【作者】 王全溪；

【导师】 李国平； 吴宝成；

【作者基本信息】 福建农林大学 ， 临床兽医学， 2004， 硕士

【摘要】 番鸭呼肠孤病毒病主要发生于番鸭，夏季多发，发病日龄以4～45日龄为主，发病率达20％～90％，应激或混合感染下，死亡率可达90％以上。国内外未见有该病血清学诊断方法的报导。因此，临床上建立一种切实可行且快速的诊断方法越来越受到国内水禽养殖业的关注。本研究以此为出发点，建立了快速、简便、准确，而且能够在基层应用的间接ELISA检测病毒抗体和反向乳胶凝集试验检测病毒抗原两种血清学诊断方法。 本课题以本实验室分离鉴定保存的番鸭呼肠孤病毒B3分离株为研究材料，开展了番鸭呼肠孤病毒病间接ELISA和反向乳胶凝集试验两种血清学诊断方法的研究与应用工作。 1．采用番鸭胚成纤维细胞成功的培养了番鸭呼肠孤病毒，感染细胞产生了明显的CPE病变，并进行病毒的扩大培养，细胞培养物经二次差速离心和硫酸铵沉淀制备了所需的诊断抗原，病毒蛋白含量为5.628mg／ml。 2．采用细胞培养的番鸭呼肠孤病毒，按所设定的免疫程序免疫成年公番鸭，获得琼扩效价为1：16的阳性血清，经粗提和纯化获得了蛋白含量为4.242mg／ml的一抗IgG。同时采集健康番鸭的血清，纯化后获得IgG，按所设定的免疫程序免疫家兔，获得琼扩效价为1：8的兔抗番鸭二抗血清，并纯化得到蛋白含量为5.395mg／ml的二抗IgG。采用改良的过碘酸钠法成功地把HRP标记在纯化的二抗IgG上，结果表明，酶结合率=0.5312，标记率=0.6909，符合ELISA的要求。 3．用硫酸铵粗提的番鸭呼肠孤病毒作为包被抗原和HRP酶标记的兔抗番鸭二抗，成功地建立了检测番鸭呼肠孤病毒病抗体的间接ELISA法。试验结果表明，包被抗原1：80(0.0704mg／ml)稀释，待检血清1：40稀释，酶标二抗1：200稀释，是本方法最佳的工作浓度。应用研究结果表明，该法不与其它病原发生交叉反应，阻断效果明显，检测番鸭呼肠孤病毒抗体可达1：800比琼扩试验高50倍，ELISA测定临床采集的血清阳性率达70％，琼扩试验阳性率37.5％，两者符合率67.5％，批内批间重复结果稳定。因此，间接ELISA法检测番鸭呼肠病毒抗体特异性高、敏感性强、重复性好、质量稳定、结果可靠，适合于临床流行病调查。 4．用纯化的抗番鸭呼肠孤病毒抗体致敏乳胶成功地建立了反向乳胶凝集试验检测番鸭呼肠孤病毒抗原的方法。通过试验，确定了抗体致敏乳胶的最佳浓度为0.4242 mg／ml，最佳致敏温度37℃，最佳致敏时间2h。应用研究结果表明，抗体致敏乳胶无自凝性，特异性强，重复性好，质量可靠。人工攻毒雏番鸭，用本方法可于攻毒后的第三天粪便中检测到病毒抗原，直至攻毒鸭全部死亡都可从粪便中检测到病毒，可见在感染早期，病鸭即可从粪便排毒，且持续较长时间，及时无害化处理粪便对防制本病有重大意义。攻毒1日龄雏番鸭，剖检无菌采集取心、肝、脾按常规方法提取病毒抗原，检测结果表明，脾脏在攻毒后第4天3只中有2只结果阳性，第5天2只死亡鸭中有一只肝脏结果阳性，此后的病死鸭脾和肝番鸭呼肠孤病毒检测都阳性，而心脏处理物未见有阳性。更多还原

【Abstract】 Indirect Enzyme-Linked Immunosorbent Assay(ELISA) and Latex Agglutination Test(LAT) were studied and applied to diagnose the muscovy duck reovirus disease. The muscovy duck reovirus , which was separated and appraised by professor Wu,was use in the two test.1. The virus was cultivated in the cell of muscovy duck embryo. The antigen was prepared for ELISA and LAT by centrifugation and precipitation. TCIDso of the virus was detected and calculated. The result was that per millimeter cell fluid consisted of 56990 pieces of TCIDso. The virus protein content was 5.628mg/ml.2. The first polyclone-antibody of the virus and the second polyclone-antibody were prepared. The healthy muscovy ducks were immunized four times(one time per ten days). The result indicated that the positive price was as much as 1:16. The positive serum was purified by precipitation and ion -exchange chromatography. The IgG protein content was detected as much as 4.242mg/ml. On the other, the anti-muscovy duck poly-antibody of rabbit was prepared, which positive price was 1:8 and the protein content was 5.395mg/ml. Then the HRP was marked to the purity rabbit anti-muscovy duck IgG. The marked rate was 0.6909.3. The reaction condition of indirect ELISA was succeeded in developing through a lot of experiment. The results indicated that diluting the antigen to 0.0704mg/ml and the serum to 1:40 and the marked IgG to 1:200 were the best reaction condition. The antigen’s specificity , sensitivity and stability were tested. The result meaned that the Indirect ELISA had good speciality, sensivity and stability. It is suitable for serological epidemiological survey and serological diagnosis of muscovy duck reovirus disease.4. The reaction condition of Latex Agglutination Test (LAT) was soluted through a lot of experiment. The best suitable condition for coating the latex was determined by titration test. The result was that antibody content 0.4242 mg/ml, reaction temperature 37 C and reaction time 2 hours were the best reaction conditions o The LAT had good speciality, sensivity and stability. The LAT detected the Muscovy Duck Reovirus from the stool of the infection duck on the third day after the artificial infection. And it also detected the virus from the spleen and the liver on the fourth and the fifth day after infection. But it was negative from the heart of the dead duck.更多还原