【作者】 刘晓丽；

【导师】 程国富；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 Ⅰ型鸭病毒性鸭肝炎(duck hepatitis virus,DVH)是雏鸭的一种高度致死性的、传播迅速的病毒性疾病。感染Ⅰ型鸭肝炎病毒(DHV—Ⅰ)的雏鸭发病率和死亡率很高,免疫接种是目前预防和控制鸭肝炎的主要手段。但是,DHV仍然时有发生,给养鸭业造成了巨大的经济损失。目前临床上急需简便快捷的检测鸭肝炎病毒抗体水平的检测方法,作为DHV抗体监测参考标准,指导养鸭生产。本研究旨在优化本实验室已建立的DHV—Ⅰ抗体乳胶凝集试验方法,观测蛋鸭和雏鸭的DHV—Ⅰ抗体变化曲线。具体研究成果如下:1.采用差速超高速离心的方法纯化病毒,用磷钨酸负染电镜观察、鉴定,可见清晰完整的DHV—Ⅰ病毒粒子,有个别病毒的的核酸部分尚未降解,病毒粒子直径约为20-40nm,核衣壳为对称的二十面体。2.将经过纯化病毒灭活后用于致敏聚苯乙烯乳胶,优化检测DHV-Ⅰ抗体的LAT方法。血清抗体的检测标准为:用倍比稀释的试验鸭血清与致敏后的乳胶进行反应,能够在3-5分钟内产生肉眼可见颗粒。3.用LAT方法观察经过四次鸭肝炎弱毒疫苗免疫的蛋鸭血清中的DHV—Ⅰ抗体,得到了高免蛋鸭随时间变化的DHV—Ⅰ抗体变化曲线。研究发现第二次免疫后10天到第四次免疫后10天抗体水平较高,抗体效价维持在8以上,第四次免疫后20天抗体水平急剧下降至2左右。因此,如要获得高免卵黄抗体,需对蛋鸭进行多次免疫并适时检测其抗体水平。4.用LAT方法监测无母源抗体雏鸭的DHV—Ⅰ抗体变化,获得了无母源抗体雏鸭免疫后随时间变化的DHV—Ⅰ抗体变化规律,研究发现,无母源抗体的雏鸭经DHV—Ⅰ弱毒疫苗免疫后一个星期抗体水平达到最高,可达到7.1,之后略有下降,直到免疫后20天抗体效价维持在5左右,而注射卵黄抗体的雏鸭在注射抗体后的第3天抗体水平达到最高,可达7.7,免疫后7天抗体水平逐渐下降至5左右。因此,对人工主动免疫和被动免疫进行比较发现DHV—Ⅰ弱毒疫苗免疫更适用于雏鸭早期免疫,而高免DHV—Ⅰ卵黄抗体则适用于DVH的治疗和紧急预防。5.用LAT方法监测有母源抗体雏鸭的DHV—Ⅰ抗体变化,获得了有母源抗体雏鸭免疫后随时间变化的DHV—Ⅰ抗体变化规律,研究发现,有母源抗体的雏鸭无论是1日龄免疫还是免疫后人工感染DHV—Ⅰ强毒,其抗体水平一直下降,免疫后一个月下降至4.5左右。DHV—Ⅰ弱毒疫苗免疫不干扰母源抗体的作用,还有助于提高雏鸭的DHV—Ⅰ抗体水平。另外,研究中还初步获得了有母源抗体雏鸭的保护力为乳胶凝集价应为8,即抗体稀释度为1:2~8。更多还原

【Abstract】 Duck viral hepatitis (DVH) caused by duck hepatitis virus-I is highly mortal disease and spread rapidly in ducklings. The ducklings infected DHV- I has high morbidity and mortality, and injecting vaccine is main method to prevent and control DVH at present. However, DHV still happens in ducks at times, and results in great damage to duck industry. If there is a way that can detect DVH antibody level simply and quickly, and also can apply in various research levels, and provide reference standard for DVH antibody detection, which will help the duck keepers to decrease their economic loss suffered by DVH. This research was conducted to optimize the DVH antibody latex agglutination test based on the method of our laboratory, and develop a method to detect DVH antibody in quantity. Besides, we can use it to observe the DVH antibody curve of breed ducks and ducklings, which can aslo help to make decision for the duck industry. The results followwed in detail:1. The virus was purified by Differential ultracentrifugation, and was done negative staining using phosphotungstic acid .Complete and lampros DVH- I virion was observed and indentified through electron microscope. Nucleic acid of several virion was not degradated yet, and diameter of virion was about 20-40nm, neucleocapsid was meristic icosahedron.2. The DHV- I antibody quantitative level was detected with the optimize DVH antibody latex agglutination test in clinic, and the DHV - I purified was allergized polystyrene latex. The detailed method is: the serum of ducks diluted multiple bind to the optimized latex, the titer of serum was the maximum deliquation that was seen some granulations.3. The DHV- I antibody quantitative level was detected with the optimize DVH antibody latex agglutination test in clinic, and the serum DHV- I antibody curve depend on time was obtained from the egg-ducks immunized with the weak DVH vaccine four times. The result showed: the antibody level of the second immunization and the fourth immunization kept much higher after 10 days coppared with ?, the titer was above 8; but rapidly decreased to 2 after 20 days in fourth immunization. Therefore, if we want to obtain egg yolk antibody, we need to immune the breed ducks several times, and also detect the antibody level in time.4. The DHV- I antibody quantitative level of the ducklings without maternal antibody was detected with the optimize DVH antibody latex agglutination test, and DHV- I antibody curve of ducklings without maternal antibody depend on time was obtained. The results showed: the antibody level of the ducklings without maternal antibody rised to peak value after seven days immunized with low virulent DHV- I vaccine, decreased a little after that, and the titer was about 5 after twenty days; however, the antibody level rised to peak value after three days immunized with egg yolk antibody, and decreased to about 5 after seven days. That’s mean: compared with artificial active immunity and passive immunity, I found DHV- I vaccine was fit to be used in the nonage of ducklings, and DHV- I yolk antibody can be used to treat or emergency prevention.5. The DHV- I antibody quantitative level of the ducklings with maternal antibody was detected with the optimize DVH antibody latex agglutination test, and DHV - I antibody curve of ducklings with maternal antibody depend on time was obtained. The result indicated whatever immunized after 1 day or infected with high virulent DHV - I after immunization, the antibody of duckling with maternal antibody kept on decreasing, and was only about 4.5 after thirty days. Meanwhile, DHV- I vaccine was beneficial to increase the DHV- I level of ducklings rather than interfere the effect of maternal antibody. In addition, the protection of ducklings with maternal antibody should be 8 for latex agglutination value, it mean antibody dilution was 1:2~8.更多还原