【作者】 宋卉；

【导师】 彭克美；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 博士

【摘要】 鸵鸟原产于非洲沙漠地带，人类集约化养殖鸵鸟的历史较其它动物如鸡、牛、猪及马属动物等短的多，并且由于鸵鸟形体高大，活动范围广，给疾病研究工作的开展带来诸多不便，因此，人们对鸵鸟许多疾病的研究目前还不完善，远不如对其它动物的疫病防治进行的深入和全面。新城疫是国内外鸵鸟养殖业最主要的传染病之一，雏鸟的发病率高达50％，成鸟发病率为3％～6％，鸵鸟因为经常进行户外活动，不可避免的要与其他野鸟或家禽接触，这可能会导致NDV在鸵鸟和其它动物之间相互传染。鉴于此，本研究以雏鸵鸟为试验动物，分为攻毒组(皮下注射鸡新城疫标准毒株F₄₈E₉)和对照组，利用透射电镜技术、放射免疫测定、组织化学法、免疫组化定位表达及原位缺口末端标记等多种方法，从亚细胞、组织细胞及细胞因子等方面系统研究雏鸵鸟免疫器官的结构特点及免疫细胞、神经免疫调控和细胞凋亡在鸡源NDV人工感染雏鸵鸟引起的机体免疫反应中的作用。主要研究工作和结果包括以下几个方面：1雏鸵鸟免疫器官的形态学特点本试验以对照组8W健康非洲雏鸵鸟为试验动物，通过光镜和电镜样本制作，采用HE、Feulgen-Methylene blue及改良Weigert法染色，研究雏鸵鸟免疫器官结构特点。结果显示：雏鸵鸟胸腺数量少，仅分布在颈后部两侧，聚集成团，髓质内有较典型的Hassall氏小体。腔上囊在泄殖腔的上方形成顶壁和侧壁，包着整个泄殖腔，不形成真正独立的囊腔，囊腔皱襞表面有密集分布的乳头状突起，每个乳头固有层内有单个腔上囊小结分布。脾脏实质内有大量类似椭球的结构，与周围淋巴组织之间形成均质淡染的嗜酸性物质。经特殊染色显示及电镜观察发现该嗜酸性物质含有胶原纤维的成分，此结构在关于其他动物脾脏的文献中未见报道。表明，鸵鸟免疫器官的外形及结构特点与其他动物之间存在较明显的差异。2免疫细胞在雏鸵鸟抵抗鸡源NDV防御反应中的免疫调节作用将试验动物随机分为攻毒组和对照组。利用组织化学Feulgen-Methylene blue和改良Toluidine blue法分别对试验雏鸵鸟免疫器官内浆细胞（Plasma cell，PC）和肥大细胞（Mast cell，MC）进行特殊染色。结果显示如下：（1）脾脏内PC主要位于红髓。胸腺皮质部及皮质与髓质交界处PC数量较多，密集分布。腔上囊内PC散在分布于整个腔上囊小结。雏鸵鸟接种鸡源NDV1～7d，脾脏内PC数量明显低于对照组（P＜0.01）。胸腺髓质部PC数目从病毒接种第3d开始急剧下降，第5d降至最低（P＜0.01），之后，PC开始增多。腔上囊小结内PC数量先降低后增加，至第9d恢复与对照组相当。上述结果表明，免疫器官内PC数量多，分布广；机体感染NDV，各免疫器官内PC数量减少，导致抗体生成和分泌降低，抑制体液免疫应答。（2）胸腺皮-髓交界处和髓质部MC数量较多，密集分布。腔上囊内MC主要分布在黏膜上皮，而脾脏内MC主要位于浅层实质，聚集分布。胸腺和腔上囊MC胞质内多含紫红色颗粒，脾脏内MC呈蓝紫色，而同一器官不同部位MC的形态、大小也不同。雏鸵鸟人工感染鸡源NDV，脾脏内MC数量总体呈下降趋势，胸腺和腔上囊内MC均在病毒接种后3d明显减少，并分别于第3d和第5d降至最低，与对照组之间差异极显著（P＜0.01）。以上结果表明，雏鸵鸟免疫器官内MC存在异质性；在病毒感染不同阶段，MC数量的动态变化提示MC在雏鸵鸟抗鸡源NDV的防御反应及机体的免疫调节过程中发挥重要作用。3鸡源NDV人工感染雏鸵鸟对神经免疫调节的影响采用免疫组织化学SABC法及放射免疫技术，对5-HT在鸵鸟免疫器官内的定位分布及病毒接种期间攻毒组和对照组鸵鸟免疫器官内5-HT表达和血清中IL-2水平的动态变化进行研究。发现，脾脏红髓内5-HT免疫反应阳性细胞（5-HTir）数量最多，阳性产物表达最强。胸腺内5-HTir主要分布于皮质和髓质交界处的血管周围及胸腺小叶间结缔组织。腔上囊小结内5-HTir数量极少，散在分布于髓质。人工感染鸡源NDV，脾脏内5-HTir数目急剧下降，与对照组差异极显著（P＜0.01），且阳性产物表达减弱。胸腺内5-HTir数量在病毒接种期间整体呈上升趋势，阳性表达产物较对照组加强（P＜0.05）。腔上囊内5-HTir在病毒接种初期减少，随后开始增加。血清中IL-2水平在病毒接种后第1d即开始下降，之后有所回升，但与对照组仍差异极显著（P＜0.01）。结果表明，脾脏是免疫器官合成外周5-HT的重要场所，5-HT数量的动态变化提示5-HT参与了ND的发病过程，其数量及表达强弱与机体的病变进程有关；IL-2在鸡源NDV感染雏鸵鸟引起的免疫应答反应中发挥重要作用，攻毒组鸵鸟血清中IL-2水平也与机体组织的损伤及疾病的病变程度密切相关。4鸡源NDV人工感染雏鸵鸟对细胞凋亡的影响利用TUNEL法检测攻毒组和对照组鸵鸟免疫器官及中枢神经系统内的细胞凋亡。数据统计分析显示，攻毒组鸵鸟免疫器官内凋亡细胞在病毒感染早期数量增加，随后数量减少。脾脏内淋巴细胞在NDV接种后5～7d凋亡细胞数量增加最明显（P＜0.01）。胸腺内淋巴细胞在病毒接种期间凋亡细胞数量均高于对照组，并且在病毒接种后1d即可检测到大量凋亡的淋巴细胞（P＜0.01）。腔上囊内凋亡细胞在病毒接种后1～7d均与对照组差异极显著（P＜0.01），除淋巴细胞凋亡外，还可检测到大量凋亡的黏膜上皮细胞。小脑内神经细胞凋亡主要发生在颗粒层颗粒细胞，感染鸡源NDV的雏鸵鸟小脑颗粒细胞凋亡数目大量增加（P＜0.01），浦肯野细胞也出现凋亡。大脑皮质部和脊髓白质内凋亡神经细胞数量均较对照组显著增加（P＜0.01）。上述研究结果表明，鸡源NDV能诱导雏鸵鸟免疫器官内淋巴细胞和中枢神经系统内神经的凋亡；感染鸵鸟腔上囊小结内黏膜上皮细胞的大量凋亡，提示可能参与了雏鸵鸟黏膜免疫屏障功能障碍的病理过程；脑内神经细胞凋亡存在区域选择性。更多还原

【Abstract】 Ostrich originated from African desert. A short intensive cultural history comparedwith fowl, cattle, pig and equus animals, big body configuration and extensive territory,all these factors cause ostrich disease research work developed inconveniently. Theostrich disease studies are still imperfect, not as profound as other animals in diseaseprevention and control. New castle disease (ND) is one of the most serious infectiondiseases that damage ostrich cultivation. The highest disease incidence of ostrich chicks is50%, the grown-up ostrich is 3%～6%. Often activating outdoors, ostrich inevitablycontacts with other wild birds or fowls, and cross infection of NDV between ostrich andother animals may be happened. In this research, ostrich chicks were divided intochallenge and control groups at random. Using the methods of transmission electronmicroscopy, radioactive immunoassay, histochemical method, immunohistochemistry andterminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), thestructural characteristics of ostrich chicks’ immune organs and the role of immunologiccell, neuroimmunomodulation and apoptosis in the immune reaction after artificiallychallenged with chicken NDV were illustrated. The main research results are as follows:1. Morphological characteristics of ostrich chicks’ immune organs8-weeks-old healthy ostrich chicks were selected from control group. The structuralcharacteristics of ostrich chicks’ immune organs was detail observed using transmissionelectron microscopy, histochemical methods (Feulgen-Methylene blue staining andreforming Weigert staining) and HE staining. The results indicated the number of thymusis little, distributed the back of neck along two sides and clustered together. There weretypical Hassall corpuscles in the medulla of thymus. The cloacal bursa structured thehanging and lateral walls of cloaca, enveloped the cloaca and did not form the trulyindependent bursa. There densely distributed convex papilates on the surface of foldsinside bursa, with one bursa nodule in lamina propria of each convex papilate. There weremany round or ellipse structures, similar to ellipsoid, in the parenchyma of spleen. Thehomogeneous texture between ellipsoid analog and circum-lymph tissue was acidophiliamaterial, which were collagen fibers by specific staining and transmission electron microscopy. And it was not found in other animals. These results demonstrate there areobvious differences in the immune organs between ostrich chicks and other poultries.2. The immunological regulation function of immunologic cell in the defencereaction to counteract chicken NDVThe experimental animals were divided into challenge and control groups at random.The histochemical methods, Feulgen-Methylene blue staining and reforming Weigertstaining were used to study the plasma cell (PC) and mast cell (MC) in immune organs ofostrich chicks. The results are as follows:(1) In spleen the PCs were mainly in the medulla. The PCs in thymus distributedintensively and the number of it was most in cortex and the boundary of cortex andmedulla. The PCs in cloacal bursa were scattered in the whole bursa nodules. Afterartificially infected with chicken NDV within 1～7days, the number of PCs in spleen ofchallenge group reduced significantly compared with control group. The PCs in thethymic medulla decreased sharply at 3 days after challenged, reached minimum at 5 days(P＜0.01), then increased again. The amount of PCs in cloacal bursa reduced first, thencame back, and was equivalent to that of PCs in control group at 9 days. Those indicatePCs in immune organs are multitude and distribute widely. The number of PCs inimmune organs after artificially infected with chicken NDV is obviously less than that ofPCs in control group, as induces the loss of antibody formation and secretion, and inhibitsthe humoral immunoresponse.(2) The MCs in thymus distributed intensively and the number of it was most inmedulla and the boundary of cortex and medulla. In cloacal bursa the MCs were mainlyin the mucosa epithelium. The MCs in spleen were mainly intensively distributed in thesuperficial lamella of parenchyma. The kytoplasm of MC in thymus and cloacal bursacontained prunosus granula, while MC in spleen was stained amethyst. The shape、sizeand gray varies of MC were different in different location of the same organ. Afterartificially infected with chicken NDV, there was a decreasing trend of the number ofMCs in spleen. The MCs in thymus of challenge group, as well as in cloacal bursa, weresignificantly fewer than that of control group at 3 days. The results show the MC inimmune organs of ostrich chicks has heterogeneity. The dynamic changes of MC indicate that MC plays a marked role in the defence reaction and immunomodulation during theartificial infection with chicken NDV.3. Effect on neuroimmunomodulation after artificially infected with chicken NDVThe study was on the distribution of 5-HT immunoreactivity (5-HTir) positive cellsin immune organs of ostrich chicks, the dynamic changes on the expression of 5-HT andthe level of IL-2 in serum after artificially infected with chicken NDV, usingimmunohistochemistry, SABC, and radioactive immunoassay. The results showed the5-HTir positive cells in spleen intensively located in red pulp, and the expression ofpositive products was stronger. In thymus the 5-HTir positive cells mainly distributed theperiphery of blood vessel and the connective tissue in lobule of thymus. The 5-HTirpositive cells in cloacal bursa were scattered in the medulla of bursa nodules. Duringinfection, the amount of 5-HTir positive cells in spleen decreased sharply and wassignificantly lower than that of control group. And the expression of positive productsweakened. While there was an increasing trend of the number of 5-HTir positive cells inthymus, the expression of positive products strengthened. The 5-HTir positive cells incloacal bursa reduced at the early stage of the artificial infection, and began increasingagain. The level of IL-2 in serum slightly decreased at 1 day after challenged then cameback, but it was still significantly different compared with control group. All the datasuggest that the spleen is the important location where the peripheral 5-HT produced inimmune organs. The dynamic changes of 5-HTir positive cells indicate that 5-HTparticipate the nosogenesis of ND. The quantity and expression of 5-HTir is relevant topathological changes. IL-2 plays the important role in the immune response to counteractchicken NDV. The level of IL-2 in serum of challenge group correlates closely with thedamage of organism and the development of disease.4. The effect of chicken NDV on apoptosis in immune organs and central nervoussystem of ostrich chicksThe TUNEL was used to detect the apoptosis of nervous cells in central nervoussystem and lymph cells in immune organs of challenge and control group. The data bystatistical analysis showed the apoptosis in immune organs of challenge group obviouslyincreased during the first stage, then delined. In spleen the apoptosis increased significantly within 5～7 days after infection compared with control group. In thymus thenumber of apoptosis increased significantly at 1 day after challenged, and was alwayshigher than that of control group. The apoptosis of lymph cell in cloacal bursa increasedsignificantly within 1～7 days, besides, a great quantity of apoptosis of mucosaendothelial cells. In cerebellum the apoptosis of nerve cell mainly occurred in thegranular cells of granular layer. The number of the apoptosis in cerebellum of ostrichchicks, as well as that of the cerebral cortex and white matter of spinal cord, increasedsignificantly after artificially infected with chicken NDV. And the positive products ofTUNEL also took place in Purkinje cells of challenge group. The results elucidate thatchicken NDV can induce the apoptosis of nervous cells in central nervous system andlymph cells in immune organs of ostrich chicks. A large number of apoptosis of mucosaendothelial cells after challenged indicates it maybe participate the pathological changesof mucosa immunologic barrier. The apoptosis of nerve cell in brain has regioselectivity.更多还原