多杀性巴氏杆菌分型分析及其感染鸡IL-1β，IL-6转录变化研究

发布时间：2018-03-17 00:16

  本文选题：多杀性巴氏杆菌　切入点：多杀性巴氏杆菌分型　出处：《东北农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：多杀性巴氏杆菌(Pasteurella multocida,P.multocida),能够引起人和动物共同感染,引起禽霍乱,猪萎缩性鼻炎、猪肺疫等巴氏杆菌病。巴氏杆菌病在世界各个地区均可暴发,对家畜家禽及养殖业有很大的影响。多杀性巴氏杆菌有多种血清型,不同动物来源的多杀性巴氏杆菌分离株的基因组存在差异,不同毒力菌株在基因组上也存在差异。本研究以国内部分地区36株P.multicida为研究对象,利用荚膜多重PCR、脂多糖多重PCR方法进行分型鉴定,并通过多位点序列分型方法对菌株进行遗传演化分析。结果显示36株P.multicida包括A,B和D 3种荚膜血清型及L1,L2,L3和L6 4种脂多糖基因型。禽源P.multicida以荚膜A型,脂多糖L1型为主,其中禽源菌株C48-1,Pm72-4,Pm731均为荚膜A型,脂多糖L1型。多位点序列分型技术将多其分为ST129,ST8,ST58,ST5,ST13,ST50和ST122等7种ST型,禽源P.multicida主要为ST129型。20只3月龄SPF鸡随机分为4组,实验组分别感染荚膜型和脂多糖型相同的禽源多杀性巴氏杆菌菌株C48-1,50 CFU/0.1mL;Pm72-4,2×104 CFU/0.1mL;Pm731,2×109 CFU/0.1mL各0.1mL,对照组注射0.1mL PBS。每个实验组分别在感染后剖杀死亡鸡,未死亡的鸡在14d时全部剖杀,迅速取脾脏样品用于RNA提取。采用SYBR Green染料实时荧光定量PCR检测实验组和对照组TLR4、My D88、IL-1β和IL-6基因mRNA相对表达量的变化,通过基因表达量变化研究感染多杀性巴氏杆菌后IL-1β和IL-6转录变化。结果显示:感染菌株C48-1后5只鸡全部死亡,感染Pm72-4菌株后有2只鸡存活,感染Pm731菌株和注射PBS后5只鸡全部存活。表明C48-1菌株对SPF鸡有很强的致病性,属于强毒株;菌株Pm72-4对SPF鸡有致病性,但毒力较弱;菌株Pm731对SPF鸡不致死,属于弱毒株。荧光定量检测TLR4、My D88、IL-1β和IL-6的表达量结果为,与对照组相比较强毒株C48-1感染后,TLR4、MyD88和IL-6的表达量均上调,且差异显著(P0.05),而Pm72-4和Pm731感染组与对照组相比差异不显著;相对于对照组感染C48-1、Pm72-4和Pm731后产生IL-1β的量均上调,但差异均不显著(P0.05)。表明感染多杀性巴氏杆菌强毒株C48-1后激活了细胞因子IL-6,但不能激活IL-1β,感染多杀性巴氏杆菌弱毒株Pm72-4和Pm731后不能激活IL-1β和IL-6。该研究为多杀性巴氏杆菌不同菌株毒力差异的分子机制的研究提供基础。
[Abstract]:Pasteurella multocida P. multocida, which can cause both human and animal infections, avian cholera, atrophic rhinitis in pigs, pulmonary disease in pigs, and other Pasteurella multocida. Pasteurella multocida can occur in all parts of the world. There are many serotypes of Pasteurella multocida. The genomes of Pasteurella multocida isolates from different animal sources are different. In this study, 36 P.multicida strains in some parts of China were used as the research objects, the capsule multiple PCRs and lipopolysaccharide multiple PCR methods were used to identify the genotypes of P. multicida strains. The genetic evolution of 36 P.multicida strains was analyzed by multilocus sequence typing. The results showed that 36 P.multicida strains were composed of three kinds of capsule serotypes (AHB and D) and four kinds of lipopolysaccharide genotypes (L1, L2, L3 and L6). Avian P.multicida was composed of capsule A type and lipopolysaccharide L1 type. Avian strain C48-1, Pm72-4, Pm731 were all capsular A type and lipopolysaccharide L1 type. They were divided into 7 ST-types by multi-locus sequence typing technique, including ST129FN, ST58S5, ST13ST50 and ST122, and P.multicida was mainly ST129 type. 20 3-month-old SPF chickens were randomly divided into 4 groups. The experimental group was infected with Pm 72-410 2 脳 104CFU / 0.1 mL Pm731G / 2 脳 10 ~ 9 CFU/0.1mL respectively, and the control group was injected with 0.1 mL PBSs. The dead chickens were killed in each experimental group after infection, and all the chickens without death were killed at the 14th day after infection, and all the chickens were killed at 14 days after the infection, and the control group was injected with 0.1 mL PBSs, and the control group was injected with 0.1 mL PBSs respectively, and the control group was injected with 0.1 mL PBSs respectively, and all the chickens without death were killed at 14 days after infection. The spleen samples were extracted quickly for RNA extraction, and the relative expression of TLR4My D88 尾 and IL-6 gene mRNA was detected by real-time quantitative PCR with SYBR Green dye in experimental group and control group. The changes of IL-1 尾 and IL-6 transcription after infection with Pasteurella multocida were studied by gene expression changes. The results showed that all five chickens died after infection with C48-1, and two chickens survived after infection with Pm72-4 strain. The results showed that C48-1 strain had strong pathogenicity to SPF chicken and belonged to virulent strain, Pm72-4 strain had pathogenicity to SPF chicken, but its virulence was weak, and strain Pm731 did not kill SPF chicken, but all of them survived after PBS injection, the results showed that C48-1 strain had strong pathogenicity to SPF chicken and was virulent to SPF chicken. The results of fluorescence quantitative detection of IL-1 尾 and IL-6 in TLR4 / MyD88 were as follows: compared with the control group, the expression levels of MyD88 and IL-6 in TLR48-1 strain were all up-regulated, and the difference was significant (P0.05A), but there was no significant difference between Pm72-4 and Pm731 infection group and the control group. Compared with the control group, the amount of IL-1 尾 produced after infection with C48-1 pm72-4 and Pm731 was up-regulated. The results showed that the cytokines IL-6 were activated after infection with C48-1 strain of Pasteurella multocida, but IL-1 尾 could not be activated, but IL-1 尾 and IL-6 could not be activated after infection with Pm72-4 and Pm731 strains of Pasteurella multocida. The molecular mechanism of the virulence difference of different strains of bacilli provides the basis for the study.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61
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本文编号：1622310