Goose Parvovirus Attenuation By Serial Passage And Colloidal Gold Immunochromatographic Assay Establishment For Virus Detection

Goose parvovirus infection is caused by goose parvovirus(GPV), which is an acute or subacute sepsis in 4 to 20 days-old goslings characterized by high infectiousness and exudative inflammatory bowel disease. This disease spreads rapidly and has a high fatality rate, the mortality rate of goslings within 5 days-old is above 95%. The transmission and prevalence of goose parvovirus infection has caused severe economic losses and restricted the development of goose industry.A pathogenic agent isolated from goslings with pathogenic changes and clinical symptoms of Derzsy’s disease by goose embryo, it was identified as goose parvovirus and named as YG strain after PCR detection, indirect immunofluorescence assay(IFA) detection and animal regression experiment. To obtain attenuated vaccine strain, GPV YG strain was propagated alternately in goose embryo fibroblasts(GEF) and duck embryo fibroblasts(DEF). GEF infected with YG strain began to induce cytopathogenic effect after 12 passages. IFA showed that the numbers of infected cells with green fluorescence increased with the time of infection, and the highest number of positive cells were obtained at 96 h post-infection. Virus titers increased with serial passages going and virus titer reached at 106.5TCID50/m L after passage 50. Compared to parental virus, the mortality and morbidity of goslings decreased obviously after infected with cell-adapted YG strain. There were no observed characterized pathogenic changes or clinical symptoms of Derzsy’s disease in goslings after infected with cell-adapted YG strain. Faeces of goslings which inoculated by GPV F50 were detected by PCR, the results of faeces defecated at 3 to 21 days post-inoculation were GPV positive, it shows that the attenuated GPV can propagate in susceptible goslings. Goslings began to produce neutralization antibody after inoculated by cell-adapted YG strain, and the antibody titer increased with the time of inoculation. The antibody titer was 10-2.55/0.2m L at 4 weeks post-inoculation, then it decreased slightly. To identify genetic changes of the attenuated GPV, the complete viral genomes of GPV F50 and its parental virus F0 were sequenced. The results show that there were 89 nucleotide and 19 amino acids mutations between them. There were 5 amino acids mutations in the non-structural protein and 14 amino acids mutations in the structural protein. The nucleotide mutations were in the region of structural protein gene and there were two 9nt nucleotide insertions in the 5’ and 3’ non-coding region, respectively.Moreover, in order to develop the colloidal gold immunochromatographic(GICA) strip for rapid and simple detection of GPV, purified monoclonal antibody(m Ab) 2D5 against GPV VP3 protein was labeled with colloidal gold particles. The purified m Ab 4A8 and goat anti-mouse Ig G antibody were blotted on the nitrocellulose membrane as the test line and control line, respectively. The developed strips specifically detected GPV with a detection limit of 104.15TCID50/m L; the results of identical batch and different batches of strips detecting 5 positive samples were same; the positive and negative corresponding rate of strips for detecting 80 clinical samples was 96.49% and 100%, respectively.A GPV named as YG strain was isolated and identified successfully. After propagated alternately in GEF and DEF, cell-adapted YG strain with low virulence was obtained. The established GICA strip has a good specificity, sensitivity, and reproducibility, which provides a technique for rapidly detecting goose parvovirus infection.