Rapid Dose Detection Method Development And Clinical Immune Effects Comparison Of Classical Swine Fever Vaccine From Different Manufacturers

Classical swine fever(CSF)is a severe infectious disease caused by classical swine fever virus(CSFV),and vaccination is an effectual measure to prevent this disease.There are many manufacturers that produce the CSF vaccine,but there are evident differences in the doses of the vaccine from different manufacturers and may take effects on the clinical immune effects.In this study,a fluorescent quantitative(FQ-)PCR method for quick detection of the dose of the CSF vaccine was developed to determine the virus load of the CSFV vaccine used commonly in Beijing,Tianjin and Hebei from six different manufacturers,the profile of antibodies against the CSFV in sera of pigs immunized with the CSF vaccine with relatively high and low dose from two manufacturers,and the differences in clinical immune effects between the two kinds of CSF vaccines were analyzed.A fragment of 200 base pair specific to the HCLV genome was amplified by RT-PCR and cloned to a T vector,and then a recombinant plasmid with the gene fragment was constructed.The recombinant plasmid was diluted serially as templates for SYBR Green Ⅰ FQ-PCR to draw a standard curve for quantitative detection of HCLV.During the templates concentration of(6.4×10~7~6.4×10~2)copies per microliter,the amplification efficacy was 92.2%,the coefficient of determination for the developed standard curve was 0.9997.The accurate sensitivity of the generated assay was 6.4×10~2 copies per microliter,and its coefficient of variation was less than 2% in the reproducible assays.The detection results of bovine viral diarrhea virus,porcine reproductive and respiratory syndrome virus and other epidemic swine RNA viruses with this assay were all negative.The results indicated that a FQ-PCR method for quantitative detection of HCLV with good specificity,sensitivity and repeatability was established.The virus load of each doses of the CSF vaccine used commonly in Beijing,Tianjin and Hebei from six different manufacturers were detected with this FQ-PCR method,and the results demonstrated that there were significant differences in the load of the HCLV from different manufacturers.The CSF vaccine E with relatively high dose and the CSF vaccine A with relatively low dose were selected to carry out a clinical immunization test in a pig farm with 900 basic sows.Sixty non-pregnant sows after weaning were divided randomly into 2 groups averagely,each was immunized with the CSF vaccine E(group E)or the CSF vaccine A(group A),and the second vaccination was conducted 6months later.After the parturition of sows,their offspring were immunized with the related CSF vaccine at 20 and 60 days of age,respectively.The sera of sows at non-pregnancy,55 days post pregnancy and14 days post parturition and the sera of piglets at 14,35,and 56 days and the sera of growing pigs at 77,98,119 and 140 days of age were collected,and the antibodies against CSFV in sera were determined with blocking ELISA respectively.The results showed that there was no difference between the two groups at the phase of the non-pregnant sows after weaning.At the other experiment period,the antibody blocking rate of CSFV in group E were all higher than those in group A,and the differences were all significant(P<0.05);the coefficient of variation of the antibody blocking rate of CSFV in groupE were all less than those in group A except the piglets aged 35 and 56 days.The positive rate of CSFV antibody in group E were all higher than those in group A,and the difference was significant(P<0.05)at the piglets aged 35 days.The antibody results indicated that the data of antibody to CSF vaccine in group E were better than those in group A,which revealed that the clinical immune effects of the CSF vaccine E was better than the vaccine A,and suggesting that the clinical immune effects of the CSFV vaccine with relatively high dose was better than that with relatively lows.In summary,a FQ-PCR method for quick detection the dose of the CSF vaccine was established and the differences in the dose of the CSF vaccines from different manufacturers were obvious,the clinical immune effects of the CSF vaccine with relatively high dose was better than that with relatively lows.These results provided an experimental base for the clinical use of the CSF vaccine with dose difference from different manufacturers.