Isolation And Identification Of Canine Distemper Virus And Establishment Of Indirect Agglutination Test For The Detection Of Its Antibody

Canine Distemper virus(CDV)is the pathogen of canine distemper in fur animals.Taking the pet dogs for example,vaccination is currently the main measure to prevent and treat the disease.Due to the differences of vaccine antigenicity,the interference of high level maternal antibody or immunized antibody,or low level immunized antibody,cause the failure of immunization and disease.The rational immune timing should be based on the results of antibody level detection.Enzyme-linked immunosorbent assay,serum neutralization test,indirect immunofluorescence test and so on have been used in the detection of CDV antibodies in clinic.However,these detection methods are complicated to operate and special instruments are needed,which affect the popularity of the antibody detedtion Therefore,isolation of local prevalent CDVand analysis of their genetic evolution of antigenicity are necessary for choosing right vaccine;and to establish a simple,specific,for single serum sample and economical antibody detection method is also necessary and is of great significance to prevent canine distemper effectively.In this study,MDCK cells were inoculated with the conditioning fluid of intestinal content forma dead dog clinically diagnosed as CDV.After subculture and electron microscope observation,indirect immunofluorescence,detection of RT-PCR,we confirmed that a strain of CDV was isolated and named CDV-DN17-1.The nucleotide sequence of H gene was analyzed,the nucleotide homology of H gene was 99.8% with that of HeB(09)3 strain which was the most similar,and the highest homology was 96.1% with CDV vaccine strain Rockborn.The phylogenetic tree analysis the evolvement of H gene of isolated strain was most closely related to HeB(09)3 strain and belonged to Asia-1 type.The neutralization test of canine serum of immune dogs and isolated virus was carried out.The results showed that the CDV antibody produced by the current vaccine strain have neutralization to isolated strain CDV.According to the analysis of CDV-DN17-1 H gene sequence,two segments from antigen region of H gene were cloned in this study,two recombinant proteins,rCDV-SDH1 and rCDV-SDH2,were obtained by prokaryotic expression,and antiserum were prepared by immunizing rabbits.Neutralization test showed that the neutralization value of serum antibody prepared by recombinant protein rCDV-SDH1 was 1: 35,which was higher than that of serum antibody prepared by rCDVSDH2.Therefore,rCDV-SDH1 was chosen as the detection antigen of agglutination test.The purified rCDV-SDH1 protein was coupled with red nanospheres of polystyrene with carboxyl,after optimization of coupling conditions,sensitized and immunized colored nanoparticles were prepared.We consider the sensitive microspheres as agglutinogen and the positive serum of CDV as agglutinin.The indirect agglutination test for CDV antibody was established.We ensured the positive criteria of the indirect agglutination test by comparing the test results of the serum samples with the commercial kits,the serum that can cause agglutination of the sensitized microspheres(++)is a positive serum.This method has good specificity,only CDV positive serum can react agglutination;the results of agglutination test are all positive for positive serum which their detection value of kit are equal or greater than 3;the agglutination results with sensitized microspheres that are stored for 1 to 3 months at 4 ? remain unchanged.The results of preliminary testing for 40 clinical serum samples showed that the indirect agglutination test established in this study and the detection result of kit was 100% compliance.In this study,a strain of CDV was isolated from the intestinal content of deaddog,and the variation of H gene in the current epidemic strain was preliminarily analyzed,which provided a reference for choosine right vaccine;the indirect agglutination test for detecting CDV antibody established in this study was specific,simple to handle and easy to judge the reslts,it can be used not only for the centralized detection of a large number of samples,but also for a single serum sample of clinicl dog in time.