Isolation And Identification Of Bovine Parainfluenza Virus Type3 And Establishment Of Indirect ELISA Method And Investigation Of Epidemiology

Bovine parainfluenza is an acute,contiguous,respiratory tract infectious disease,caused by infectious bovine parainfluenza virus type 3 (BPIV3), which has brought about enormous disservice for the cattle industry. In the mid June of 2008, a bovine disease, likely due to infectious bovine parainfluenza (BPIV3),infection was widely spread in cattle herds of Heilongjiang province.To isolate BPIV3,samples were collected from sick cattle. Using purified recombinant N protein established a indirect ELISA methods and make a preliminary epidemiologic survey by it. Specific primers were designed and synthesized according to the reported nucleocapsid (N) protein gene seguence from Bovine parainfluenza virus type3 in GenBankTM. Total RNA was extracted from the supernatant of cell culture from BN-1 strain. The size of 704 bp fragment was amplified.Make use of specificiry,sensibility and repeatability established the RT-PCR of Bovine parainflunza virus type 3.Clinical samples were collected from nasal secretions of an invasive bovine with high fever. The samples were cultured in MDBK cell line and isolated a strain virus. Physicochemically,RT-PCR and sequence analysis the segregation strain is bovine parainflunza virus type 3.Total RNA was extracted from the supernatant of cell culture. Specific primers were designed and synthesized according to the reported nucleocapsid (N) protein gene seguence from bovine parainfluenza virus type 3 in GenBankTM. The gene of N protein was amplified by RT-PCR from the total RNA, then cloned into pMD18-T vector. The positive plasmid pMD18-T-N was got by identification. Then the gene of N protein was subcloned into the downstream of the pGEX-6P-1 vector named as pGEX-6P-1-N. The nucleotide identity of the N genes between BPIV3 strains in GenBankTM database and the isolates ranged from 83.5% to 99.4%. The positive plasmid was transformed into the optimization competent E. coli BL21(DE3) and induced by 1mmol/L IPTG. SDS-PAGE result showed that the gene of N protein was expressed at high level in prokaryotic expression system. Additionally, the western blot assay proved the recombinant N protein of BPIV3 DQ strain having good reactionogenicity.Taking the purified recombinant N protein as antigen, We established indirect ELISA method for detecting BPIV3 antibody. To validate the assays, the ELISA was performed with standard positive and negative sera of BPIV3. Modified reaction condition of ELISA:the optimal coating antigen for microplate was 7.5μg/mL; Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The best serum dilution buffer was 5% skim milk, and the optimal dilution of sera samples was 1:100. The working concentration of HRP-labeled rabbit anti-bovine IgG was 1:5000. There is no any cross reaction between the antigen and the positive serum of IBRV, BRSV,BCV. The results showed that the specificity of the developed indirect ELISA. A Total of 603 sera of cattle, which obtained from some area in Heilongjiang province were tested by developed indirect ELISA method. The result indicated that the overall seroprevalence of BPIV3 in cattle in some regions above in Heilongjiang provienc is 78.44%.Results of this investigation is the first time show that the bovine parainfluenza virus type 3 is existence in China. The research is usefull for epidemiology,immunology,mechanism of pathopoiesis,immunoprophylaxis and it can be a technique support for the rapid diagnosis and prevalence survey of BPIV3 in china.