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

Bovine respiratory disease complex (BRD) is the leading cause of morbidity and mortality worldwide, and contributes to greatly economic loss. Parainfluenza virus type 3(BPV-3)is one of the major etiological agents. Objective of the study was to isolate BPIV-3 from the dairy cattle with BRDC, as well to establish indirect Enzyem Linker Immuneasorbent Assay (ELISA) with the recombination HN protein as the antigen. The dynamic of antibody titer in the infected dairy cattle was measured by viral neutralization assay and indirect ELISA.The optimized method for monitoring neutralization antibody was set up.The milk and blenna narium of fever symptomatic dairy cattle were inoculated into MDBK monolayer cells to isolate virus.The virus was identified by presence of the CPE and electron microscope, physical and chemical characteristic of virion particles, hemagglutination test, viral neutralization assay and RT-PCR methods. A pair of special primers for expressing HN protein was thesized and the HN gene was amplified by RT-PCR method followed by construction of the expression vector PQE30-HN. Recombination protein HN was expressed in E. coli XL1blue by addition of IPTG and was confirmed by SDS-PAGE and Western Blotting. The indirect ELISA was established under optimized parameters including antigen concentration, antigen coating period, blocking buffer, sensitivity and reproducibility。The virus from cattle could be neutralized by BPIV-3 positive serum and gene sequence shared 99.8% identity with counterpart of BPIV-3.Result of neutralizing antibodies monitor showed that titer of antibody in rehabilitation was four times higher than it in acute stage. Expression vector of pQE30-HN was successfully constructed in E.coli XL1blue host cells and 40 kDa recombinant protein was generated upon induction of IPTG at 37℃.The activity of recombinant protein was confirmed by Western-blotting using BPIV-3 positive serum, concentration of antigen(6μg/ml), serum dilution(1:50), blocking solution(5% skimmed milk powder), time of blocking(37℃60min), concentration of second anti(1:10000) and critical value(OD450 0.297) were set under optimization parameters which may be the key factor in ELISA.The recombinant protein reacted with serum against BPIV3,but failed to react with sera against bovine coronavirus, bovine coital exanthema virus and bovine respiratory syncytial virus respectively. Sensitivity test of indect ELISA conferred 2-4 tites higher than virus neutralization test. Antibody monitor in cattle population suggested that neutralizing antibody titers lay at range of 1:32-1:128 for 6-8 months, then it declined to 1:8-1:16. In this study, BPIV-3 was isolated, and certified as one of pathogens of BRDC in China. An accurate indirect ELISA was developed using recombinant HN protein expressed in E.coli, which could be used to conduct epidemic study.