Identification Of Duck Hepatitis Virus Isolates And Development Of Egg-yolk Antibodies

Duck virus hepatitis was caused by duck hepatitis virus which belongs to picornaviridae enterovirus. It was an acute infection disease, which were characterized by acute incidence, short pathogenesis and high mortality. Spasm was typical feature of the disease. The main pathology of the disease was enlargement and hyperemia of the liver. Duck virus hepatitis was caused by three kinds of virus which were DHV-Ⅰ,DHV-Ⅱand DHV-Ⅲ. There was no antigen dependability among the three kinds of type. The major virus strain in China was duck virus hepatitis typeⅠ. However, new disease which like duck virus hepatitis successively broke out in duck flows which were inoculated with DVH typeⅠvaccine or egg yolk antibodies against DVH typeⅠin recent years. It was doubtful of an variant of DHV typeⅠor new type DVH. It was the biggest trouble maker to the development of the poultry industry.Biology character,serotype and toxicity of a strain of suspected duck hepatitis virus were tested ,which was separated from clinical dead duck having the symptom of duck virus hepatitis. RT-PCR method was established to identify the virus by type 1 and novel DHV primers respectively. Neutralization test was also carried out with type 1 and novel DHV positive serum. Furthermore, tests of duckling toxicity and inoculation with the virus to duck embryo liver cell were also done according to the ELD50 and LD50. The liver and spleen from the dead duckling inoculated with the virus were prepared for tissue slices, and the histopathological changes were observed clearly. Results indicated that:A round non-enveloped virus showing the size about 40nm could be observed by electron microscopy. Expected fragment about 705bp matching with novel DHV was amplified by RT-PCR, and ELD50 of the virus was 10-5.7/0.2mL. The isolated virus could not be neutralized by type 1 DHV positive serum, while neutralizing titer of novel DHV positive serum to the isolated virus was about 1:200. Typical symptoms and CPE could also be observed obviously when inoculated the isolated virus to ducklings and duck embryo liver cell. All the results showed that the identified novel DHV could be development and application as a vaccine candidate strain. The isolated virus was used for preparation of DHV tissue deactivation vaccine. High layers was injected by the vaccine, and their eggs were used to prepare highly immune yolk antibody. Antibody concentration was determined by neutralization test. The extraction method of yolk antibody and the save methods of yolk antibody had also been examined. Reasonable immune program was that the vaccine was injected in neck subcutaneous with 2.0mL for the first time; one week later, the vaccine was injected in left groin with 2.5mL for the second time; one week later, the vaccine was injected in right groin with 2.5mL for the third time. The eggs were qualified and collected from 5th to 8th after the first time immune. The optimized extraction method of yolk antibody was the method using isotonic Na chloride. The optimized preserving methods of yolk antibody were lyophilized or saved in -20℃.The yolk antibody were used for treatment of DVH. Results indicated that:When antibody titer reached 27,it had a good treatment. It could also saved ducking which had been injected by 10 000LD50DHV. Duckling which had used the yolk antibody, could be safe through susceptible period. The best method of treatment was injected in muscle. Effection on protection from DHV depends on the time of treatment: the sooner the better, especially with in 24 hour .also,this jolk antibody has a good therapeutic efficacy on Cohabitation infectionThe test focused on the development and preparation of DHV vaccine & yolk antibody at the basis on a clinical case of DVH, has provided a technical and theoretical tool for lacal disease result from DHV.