Research On Diagnosis And Control Of Bovine Viral Diarrhea In The Intensive Beef Farms In Henan Province

Bovine viral diarrhea (BVD) is one of the key infectious diseases caused by bovine viral diarrhea virus(BVDV), which mainly infects cattle, sheep, deer, swine, etc. Its clinical symptoms are characterizcd by fever, mucous membrane erosion, ulcer, leucopoenia, diarrhea, cough, abortion or congenitally deformed babies. Every year the number of cattle dying of BVD is no less than 5 million in the world, accounting for 0.5-1% of the total number of cattle. With the quick development of cattle industry and the introduction of foreign cattle breeds, the occurrence and spread of BVD are also increasing in China. As early as 1960s, the disease was found in areas such as Yunnan, Shandong, and Sichuan Province in China. It also emerges in Henan Province at present. In order to set up a specific and sensitive diagnosis assay, control its spreading and decrease the loss, researches have been made on the following aspects in the thesis:1. Research on Epidemiology of BVD in Henan Province671 blood samples were collected randomly from cattle farms of 15 counties in Henan Province such as Tanghe, Neixiang, etc.and 645 blood samples were collected from the intensive beef farms of Nanyang, Zhoukou and Anyang city. BVD antibody and BVDV antigen in these samples were detected respectively by BVD ELISA Test Kits, and the results showed that the positive rate of BVD antibody was 22.95%(154/671),while that BVDV antigen 15.50%(100/645). Therefore,it indicates that BVD extensively exists in Henan Province.2. Isolation and Identification of BVDV regional strains in Henan Province6 specimen collected from the doubtful BVD cases on the intensive beef farms of different areas in Henan Province were, inoculated in MDBK cells, 4 generations were reproduced blindly, and two virus strains that could cause cytopathic effect changes were acquired. Through electronics microscope observation, physical and chemistry characteristics identification,Agar gel precipitation (AGP), and Neutralization test(NT), it was confirmed that the two virus strains, named HN-1 and HN-2 strain were BVDV,. The animal regression test also verified that the two virus isolations were BVDV.3.The development of hybridoma cell strains excreting monoclonal antibody against BVDVThe BVDV HN-1 was reproduced in MDBK cells. The virus was centrifugated and purified. BALB/c mice were immunized with the purified BVDV. Using lymphocyte hybridoma technique, mouse spleen cells were fused with myelomas cells which was named NS0. Four hybridoma cell strains, named 3A8 3C7 3C11 3E9, were obtained by indirect ELISA and tri-time limited dilution. By evaluating, three strains belonged to IgGl subset, and the other one was IgG2a subset. The average chromatosome number of hybridoma cells were 99 strips. The monoclonal antibodies secreted by 3A8 3C7 3C11 and 3E9 strains had no cross reactions with BCV and BRV. The monoclonal antibody secreted by 3A8 3C7 3E9 strains had cross reactions to HCV BDV, but the monoclonal antibody secreted by 3C11 strain had no cross reactions to HCV and BDV. The specificity of monoclonal antibody secreted by 3C11 strain was better. ELISA titers of monoclonal antibodys which were existed in superior schedule of culture of hybridoma cells and ascites of mice were 1:1000 and 1 : 200000 respectively. After continuous cultivated to 20 generations, the hybridoma cell strains could still excrete antibody stablely. The monoclonal antibody was very important to the investigating BVDV and creating pykno- diagnosis method.4. The establishment and application of monoclonal antibodies double sandwich ELISA detecting bovine viral diarrhea virus3C11 which could secrete McAb stabely was anabiosised. Then the ascites were produced and the IgG was purified by the common method. Applying the rabbit-anti-BVDV IgG and McAb, the double sandwich enzyme-linked immunosorbent assay (ELISA) for detecting bovine viral diarrhea Virus (BVDV) was developed. Comparied with Virus Isolation and Neutralization Test, the positive coincidence rates were 86.67 % and 93.33 % respectively and the negative coinc...