Isolation And Identification Of Canine Parvovirus And Establishment Of Indirect Agglutination Test For Detecting Its Antibody

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV).The main clinical signs of this disease are fever,emesis,hemorrhagic colitis,leukopenia and myocarditis of puppies.At present,immunization is the main measure to prevent and control this disease.In recent years,reports of CPV infection continue to emerge due to differences in the antigenicity of vaccines,the interference of high level maternal antibody and immune antibody,or the low level immune antibody.Therefore,we should regularly isolate the local CPV and analyze the genetic evolution of their antigenicity,understand the level of puppies’ maternal antibodies of CPV in time and the immune dogs’ antibody levels of parvovirus,determine the optimal time of vaccination.These are of great significance for the prevention and control of CPV.In this study,we inoculated processed excrement from dogs suspected of being infected with CPV to F81 cells.After subculture,the CPV virus which named CPV-DN17-1 was isolated and identified by electron microscope observation,indirect immunofluorescence,PCR,and Hemagglutination identification.We cloned and sequenced the VP2 gene of the CPV CPV-DN17-1,and compared with VP2 genes of other twenty-four CPV strains,including vaccine strain,the domestic and foreign isolates recorded in GenBank.The results showed that the nucleotide homology between the isolate and the vaccine strains exist differences,which was 95.6%~98.8%.There were no significant differences of nucleotide homology between the domestic isolates and abroad isolates,which was 98.7%~99.8%.The amino acid homology between the isolate and HRB-e2(Harbin)was 100%,and the other strains were 97.9%~99.4%.Phylogenetic tree analysis showed that virus isolate was distantly related to vaccine strains,foreign isolates,other species of parvoviruses were distant,and the isolatewas closely to domestic isolates,indicating that the isolate strain was not derived from vaccine strains.We compared the amino acid array of virus isolate with the reference strains in order to analyze the sites of AA mutation of VP2,then the genotype of the isolate was identified as new CPV-2a;compared the isolate with the vaccine strains through antigenicity analysis,we found that there is no obvious change in main epitope.In this study,two pairs of primers sVP2-F1/R1 and sVP2-F2/R2 of VP2 recombination gene were designed and synthesized in the coding region of the major antigenic epitopes of CPV-DN17-1 according to references and analysed by DNAStar-Protein software,and then recombinant plasmids pGEX-VP2-S1 and pGEX-VP2-S2 were constructed.Recombinant proteins named rVP2-S1 and rVP2-S2 were induced,expressed and identified.The polyclonal antibody prepared by using the recombinant protein was detected by ELISA and neutralization test.The results showed that the Indirect-ELISA titer of recombinant protein rVP2-S1 was 1:12800,and the neutralization titer was 1:128;the Indirect-ELISA titer of recombinant protein rVP2-S2 was 1:6400,and the neutralizing titer was 1:54.The red nanospheres of polystyrene with carboxyl were sensitized by recombinant protein rVP2-S1.The sensitization conditions were optimized by screening the allergenic protein concentration,sensitization buffer,and EDC content.The results showed that the agglutination of sensitized colored microspheres and CPV-positive serum was the most significant when the nanospheres were 25 μL(5% w/v),the amount of allergenic protein was 0.3 mg,EDC was 0.015 g,and acetic acid buffer was 500 μL.The agglutination result was calibrated by the test results of the kit.The criterion for the determination of the results was that if sensitized microspheres were more than 50% aggregated,the serum was positive,which means this serum antibody have immune protective effect on dogs.The result was negative if the agglutination was less than 50%,which means this serum have no immune protection.The experiment proved that the method is specific,sensitive,reproducible and stable.The coincidence rate was 97.5% between the established method and kit by detecting 40 serum samples collected from clinic.In this study,we established an indirect agglutination test for CPV antibody detection,this methodhas good specificity,sensitivity,stability,and can meet the clinical needs for detectingsingle serum sample.It has significant value in clinical application.