coli were isolated. Identification of the phylogenetic types of the isolated strains confirmed that all 7 strains of Escherichia coli belong to B2 type. Two strains of Escherichia coli named E17 and E4 were selected for animal pathogenicity test. The results showed that the two strains were pathogenicity. In order to understand the difference of drug sensitivity of 7 strains of duck Escherichia coli, the drug sensitivity of 7 strains of Escherichia coli isolated from duck was tested by kirby-Bauer disk method. The results showed that all 7 strains of Escherichia coli had multiple drug resistance. The least resistant to 5 drugs and the most resistant to 21 drugs. Imipenem, furantoin and furazolidone can be used as Gao Min drugs for the prevention and treatment of duck Escherichia coli. 12 virulence genes of 7 isolates were detected. The results showed that all the other 4 strains contained 12 virulence genes except the stx gene of E14 E17 EB. 2. According to the published APEC iss,cvaC,iucD,irp-2,iroN,tsh gene sequence in Genbank, six pairs of specific primers were designed with Primer 5.0 software to establish a multiplex PCR detection method for duck APEC. The specificity and sensitivity of the multiplex PCR method were tested. The results show that the method is specific, the minimum DNA nucleic acid amount detected by amplification is 100 pg/ 渭 L, and the minimal detected colony number is 105 CFU.. The multiplex PCR was used to detect 25 strains of APEC (all belong to B2 type) and 79 strains of non-pathogenic Escherichia coli (31.65% belong to group A, 50.63% belong to group B1, 1.27% belong to group B2 and 16.46% belong to group D). The results showed that when the isolates contained 3 or more of the above 6 virulence genes, the probability was 92.0% that the isolates were pathogenicity, and the error rate was 8.0%. The established multiplex PCR method can be used to detect APEC. easily and quickly. This study will provide some support for the further study of APEC in Xuzhou, Jiangsu Province.
【學(xué)位授予單位】：牡丹江師范學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61

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