【作者】 张志敏；

【导师】 于三科；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2005， 硕士

【摘要】 鸡球虫病是由复顶器门球虫寄生于鸡肠道上皮细胞内引起的一种原虫病,是以减轻体重和降低饲料转化率为主要特征的危害极其严重的全球性寄生虫病。全世界每年因球虫病造成的经济损失约为20 亿美元。目前控制鸡球虫病仍然是以药物防治为主,但由于耐药虫株的不断出现以及药物残留等问题,使药物开发和药物防治受到极大的限制。用早熟系卵囊研制成的弱毒活虫苗是目前应用范围较为广泛的球虫疫苗,在免疫预防球虫病中发挥了极其重要的作用。在鸡艾美耳属的各种球虫中,柔嫩艾美耳球虫(Eimeria tenella)是致病性最强的一个种。本试验通过对陕西省杨凌区鸡E. tenella 早熟株选育并对其生物学进行研究,为球虫早熟苗的研制奠定基础;提取E. tenella 基因组DNA是从分子水平上深入研究该病的基础。本文包括以下两方面的内容。　第一部分:鸡E. tenella 早熟株选育及生物学特性研究。以陕西省杨凌区鸡E. tenella 孢子化卵囊为虫种,采用早熟系虫株选育的方法,反复收集感染后最先排出的球虫卵囊,选育出E. tenella YL 早熟株并对早熟株和母株的潜隐期、致病性、免疫原性及免疫保护力等进行了比较。结果表明:经18 代选育的E. tenella YL 早熟株的潜隐期比母株提前了24 h,卵囊的形态与母株相似,但没有母株卵囊大;用不同剂量球虫卵囊分别感染鸡时发现,早熟株的繁殖能力低于母株,每羽鸡所排卵囊量与剂量呈正相关,而每个卵囊的繁殖能力与剂量呈负相关;从试验鸡的临床症状、死亡率及盲肠病变记分等方面分析,YL 早熟株的致病性明显低于母株;在同等剂量免疫下,早熟株的免疫原性好于母株,早熟株和母株免疫组对再攻虫时的卵囊产量都有明显的抑制作用,但早熟株免疫组的抑制作用高于母株; 2 次免疫后,早熟株和母株免疫组都有较高的免疫保护力,攻虫后的卵囊抑制率均在91% 以上。　第二部分:艾美耳球虫基因组DNA 的提取。以　E. tenella 孢子化卵囊为材料,采用氯化苄法和常规提取　DNA 的方法提取了艾美耳球虫基因组　DNA。氯化苄在　65℃条件下与蛋白质和几丁质等发生反应,能够有效地破坏球虫卵囊壁,可用于大规模提取基因组　DNA;常规方法提取　DNA 的程序是用蛋白酶　K 消化处理,用酚∕氯仿抽提,再用氯仿抽提,部分步骤需要重复。通过对比试验,结果表明:这两种方法都可用于鸡球虫基因组　DNA 的提取,所获得的　DNA 数量和质量都很高,可用于各种分子生物学实验。但氯化苄法具有简便、快速、经济实用的特点,更适用于球虫卵囊大量样品　DNA 的快速提取。更多还原

【Abstract】 Avian coccidiosis is a parasitic protozoan belonging to the Phylum Apicomplexa. The pathogens invade the gut epithelium and result in coccidiosis in poultry throughout the world. This debilitating disease is characterized by poor weight gain and inefficient feed conversion. The annual worldwide cost is estimated to be about $2 billion. Eimeria tenella is the most pathogenic species of Eimeria. Currently, the disease is controlled by anticoccidial feed additives and it has played a major role in the growth of poultry industry, but some degree of resistance to all anticoccidial drugs has development and the reports of drug-residue in food have heightened the consumer’s concerns. Live vaccine, consisting of either virulent coccidial strains or live attenuated parasites of several species, is proved to be effective in preventing coccidial infection. We have selected the precocious strain and studied the characteristics of E. tenella in Yangling District of Shaanxi province. Extraction the genomic DNA from E. tenella provided the basic material for further research in molecular biological study. This paper contains mainly two parts. ①Selecting the precocious strain and studying the bio-characteristics. A precocious strain of E. tenella was selected by repeated passages of oocysts that were early collected from faeces of previously infected chickens. We have studied on the pathogenicity、prepatent period、immunogenicity　and so on compared with the parental strain. The precocious strain had the following characteristics:the prepatent period of oocysts was 24 hours earlier than that of parental strain;The precocious strain was smaller in size, but it was similar to the parental in morphology;The pathogenicity and the reproductive capacity of precocious strain were all decreased in comparison with the parental strain. The immunogenicity was better than that of parental strain. After two times immunity the immunological protection of both strains was both high, and the ratio of occyst productive inhibition was all above 91%.　②Extracting the genomic DNA from E. tenella sporulated oocyst. In order to looking for a suitable method for extracting high quality genomic DNA from E. tenella sporulated oocysts, the Benzyl Chloride method was used in comparison with the usual method. Benzyl Chloride can efficiently break sporulated oocyst wall at 65℃. The results indicated that, the two methods were all good for extracting genomic DNA. The quantity and quality of the DNA extracted by two methods was all high. But the Benzyl Chloride method was not only simple and fast, but also not expensive. Therefore, the Benzyl Chloride method is a suitable method for large-scale preparation of genomic DNA from E. tenella sporulated oocysts .更多还原