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  本文選題：多殺性巴氏桿菌　切入點：多殺性巴氏桿菌分型　出處：《東北農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：多殺性巴氏桿菌(Pasteurella multocida,P.multocida),能夠引起人和動物共同感染,引起禽霍亂,豬萎縮性鼻炎、豬肺疫等巴氏桿菌病。巴氏桿菌病在世界各個地區(qū)均可暴發(fā),對家畜家禽及養(yǎng)殖業(yè)有很大的影響。多殺性巴氏桿菌有多種血清型,不同動物來源的多殺性巴氏桿菌分離株的基因組存在差異,不同毒力菌株在基因組上也存在差異。本研究以國內(nèi)部分地區(qū)36株P(guān).multicida為研究對象,利用莢膜多重PCR、脂多糖多重PCR方法進行分型鑒定,并通過多位點序列分型方法對菌株進行遺傳演化分析。結(jié)果顯示36株P(guān).multicida包括A,B和D 3種莢膜血清型及L1,L2,L3和L6 4種脂多糖基因型。禽源P.multicida以莢膜A型,脂多糖L1型為主,其中禽源菌株C48-1,Pm72-4,Pm731均為莢膜A型,脂多糖L1型。多位點序列分型技術(shù)將多其分為ST129,ST8,ST58,ST5,ST13,ST50和ST122等7種ST型,禽源P.multicida主要為ST129型。20只3月齡SPF雞隨機分為4組,實驗組分別感染莢膜型和脂多糖型相同的禽源多殺性巴氏桿菌菌株C48-1,50 CFU/0.1mL;Pm72-4,2×104 CFU/0.1mL;Pm731,2×109 CFU/0.1mL各0.1mL,對照組注射0.1mL PBS。每個實驗組分別在感染后剖殺死亡雞,未死亡的雞在14d時全部剖殺,迅速取脾臟樣品用于RNA提取。采用SYBR Green染料實時熒光定量PCR檢測實驗組和對照組TLR4、My D88、IL-1β和IL-6基因mRNA相對表達量的變化,通過基因表達量變化研究感染多殺性巴氏桿菌后IL-1β和IL-6轉(zhuǎn)錄變化。結(jié)果顯示:感染菌株C48-1后5只雞全部死亡,感染Pm72-4菌株后有2只雞存活,感染Pm731菌株和注射PBS后5只雞全部存活。表明C48-1菌株對SPF雞有很強的致病性,屬于強毒株;菌株P(guān)m72-4對SPF雞有致病性,但毒力較弱;菌株P(guān)m731對SPF雞不致死,屬于弱毒株。熒光定量檢測TLR4、My D88、IL-1β和IL-6的表達量結(jié)果為,與對照組相比較強毒株C48-1感染后,TLR4、MyD88和IL-6的表達量均上調(diào),且差異顯著(P0.05),而Pm72-4和Pm731感染組與對照組相比差異不顯著;相對于對照組感染C48-1、Pm72-4和Pm731后產(chǎn)生IL-1β的量均上調(diào),但差異均不顯著(P0.05)。表明感染多殺性巴氏桿菌強毒株C48-1后激活了細胞因子IL-6,但不能激活I(lǐng)L-1β,感染多殺性巴氏桿菌弱毒株P(guān)m72-4和Pm731后不能激活I(lǐng)L-1β和IL-6。該研究為多殺性巴氏桿菌不同菌株毒力差異的分子機制的研究提供基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.61
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本文編號：1622310