Isolation of Newcastle Disease Virus and Its Establishment of Immunohistochemistry Assay
|School||Jilin Agricultural University|
|Course||Basic Veterinary Science|
|Keywords||Goose NDV isolation and identification pathological changes immunohistochemistry|
ND mainly infected birds, which is a sick type of highly contagious deadly infectious diseases.Because of disease acute, high death rate, the development of the poultry industry pose a serious threat. The World Organization for Animal Health (OIE) reported the disease as a statutory requirement. NDV infection of different types of wild birds generally have different clinical manifestations. Conditions in captivity, the species of birds can be infected with NDV and disease. ND on geese, ducks and other waterfowl in the pathogenicity of major changes have taken place in China, and its related public health problems should also be highly concerned about.The incidence collect diseased geese of a zoo in Jilin Province. By the way of isolating SPF chick embryo, morphological observation of electron microscopy, HA and HI identification, we designed a pair of primers, which in the conserved region of gene F in NDV. Then identified it as ND virus by the way of RT-PCR, named NDV JL/1/10. The virulence of the isolates were determined, so value of ELD50, MDT, ICPI and IVPI were 10-7.25,58.5h,1.84 and 2.18. In terms of biological characteristics which show that the Newcastle disease virus is of strain virulent.The rabbit anti-NDV serum had been prepared successfully, and the antibody titers could reach to 1:27. Preparation of rabbit anti-NDV hyperimmune serum, by bitterness-saturated ammonium sulfate precipitation and DEAE-52 cellulose anion exchange method which combines the purified antibodies. The antibodies with high purity and specificity were observed at the band of about 50KD by SDS-PAGE electrophoresis. Establishing and improving the SP immunohistochemical method of Newcastle disease virus in tissue distribution Orientation, optimizating of immunohistochemical methods in a variety of conditions. Trialing to make sure that 0.01M PH6.0 citric acid buffer is antigen retrieval solution and that antigen retrieval method for high temperature and pressure. Using 0.01M pH7.4 PBS-T as washing liquid, 0.05%Tween-20 as antibody dilution. The working concentration of the first antibody is 1:100. Then we established strong stabilited, high sensitivited, high reproducibilited method for immunohistochemistry.Pathological changes in the geese is about gross and histological lesions from a dozen organs involved of liver, spleen, lung, glandular stomach, small intestine, etc. General disease showed that glandular stomach, small intestine, cecum mucosal bleeding, which are obviously. Histological showed that liver, spleen, pancreas and small intestine with severe pathological changes of necrosis. The glomerular decline sharp, with various epithelial cells and lymphocytes significantly degeneration and necrosis. SP immunohistochemical method was established and optimized, we viraled antigen distribution in various tissues was detected compared with H.E. staining from histopathology. The immunohistochemistry showed that NDV infection was positive in many geese organs (e.g. respiratory system, digestive system, and immune system). The detection rate of NDV infection in trachea, lung, glandular stomach, thymus, intestine, spleen et. al is higher than other organs. Viral antigens mainly exist in the cytoplasm of various epithelial cells, liver cells, and reticular cell. Immunohistochemical staining intensity was compared with pathological changes of tissues caused by a virus. The results showed that geese source NDV could replicate in various geese organ cells, resulting in extensive tissue damage.This study provide the corresponding reference for basic research of infection in wild waterfowl in the pathogenesis of Newcastle disease and viral antigen in infected animals the distribution of the body. The tissue tropism of NDV to geese was revealed in the cellular level.