| Bovine viral diarrhea/mucosal disease (BVD/MD) is caused by infection of cattle with bovineviral diarrhea virus (BVDV).BVDV can cause Diarrhea,reproductive disorders,immunesuppression,persistent infection and severe mucosal disease.The disease mainly infected cattle,especially calves,can also infect other ruminant animals and humen beings.BVDV hasserological cross-reactions with other members of pestiviruses,it caused great difficulties fordisease diagnosis and vaccine,and has a serious impact on chinese cattle industry.At present,thereis no vaccine against BVDV.Therefore the establishment of a fast and effective method ofdiagnosis is particularly important.BVDV E2protein is an immune envelope protein,as diagnostic protein,can stimulate theproduction of neutralizing antibodies.as BVD diagnostic protein.In this study,the E2protein wassuccessfully expressed and purified by insect cells eukaryotic expression systems.Proteinconcentration was360μg/mL after the identification of indirect immunofluorescence andWestern blot.Using the recombinant and purified E2protein as antigen,an indirect ELISA wassuccessfully developed to detect antibody to BVDV.The optimal working parameters weredetermined as follows:the recombinant E2protein antigen for coating at6μg/mL,testing seradilution at1:80.the optimization of optimum sera reaction time at60min,the optimum Rabbitanti-bovine IgG/HRP concentration at1:10000,the optimum substrate reaction time at15min.The optimal coating buffer was carbonate buffer solution.The best blocking reagents was5%skim milk.Compared with foreign commercial kits,the total positive rate is91.5%.Therefore,inthis paper,BVDV E2indirect ELISA diagnostic method we have established,can be used as thedetection method of BVDV.In order to understand the dairy cow bovine viral diarrhea prevalence,1485dairy cow serumsamples were surveyed by the established BVDV antibodies indirect ELISA method in parts ofHeilongjiang in2012to2014.The results showed that the percentages of the positive serumsamples having the antibody to BVDV were59.36%,61.7%and70.33%from2012to2014,andthe average total positive rate is64.10%.The results demonstrated that the infection rate ofBVDV was increasing in Heilongjiang Province. The Statistical Analysis of dairy cows BVDprevalence in Heilongjiang found that the west higher than the east,high infection in Autumn andwinter, the backyard higher than dairy farms. |
