鸡源致病性沙门氏菌毒力基因与致病性的相关性研究

发布时间：2018-03-15 07:40

本文选题：鸡　切入点：致病性沙门氏菌　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：沙门氏菌具有广泛的宿主,是一种重要的人畜共患病的病原菌。近年来,抗菌药物在临床上的普遍使用甚至滥用,导致沙门氏菌的耐药问题越来越严重。沙门氏菌能够入侵宿主体内增殖和致病,与其毒力基因密切相关。本研究对鸡源致病性沙门氏菌进行分离鉴定,对其耐药性、致病性进行测定,对其耐药基因、毒力基因进行检测,分析耐药基因与耐药表型、毒力基因与致病性的关系,为沙门氏菌病的防治提供科学依据。采集疑似病鸡的组织病料,通过SC增菌液对沙门氏菌进行增菌培养。用XLD琼脂平板对沙门氏菌进行分离培养。选取纯培养物的单个菌落,分别进行生化鉴定、血清学鉴定和PCR鉴定等,共获得55株沙门氏菌分离株。这些菌株包含4个血清群,A群6株,B群23株,C群1株,D群25株。其中D群约占45%,为沙门氏菌优势血清群。采用CSLI推荐的K-B琼脂法测定55株沙门氏菌分离株对18种抗菌药物的敏感性。结果显示,55株沙门氏菌分离株对利福平、阿莫西林、青霉素G、苯唑西林以及头孢噻吩的耐药率达到100%;对红霉素、林可霉素、多粘菌素B、复方新诺明及呋喃妥因耐药率达到90%以上;对强力霉素、链霉素及诺氟沙星的耐药率在50%～90%之间:对庆大霉素及大观霉素的耐药率在10%以下,分别为7.27%、3.64%。利用PCR方法检测55株沙门氏菌分离株的16种耐药基因blaTEM、blaPSE、blacMY、aadA1、Aph(3)-IIa、aadA2、tetB、tetG、tetM、tetX、Sul1、Su13、qnrA、qnrB、qepA、qnrS。结果显示,55株沙门氏菌分离株中,含有3种耐药基因的菌株比例为25.45%,含有5种耐药基因的菌株比例为21.82%,含有2种耐药基因的菌株比例为21.82%,含有4种耐药基因的菌株比例为20.00%,含有1种耐药基因和7种耐药基因的菌株比例分别为3.64%和1.82%。在所检测耐药基因中,tetX的检出率最高,达到78.18%,而blaPSE、Aph(3)-Ⅱa、aadA2、qnrA、qnrB则未被检出。利用PCR方法检测55株沙门氏菌分离株的31种毒力基因(质粒毒力基因spvR、spvA、spvB、spvC、spvD;毒力岛基因invH、sipA、sipB、sopA、sopD、avrA、hilA、iacP、prgK、ssaB、ssaQ、sifA、sseL、ssrA、ttrB、mgtC、misL、rmbA、rhuM、sugR、orf319、siiD(spi4D)、siiE、spi4H、sopB、pipC)。结果显示,质粒毒力基因的检出率均在60%以上;毒力岛基因ttrB和orf319检出率最高(92.73%),而ssaQ以及sifA检出率均在10%以下。通过小鼠致病性试验检测55株沙门氏菌分离株的致病性。结果显示,所有分离株对小鼠均具有致病性,但致死的时间和剂量有所差异。随机选取其中10株进行测定,其半数致死量(LD50)介于3.18×108cfu/mL和4×107cfu/mL之间。以上研究结果表明,本研究分离鉴定获得的55株鸡源致病性沙门氏菌分离株中,D群血清型比例约为45%,为优势血清群;沙门氏菌普遍存在耐药性,多重耐药现象严重;分离菌株对小鼠具有致病性;其耐药表型与耐药基因、致病性与毒力基因之间均呈现正相关。
[Abstract]:Salmonella has a wide range of hosts and is an important pathogen of zoonosis. In recent years, antimicrobial agents have been widely used and even abused in clinic. The drug resistance of salmonella is becoming more and more serious. Salmonella can invade the host body to proliferate and cause disease, which is closely related to its virulence gene. The pathogenicity was determined, drug resistance genes and virulence genes were detected, and the relationship between drug resistance genes and phenotypes, virulence genes and pathogenicity were analyzed, which provided scientific basis for the prevention and treatment of salmonella disease. Salmonella was cultured in SC medium. Salmonella was isolated and cultured with XLD Agar plate. Single colony of pure culture was selected for biochemical identification, serological identification and PCR identification. A total of 55 Salmonella isolates were obtained, including 4 serogroup A, 6 strains, 23 strains of group B, 1 strain of group C, 25 strains of group D. among them, group D is about 45%, which is the predominant serotype of salmonella. K-B Agar method recommended by CSLI was used to determine the results. The sensitivity of 55 Salmonella isolates to 18 antimicrobial agents was determined. The results showed that 55 Salmonella isolates were resistant to rifampicin. The resistance rates of amoxicillin, penicillin G, oxacillin and cefthiophene to 100 kum; to erythromycin, lincomycin, polymyxin B, compound sulfamide and furantoin were over 90%; to doxycycline, to doxycycline, to doxycycline, to doxycycline, to doxycycline, to doxycycline, to doxycycline. The resistance rates of streptomycin and norfloxacin were between 50% and 90%. The resistance rates to gentamicin and spectinomycin were below 10%. 16 resistant genes of 55 Salmonella isolates were detected by PCR method. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 strains of Salmonella spp. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 strains of Salmonella spp. The results showed that there were 55 strains of Salmonella spp. The results showed that there were 55 isolates of Salmonella spp. The results showed that there were 55 isolates of Salmonella. The proportion of strains containing three resistant genes was 25.45um, the proportion of strains containing five resistant genes was 21.822am, the proportion of strains containing two resistant genes was 21.822um, the proportion of strains containing four resistant genes was 20.00, the proportion of strains containing one drug resistance gene and seven drug resistance genes was 20.00. The proportion of resistant genes was 3.64% and 1.82, respectively. The detection rate of tetX was the highest among the tested resistant genes. The virulence genes of 55 Salmonella isolates were detected by PCR method. 31 virulence genes (plasmid spvRspvspvAspvBspvCspvDand virulence island gene invhophisiasopAsopAsopDavrAhila) were detected by PCR method, and the results showed that the virulence was higher than 60%. The results showed that the virulence was higher than that of the other three strains of Salmonella sphaesii. The results showed that the virulence of 31 virulence genes of 55 Salmonella isolates were detected by PCR method. The detection rate of the virulence genes of plasmid spvrrsspvspvspvCspvcspvDspvDspvDV DspvDvDvDvDvDspvDvDvDvDspvDvDv of the virulent island gene invHopsiaopsis Asopa Asopa DavrAhila was detected, and the results showed that the virulence gene was more than 60%. The results showed that the virulence was more than 60%. The results showed that the virulence was higher than that of the PSEP. The detection rate of the virulence gene was higher than that of the control group. The detection rate of 31 virulence genes of 55 Salmonella isolates was determined by PCR method. The detection rate of virulence island gene ttrB and orf319 was the highest, while the detection rate of ssaQ and sifA was below 10%. The pathogenicity of 55 Salmonella isolates was detected by mouse pathogenicity test. The results showed that all isolates were pathogenicity to mice. But the lethal time and dose were different. The LD50 of 10 strains was between 3.18 脳 10 8 cfur / mL and 4 脳 10 7 cfur / mL. The results showed that the LD50 was between 3.18 脳 10 8 cfur / mL and 4 脳 10 7 cfur / mL. In this study, 55 strains of chicken pathogenic Salmonella were isolated and identified, the proportion of serotypes of group D was about 455.It was the dominant serotype of Salmonella, the drug resistance of Salmonella was common, the phenomenon of multidrug resistance was serious, the isolated strains were pathogenicity to mice. There was a positive correlation between drug resistance phenotype and drug resistance gene, pathogenicity and virulence gene.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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