【作者】 李鹏；

【导师】 夏平安； 赵桂苹；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 鸡白痢沙门氏菌病是一种急性、系统性疾病,主要危害1周龄以内雏鸡,造成高死亡率;成年鸡无明显病变,多数呈隐形感染。鸡白痢给世界禽业造成巨大的经济损失,且感染后的长期带菌及抗生素的滥用对公共卫生也产生了巨大的潜在危害。近年来研究发现,先天免疫在抵抗病菌感染中发挥着重要的作用。TLR4及其信号通路在抗沙门氏菌感染中的作用越来越受到人们的重视。研究证实,人和鼠的MyD88-依赖途径在病原识别方面作用突出,但是MyD88-依赖途径的作用在鸡上的研究很少,对其信号通路基因表达的研究也处于起步阶段。本实验选用鸡白痢沙门氏菌感染模型,人工感染SPF雏鸡,一方面从水平传播特性和组织病理学及机体免疫反应来研究其对雏鸡危害;另一方面从先天免疫的角度,探讨了机体MyD88-依赖途径在抵抗鸡白痢沙门氏菌感染中发挥的作用。研究结果如下:【试验一】240只3日龄SPF鸡按0,20%,40%,60%,80%,100%的感染率分6组进行人工感染鸡白痢沙门氏菌C79-13株,饲养于隔离仓中。于感染后1d、3d、7d、14d、21d和28d进行细菌学、组织病理学和免疫学指标测定。结果显示:(1)鸡白痢沙门氏菌不同感染率组传播速度不同。20%感染率组72h就能感染整个鸡群,感染率越高其传播速度越快,对鸡群的危害越大,造成的死亡率越高(5.4%-35%),且鸡白痢沙门氏菌可造成雏鸡生长抑制;(2)组织学病变主要以肝炎、心包炎和肺炎为主,集中在感染后3d-7d之间;(3)细菌学检测组织载菌量(bacterial burden),盲肠高于脾脏;(4)感染组血清IgG和TNF-α在3DPI时显著升高。【试验二】80只3日龄SPF鸡按随机分为两组,处理组经口注射鸡白痢沙门氏菌C79-13株悬液108cfu 0.5ml,对照组注射同剂量生理盐水。每个处理组分别在感染后1d、3d、7d和14d随机取8只鸡,用于脾脏和盲肠样品RNA提取。采用SYBR GreenⅠ染料实时荧光定量PCR(QPCR),比较处理组和对照组脾脏和盲肠组织MyD88-依赖途径信号分子TLR4、MyD88、TRAF6和NF-κB基因mRNA相对表达量的变化。结果显示:(1)脾脏组织TLR4、MyD88、TRAF6和NF-κB基因mRNA相对表达量在3DPI显著升高(P<0.05);(2)盲肠组织TLR4、MyD88和TRAF6 mRNA相对表达量在1DPI和3DPI有上调的趋势,但没有统计学意义(P>0.05);1DPI时,NF-κB基因mRNA相对表达量明显上调(P<0.05)。结果表明,鸡白痢沙门氏菌感染后,MyD88-依赖途径被激活,MyD88-依赖途径在鸡白痢沙门氏菌感染中发挥重要作用;TLR4、MyD88、TRAF6和NF-κB基因mRNA表达在脾脏和盲肠存在差异。更多还原

【Abstract】 Salmonella Pullorum infection is an acute and systemic disease, which results in a high mortality in young chickens but rare clinic symptoms in adult birds. As a fowl-specific pathogen, S. Pullorum continues to cause economic losses worldwide. The ever-increasing problems of antibiotic abuse and chronic carrier of this bacteria in affected chickens also do a potentially huge harm to public health.The innate immune system plays a critical role in host defense against infectious disease. Despite the extensive knowledge of MyD88-dependent pathway in humans and mice, little information is available for the molecules of this pathway in chickens.Trial 1 Two hundred and forty chickens were randomly divided into 6 groups of 40 each and were infected artificially with S. Pullorum on a basis of infectious rate at 0, 20%, 40%, 60% and 100% respectively. The serological, bacteriological and histopathological parameters of host were determined at day 1, 3, 7, 14, 21, and 28 post infection.The results showed that the propagation speed of S. pullorum in affected chickens was increased in proportion to the infectious rate and so do the mortality (5.4%-35%). The bacteria could spread through the entire flock from 20% to 100% within 72 h and had a suppressive impact on the growth and development of young chickens. The characteristic lesion in tissue is mainly hepatitis, pericarditis and pneumonia, which mostly occurred between 3 and 7 day post infection. The bacterial burden of cecum was higher than the spleen. The serum concentrations of IgG and TNF-αwere significantly increased at 3 day post infection (P<0.05).Trial 2 Chickens were randomly divided into 2 groups of 40 each on d 3. One group was orally inoculated with 0.5mL of the bacterial suspension containing 108 CFU S. Pullorum and the control group was given 0.5ml saline. Eight chickens from each group were randomly chosen and sacrificed at 1, 3, 7 and 14 days post-infection. The spleen and caecum were removed and a portion of these two organs was immediately placed in RNAlaterTM for the determination of relative expression level of TLR4 signal pathway molecule genes (TLR4, MyD88, TRAF6 and NF-κB).The results showed that the infected chickens had significant up-regulation of TLR4, MyD88, TRAF6 and NF-κB mRNA expression in the spleen on day 3 and NF-κB on day 1 post infection compared with control ones (P<0.05). In the caecum, the mRNA expression levels of the pathway molecules gene were numerically up-regulated in infected group but not significant compared to uninfected controls on day 1 and 3 post infection(P>0.05). In conclusion, the MyD88-dependent pathway of TLR4 plays a role in S. Pullorum infection of chickens. The expression differences for TLR4、MyD88、TRAF6 and NF-κB genes existed between the spleen and caecum in affected chickens.更多还原