【作者】 武林；

【导师】 李培英；

【作者基本信息】 安徽农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 隐孢子虫(Cryptosporidium)是人和动物腹泻的重要病原之一,也是人类艾滋病患者的主要致死因素之一。美国疾病预防控制中心将其列为B类病原。隐孢子虫病(Cryptosporidisis)是一种由隐孢子虫引起的呈世界性分布的人畜共患寄生虫病。2003年该病被列为我国重点防范的两种重要寄生虫病之一。近20年来,隐孢子虫病研究在病原学、形态学、流行病学、病原分子生物学、诊断及综合防治技术等方面取得许多重要成果;但是在治疗方面,尚未见到公认有效的疗法或药物报道,主要原因是迄今没有揭示其完整的致病机理。本课题旨在通过对安徽省某奶牛场奶牛隐孢子虫检测获取隐孢子虫感染强阳性奶牛,然后对其进行某些指标观察、检测及分析;通过这些指标的统计、分析,研究隐孢子虫感染奶牛后,对奶牛的生理生化指标及生产性能的影响。首先从安徽省某奶牛场采集381份奶牛粪样,用金胺酚-改良抗酸染色及饱和白糖漂浮后,通过显微镜观察,根据其形态、结构、大小等特征,鉴定虫种为鼠隐孢子虫(Cryptosporidium muris)。对不同年龄段奶牛隐孢子虫感染情况进行统计、分析后发现:各个年龄段奶牛均可感染隐孢子虫,但不同年龄段奶牛隐孢子虫感染情况不同;犊牛感染率显著高于其他两个年龄段的奶牛,而青年牛和成年牛间的感染率差异不显著。同时发现各年龄段奶牛隐孢子虫感染强度高低不均。其次采用蔗糖密度梯度离心法对分离的隐孢子虫卵囊进行纯化。取纯化过的卵囊沉淀,先经高速组织匀浆机匀浆,后于超声波细胞裂解仪上裂解,提取模板DNA。根据隐孢子虫18s rDNA序列,设计并合成出隐孢子虫特异性引物,扩增出540bp的片段。初始PCR产物进一步作套式PCR,扩增出一条250bp特异性的片段。随机直肠采取成年奶牛粪样64份,编号,分别用上述4种方法检测。在64份奶牛粪样中,分别采用金胺酚-改良抗酸染色法及饱和白糖溶液漂浮法判为阳性的奶牛粪样有11份及12份,这些阳性粪样经PCR和套式PCR检测均呈阳性;11342号粪样采用金胺酚-改良抗酸染色法判为阳性,而经饱和白糖溶液漂浮法判为阴性,应用PCR方法及套式PCR检测均呈阳性。经t检验,PCR和套式PCR方法检出率均明显高于病原学方法。再次临床驻点观察、检测14头隐孢子虫感染强阳性奶牛及14头健康、隐孢子虫感染阴性奶牛的体温、脉搏次数、呼吸次数、产奶量、乳房炎等指标并详细记录,分析其与隐孢子虫感染的关系。结果显示,隐孢子虫感染后奶牛体温、脉搏次数、呼吸次数未见显著性变化,说明这些临床指标不适宜作为隐孢子虫病诊断的主要指标。但是与健康、隐孢子虫感染阴性组奶牛相比,隐孢子虫感染强阳性组奶牛的产奶量下降极为显著,乳房炎感染率也显著升高。最后以10头隐孢子虫感染强阳性奶牛为试验组,另取10头隐孢子虫感染阴性、健康奶牛为对照组。采取血液及分离血清后,分别对隐孢子虫感染强阳性组奶牛和阴性、健康组奶牛进行AST、ALT、ALP、γ-GT、TP、A、A/G、BUN、血糖、肌酐、NO、SOD等生化指标的检测。发现BUN、肌酐、AST、ALT及SOD活性均没有发生显著变化,NO、γ-GT、TP及ALP均显著或极显著的升高,同时A/G值极显著的下降。结果表明隐孢子虫感染后机体的肝功能受到较为严重的损伤,肾功能及抗氧化能力尚比较正常;同时,奶牛患有一定的低糖血症,机体的产奶量由此受到较大的影响。综上所述,奶牛隐孢子虫检测结果为该场人畜共患隐孢子虫病的防治奠定了基础。对隐孢子虫感染奶牛后机体的生理生化指标及生产性能等方面的研究为揭示隐孢子虫的致病机理提供了理论依据。更多还原

【Abstract】 Cryptosporidium was one of the important zoonosis, and was the human HIV-positive person’s one of the factors of mainly causing death too. Control and prevention centre from the diseases of U.S.A.classified it as B etiology. Cryptosporidisis was one worldwild distribution parasitic disease which infected people and animals together. This disease was classified as one of the two kinds of important parasitic disease that our country took precautions against especially in 2003. The past 20 years, cryptosporidium was studied and made a lot of important achievements in many respects such as aetiology , morphology , epidemiology , pathogenic molecular biology , diagnosis and integrated control technology ,etc.; But had not seen and generally acknowledged the valid treatment or medicine report, The main reason was that have not announced its intact pathogenic mechanism so far.This subject obtained to Anhui Province some diary farm cow cryptosporidium infected the better positive cow, then carried on some indexes to observe, measure and analyze. Through analysis of these statistics, studied the impact on biochemical index of physiology and production character of the cows which cryptosporidium infected . Firstly, gathered 381 cow excrement from some diary farm Anhui Province, resisted acid dye wtih gold amine phenoland float with saturation white sugar, examined through the microscope , according to characteristics such as its shape、structure、size、etc. , determined the strain as Cryptosporidium muris.Counted and analyied infection of different age bracket cow, fought each age bracket cow could infect cryptosporidium but different age bracket cow with different situation . Calf’s infection rate was higher than the cows of other two age brackets notably, but the difference among the infection rate of young ox and adult ox was not prominent . Fought infection intensity of every age bracket cow to be high or low prices uneven at the same time. Secondly adopted the at odds with the community law of density gradient of cane sugar to purify the cryptosporidium’s egg capsule and separated. Fetched purified egg capsule precipitate , evened thick liquid first with organize high speed even thick liquid machine, cleavaged at ultrasonic cell cracked appearance, obtained the template DNA. According to the crytposporidium 18s rDNA, designed and formatef the cryptosporidium specificity the thing , amplified the snippet of 540bp out. Initial PCR further to make Nested PCR, amplified the snippet of a 250bp out.Random hollowed out 64 copies of excrement from adult cows rectum, serialed number, measured by 4 kinds of methods described above separately. At 64 copy , judged postive by amine phenols gold separately- improve resist there aren’t decoration method had 11, saturation white sugar solutions sour had 12; meanwhile PCR and Nested PCR had 12. 11342 excrement kind judged by gold amine phenol- improve , resist sour decoration method positive , floated by saturation white sugar solution law negative , emploied by PCR and Nested PCR method positive. Examined by PCR, and Nested PCR methods appeared higher than aetiology method obviously. Thirdly, observed clinically and measured 14 crytposporidium infected the strong positive cow and 14 heads, crytposporidium infect negative body temperature , pulse of cow , breathe number of times , milk yield , breast inflammation ,etc. index and detailed record, describe out the relation with crytposporidium infected.Result revealed crytpsosporidium had not caused obvious change of body temperature, pulse times , breathe times, proved which could not assist and diagnose the crytpsosporidium with these clinical indexes . But compared with the healthy situation , milk yield of cryptosporidium infect strong positive group droped extremely prominent, and the infection rate of inflammation of breast increases notably.Lastly,chosed 10 cows infected by cryptosporidium strong positive as experiment group , 10 negative , healthy cow as compared group. After separated serum, carried the detection in indexes such as AST , ALT , ALP ,γ- GT , TP , A , A/G , BUN , blood glucose , skin anhydride , NO , SOD ,etc on two groups . Fought BUN , skin anhydride , AST, ALT and SOD activity had taked place prominent change, NO ,γ- GT , TP , ALP prominent or prominent increased extremely, extremely prominent declined of A/G value at the same time . Result announced cryptosporidium infected damaged liver function of organism comparatively serious, renal function and anti-oxidant ability were also relatively sound; Meanwhile, the cow suffered from certain less sugar blood disease , and decreased the milk yield of the organism according of influenced by the fact .In a word, the result of exzaming cow cryptosporidium provided prevention and cure of Cryptosporidisis.Reasearch of physiology biochemical index and production performance on cows which infected by cryptospordium offered theoretics foundation to open out cryptospordium mechanism .更多还原