| Infection with porcine circovirus type 2(PCV2)causes immune dysfunction and immunosuppression in the body,resulting in secondary and mixed infections with other pathogens and occurrence of diseases,which give great losses to swine industry.PCV2 persistent infection caused a high positive rate of serum antibodies in pigs,and the antibodies produced by vaccination could not be effectively distinguished from those produced by natural infection.It has became the current biggest challenge of vaccine immunization prevention and control.In view of this,it was found that the existence time and state of PCV2 specific IgM antibodies play an important role in distinguishing vaccine immunity from viral infection by monitoring the dynamic changes of IgG and IgM antibodies in pigs.Based on this,the capture ELISA method for detecting PCV2 IgG antibody based on SPG and the capture ELISA method for detecting PCV2 IgM antibody with anti-pig IgM polyantibody as capture antigen were established,respectively.The diagnosis results of the two antibody detection methods can provide scientific guidance for judging the status of swine infection and the timing of vaccination.The content of the study can be divided into the following three parts:1.To analyze the regularity of IgG and IgM antibody growth and decline after PCV2 infection and immunization in pigsWe used PCV2 commercial inactivated vaccine and cell-isolated virus to immunize and infect antibody-negative pigs respectively,and collected peripheral blood and separated serum regularly after inoculation.The commercial INGEZIM IgG/IgM Kit was used to detect the serum samples.By monitoring the dynamic changes of the antibodies,it was found that the IgM antibody was first produced after immunization and infection,then transformed into IgG antibody and persisted for a long time.The duration of IgM produced by PCV2 cytotoxicity after artificial infection lasted for 20-30 d,and the duration of IgM produced by natural infection(positive control of the infection group)existed for 55 d,while the duration of IgM in the immune group inoculated with inactivated vaccine was less than 14 d.PCV2 IgM antibody production characteristics can optimize the immune process and distinguish between virus infection and vaccine immunity.The first immune should avoid IgM antibody positive phase.After 7-14 d,the IgM antibody becomes negative after immunization.If the test result is positive before the next vaccination,then a new round of PCV2 infection must occur,and additional immunization is not recommended at this time.2.A capture method for detection of PCV2 IgG based on SPG ELISA was establishedBy comparing three immunoglobulin binding proteins SPA,SPG and PPL as capture antigens,and HRP labeled PCV2 Cap as the second antibody,it was determined that SPG was the optimal coated antigen.After the reaction conditions were optimized,the cut-off value of ROC curve was 0.61,when the sensitivity and specificity were 93.9% and 90.3%,respectively.The total coincidence rate between this method and commercial INGEZIM Circovirus IgG Kit was 96.23%.3.A capture method for detection of PCV2 IgM antibody was establishedBy using anti-porcine native IgM polyantibody as coating antigen and HRP-labeled R2346 as secondary antibody,a ELISA method was established for detection of IgM antibody against PCV2.After optimizing the optimal reaction conditions,the ROC curve was used to determine the cut-off value of 0.492,the corresponding sensitivity and specificity were 96.7% and 97.2%,respectively,and the coincidence rate between IgM and INGEZIM IgM Kit kit was 92.7%.In this study,the capture ELISAs for detection of pcv2 IgG and IgM antibodies were established.It provide a simple and effective way to optimization of vaccine immunization procedures,early rapid diagnosis of PCV2 and distinction between viral infection and vaccine immunization.,which lay the reliable basis for further control of the spread of PCV2 in pig farms. |
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