【作者】 周克夫；

【导师】 林宇光；

【作者基本信息】 厦门大学 ， 动物学， 2004， 博士

【摘要】 2001年8月份至2004年7月份，作者对厦门产的柔嫩艾美尔球虫(E．tenella)的病原生物学、生活史各期形态特征、球虫的致病作用等方面进行了详细的研究，在此基础上对鸡感染从厦门获得的柔嫩艾美尔球虫{以下简称柔嫩艾美尔球虫(厦门))后血清抗体变化规律也进行了考察，并对转胸腺素α1基因蓝藻在球虫感染中的保护作用及药代动力学进行探讨，研究内容结果如下： 一、柔嫩艾美尔球虫厦门(E．tenella)病原生物学考察，此项内容包括： 1．采用单卵囊分离技术分离并增殖到柔嫩艾美尔球虫(厦门)，建立了用蔗糖梯度离心的方法纯化球虫卵囊的方法。 2．E．tenella(厦门)内生发育阶段的各期形态的研究包括采用直接涂片、石蜡组织切片和冰冻切片的方法在光学显微镜下进行观察以及采用透射电镜手段进行内生阶段形态观察。结果显示，E．tenella(厦门)具有3代裂殖体，其中第二代具有大小两型的裂殖子，吞噬细胞在裂殖子的移行和转运中起作用，裂殖子中的棒状体先于或与微线同时出现，裂殖子发育过程中出现类似蜕皮现象，裂殖生殖同时具备两种生殖分裂方式，盲肠内发育的大配子体数量是小配子数量的50倍以上。小配子具有4根鞭毛，几个小配子体分布在相邻的宿主细胞内。大配子体的疏松形的成囊体1先于致密形的成囊体2出现。经冰冻切片和苏丹黑B染色证明，大配子体内主要是脂肪性质的成分。 3．27℃下，E．tenella厦门卵囊孢子化时间为20h，潜伏期为146h，孢子化卵囊在4℃下保存4个月致病作用明显减弱，未孢子化卵囊在4℃和8℃下30d，孢子化率接近0。 二、E．tenella厦门的致病作用： 1.10000个卵囊/只的感染量，鸡的死亡率为40·60%。对10日龄以内的雏鸡危害相对大。第二次感染对1一8日龄小鸡危害大，而第二次感染对10日龄以后的小鸡危害小。 2.感染球虫的鸡血清生化指标发生明显改变，并出现明显贫血的症状。 3.电镜下发现被感染的小鸡盲肠绒毛变少、折断，被寄生的宿主细胞内线粒体数量明显减少，小配子体寄生的宿主细胞核还发生核膜损伤的现象。 三、虫感染后血清抗体消长规律的研究 1.运用冻融的方法制备了球虫抗原，建立了检测鸡感染球虫后血清抗体的ELISA方法。 2.血清中抗球虫抗体在第一次和第二次感染后的第4d均出现一个峰值，而第三次感染在7d内没有检测到抗体水平升高。 3.对3次感染球虫的鸡血清蛋白进行SDS一PAGE分析，第一次和第二次感染后7d出现14条带，增加的3条分子量分别为21kDa、33扔a和50 kDa.另外30KDa的蛋白带在第一次感染和第二次感染后7d量明显增多，而第三次感染后7d出现12条带。其中33 kDa和50KDa两条带消失。 四、转胸腺素Ql(T QI)基因蓝藻在鸡体内分布和对球虫感染中的保护作用的研究 1.建立了检测T al的EUSA方法，并检测到喂转胸腺素Ql基因蓝藻的小鸡几种器官和组织中T Ql出现升高。 2.通过实验观察发现，转基因藻在改善球虫感染的症状方面具有明显的效果，同时，在提高抗体水平方面也有一定作用。 本项研究完善了该球虫的生物学特性，补充了内生发育超微结构方面的资料，为球虫病的药物和免疫防治提供理论依据。同时，为应用转胸腺素Ql基因蓝藻作为鸡免疫增强剂提供科学依据。更多还原

【Abstract】 The present paper deals with the morphology , developmental cycle and pathogenicity of E.tenella from Xiamen, the paper also deals with the immune response of chicken to E.tenella ,and study on the effect of algae transformed T a 1 gene for anti-cbccidia, the results as follows:The study on the biology of E.tenella from Xiamen, it deals with the following contents:1. The E.tenella was separated from chiken in Xiamen using a new single-oocyst isolation technical, and the other method for purification of E.tenella was also established with discontinuous sucrose gradient. The result showed that both of these methods are effective.2. Study on the endogenous cycle of E.tenella from Ximen , the technical involves the smear of caecum mucosa, tissue section and electron microscope, the results indicated that schizont of E.tenella including 3 generations, there are 2 types in the second generation schizont, the phagocytes were important in transferring of merozoite; The rhotry(RH) of merozoite was earlier appeared than the microneme(MN) and a phenomenon of molting appeared during the period of maturation of merozoites. The number of macrogametocytes is more 50 times than that of microgametecytes. There are 4 flagellum in microgamete and there are several microgametes appeared in one region of several host cells.3. The wall-forming body 1(WF1) was loose, and earlier appeared than the wall-forming body2(WF2)that is fine and close. With Cryostate sections combining freezing, stained with Sudan black B, the result indicated that the chemical composition in macrogamet is lipoid.4. It spended about 20h at 27 for sporulation, the incubation period is146h, the pathogenicyty of sporulated oocyst is declined obviously stored at 47癈 for 4 months. When the oocysts were stored at 4 and 8 for 30 days before sporulation,the sporulation rate was nearly zero. The pathogenicity of E.tenella from Xiamen1. Oral infection with the dosage of 10, 000 oocysts, the death rate of chicken was 40%-60%. It is relatively more harmful to chickens within 10 day age than other age chickens.2. The biochemical indexes number changed in chickens after infection with E.tenella XM and showed the anaemia symptom.3. It was found that the villus of caecum of chicken reduced and broke off after infection with E.tenella XM, the host cell membrane of nucleus was damaged obviously and this damage was only found in host cell that infected with microgametocyt. The number of mitochondrion decreased obviously in host cell after infection E.tenella(Xiamen). Study on antibody to coccidia1. Preparation the the antigen of coccidian with freezing and melting, the result proved it was a good methold, and also found it was better than that of ultrasonic crushing.2. An indirect ELISA for detecting serum E.tenella -specifec antibody in chicken was successfully established, and result proved that the peak value was appeared at 4th day after infection. The biodistribution of Thymosin a 1 in chickens and the effect of algae transformed Thymosin a 1 gene.1. An indirect ELISA for detecting T a 1 was established, with this assay, the result indicted that the content of T a 1 in several organs of chickens fed with algae transformed Thymosin a 1 gene were higher than that of control chickens.2. The experiment showed that the transformed algae is effective in protecting from coccidia infection, on the other hand, it can increase the levelof anti-body to coccidian.The paper perfected the endogenous cycle about E.tenella, and provide the new stuff about the life cycle, morphology, pathology and physiology of E.tenella. As an immunopromoter, the usage of algae transformed Ta 1 gene will give a new way to prevent coccidiasis. This work is very important in theory and practical for preventing coccidiasis.更多还原