Separation And Identification Goose Parvovirus WH-11Strain And Development Of Sandwich Elisa For Goose Parvovirus

Gosling plague (GP) or Derzsy’s disease is caused by gosling plague virus (goose parvovirus). GP is a highly contagious disease in gosling and muscovy ducklings. The disease spreads fast. The morbidity and mortality are90-100%. With the growing of goslings, the morbidity and mortality declines. The day-old of maximum incidence is60days, but the symptoms are mild and the course is long and the mortality is low. The disease occurs the whole year. Because of the different of raising pattern in southern and northern China, the time of the gosling plague epidemic is different. A large surface necrosis of the small intestine and exudate cemented the pseudomembranous emboli clogging in the small intestine occurred.Currently, the disease is still the most important infectious disease in the goose industry and intensive farming Muscovy.The disease can cause significant harm.We carried on the isolation and identification of the GPV. We learned the GP epidemiological and etiological data. The study provided the basis for clinical diagnosis.We amplified the gene sequences of GPV and analyzed the sequence.The study provides the basis for studying the molecular epidemiology of goose parvovirus. In this study, we constructed the recombinant plasmid and expressed the structural protein VP3. The study conducted goose parvovirus double-antibody sandwich ELISA method.The primary purpose of the method is to provide testing services for the grass-roots farmers.The method is the basic diagnostic method of GP.We can prevent GP in time.The main research content is composed of the following experiments. We carried out the preliminary diagnosis of the suspectedcases of gosling plague from Wuhe farm based on clinical symptoms andpathological manifestations. We successfully amplified virus from goose embryoes. The hemagglutination test, agar diffusion test, PCR method, and regression test were used to confirm the goose parvovirus. This virus strain was named as GPVWH-12. We amplified and sequenced the whole genome of GPV. Sequence analysis showed that each group of virus gene sequence highly homologous. The nucleotide sequence of GPVWH-12shares the highest similarity (99.6%) with KC478066. It shares93.8%similarity with HQ891825.Phylogenetic analysis showed that GPVWH-12locates in the same branch with KC478066. The nucleotide sequence and amino acid sequence of NS1and VP1have a high homology. We analyzed the postion of the virus strain in the genetic evolution. We amplified the the genome of VP3protein to construct the recombinant plasmid PcoldI-VP3. The recombinant VP3was expressed and purified from E. coli expression system. Polyclonalantibody of VP3was prepared by immunizing rabbit. The antibody was further purified by protein G chromatography. In order to develpe double-antibody sandwich ELISA method for clinical diagnosis, we screened the coating liquid, the concentration of enzyme-labeled secondary antibody, blocking liquid and blocking time. We carried on specific testand sensitive test for identification the method and compared with PCR method further. The results showed that:Coating liquid is ddH2O. The concentration of enzyme-labeled secondary antibody is1:1500. Blocking liquid is1%gelatin. Blocking time is20min. Specifictest showed good specificity.GPV does not cross-react with gooseinfluenza. The coincidence of ELISA and PCR method was100%.