| Duck Virus Hepatitis (DVH) is an urgent and highly fatal infectious disease of young duck by Duck Hepatitis Virus. It is characterized by urgent illness, rapid spread, short disease progress and high death rate. It is caused by DHV I and spreads everywhere in the world, it was first reported in our country in 1963. It is one of the main serious diseases in duck, it exist in many places of our country. Its incident continues in high level with the rapid development of duck breeding. Based on the clinical statistic, both the occurring and death rate is over 65%, which brings great economic loss to duck breeding. Therefore, the diagnose of this disease as soon as possible is the key to treating and controlling this diseases. Therefore, the establishment of more rapid and feasible diagnostic methods is increasingly concerned by waterfowl cultivation industry in our country. In this study, we established two serological diagnosis methods, which are rapid, simple, accurate and can be used in primary level, the two methods is the Indirect ELISA that can detect viral antibody and the latex agglutination text that can detect virus antigen.In this study, we took DHV variant strain as research material, which is separated by our laboratory, studied two serological methods that are Indirect ELISA and latex Agglutination Text (LAT), and applied it into practice.1. The antigen was prepared for diagnosis by Freeze-thaw repeat. Chloroform extraction and chromatography on Sephadex G-200. The virus protein content was 6.2357mg/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 4.6573mg/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 8.0068mg/mL. Then the HRP was marked to the purity rabbit anti-muscovy duck IgG. The marked rate was 0.7464.3. 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 0.4657mg/mL, reaction temperature 37℃and reaction time 2 hours were the best reaction conditions. The LAT had good speciality, sensivity and stability.4. Adoption the competitive principle of purified antibody of anti-DHV sensitive lax and serum antibody to be tested to compete the minimum dose of antigen ,this method do not need antigens to be highly purified, avoiding the problem that DHV is very difficult to purify. The experiment results showed that the minimum dose of antigen is 1:320 (0.01949mg/mL). The adoption of the results showed that this method would not have cross reaction with other antigens, the effect of interdiction is prominent, the antibody of DHV that can be tested is 1:800, it is 50 times higher than agarose diffuse test, the serum positive rate is 62.5%, the positive rate of agarose diffuse test is 37.5%, the accordance rate of both is 75%, besides, the repetitive results of groups are steady. Accordingly, the adoption of Competitive latex Agglutination Text to test the antibody of DHV is specificity, sensitivity, repeatability, and the results are credibility, this method is fit for investigation of clinical epidemiology.5. The reaction condition of Indirect ELISA was succeeded in developing through a lot of experiment. The results indicated that diluting the antigen to 10μg/ml and the serum to 1:25 and the marked IgG to 1:800 were the best reaction condition. The adoption of the results showed that this method would not have cross reaction with other antigens, the effect of interdiction is prominent, the antibody of DHV that can be tested is 1:800, it is 50 times higher than agarose diffuse test, the serum positive rate is 70%, the positive rate of agarose diffuse test is 37.5%, the accordance rate of both is 67.5%, besides, the repetitive results of groups are steady. Accordingly, the adoption of Indirect ELISA to test the antibody of DHV is specificity, sensitivity, repeatability, and the results are credibility, this method is fit for investigation of clinical epidemiology.
|
PDF Full Text Request