| Chinese giant salamander (Andrias davidianus), namely Wawa-yu, is one of the rare animals and newly emerged culture species in China. Because of its high economic value, the artificial breeding and intensive farming scale of this species is developing fast Shaanxi, Hunan, Zhejiang, and Hubei provinces etc. in recent years. However, along with the rapid development of the artificial breeding and culture, the diseases of Chinese giant salamander have become the great concern. The viral hemorrhagic disease of giant salamander is one of the most severe diseases and have caused huge economic losses, but currently, no effective method is available for the prevention and control of the disease. Here in this study, the methods of inactivating the giant salamander iridovirus with P-propiolactone (BPL) and formalin were investigated respectively and the optimal conditions and efficacies were compared, and therefore the technology for preparing the killed vaccine has been determined. This study has laid a foundamental basis for the prevention and control of the disease by immunization. The contents and results of the study are as below:1. Inactivation of GSIV with BPL. The GSIV cell cultures was inactivated with BPL at different concentrations (0.025%,0.05%,0.1%,0.2%) and different time (24h,48h,72h and96h) at4℃, respectively. Then the efficacy of inactivation of GSIV was tested by means of bacterial culture, cell culture, viral DNA PCR amplification and artificial infection of healthy fish to determine the optimal conditions of inactivation. The SDS-PAGE and Western blotting were applied to analyze the change in structure protein and antigenicity of the inactivated virus. The results showed that the GSIV were inactivated completely with the final concentration of0.1%of BPL for72h at4℃. There were no bacteria and no cytopathic effect (CPE) observed both in bacteria and in cell culture. The PCR amplification with specific primer pair targeted the viral major capsid protein (MCP) gene generated no product. The artificial infection test in healthy giant salamanders did not induce the clinical signs of the disease. The results of SDS-PAGE and Western blotting demonstrated that the structure protein and the antigenicity of the inactivated GSIV treated with the final concentration of0.1%of BPL for72hat4℃had not significant change.2. Inactivation of GSIV with formalin. The GSIV cell cultures were inactivated with formalin at different concentration0.05%,0.1%,0.2%,0.4%and different time (24h,48h,72h and96h) at37℃. Then the efficacy of inactivation of GSIV was tested by means of bacterial culture, cell culture, viral DNA PCR amplification and artificial infection of health fish to determine the optimal conditions of inactivation. The SDS-PAGE and Western blotting were applied to analyze the change in structure protein and antigenicity of the inactivated virus. The results showed that the GSIV were inactivated completely with the final concentration of0.2%of formalin for72h at37℃. There were no bacteria and no CPE observed both in bacteria and cell culture. The PCR amplification with specific primer pair targeted the viral major capsid protein (MCP) gene generated no product. The artificial infection test in healthy giant salamanders did not induce the clinical signs of the disease. The results of SDS-PAGE and Western blotting demonstrated that the structure protein had obvious damage.3. Studies on immunological effect of the GSIV inactived vaccine. The giant salamander can get a better antibody level after injected two kinds of inactivated vaccine, and the strongest neutralizing capability was on21d. But the antibody level of BPL inactivated vaccine was better than the formalin. Relatively immune protection results can be seen that the BPL inactivated vaccine group received a better immune protection effect. The study of two kinds of inactived vaccine immune effect shown that the BPL destructive effect of viral antigen less than the formalin, so the BPL inactived vaccine kept better immunogenicity. |
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