| Duck-origin goose parvovirus(D-GPV)belongs to Parvovirinae,a member of Parvovirinaes,whose genome is single-stranded DNA.D-GPV infected cherry valley duck caused dysplasia,tongue outreach,growth retardation as the main feature of the epidemic disease,according to clinical symptoms was initially named duck atrophy dwarfism syndrome(BADS).Since in 2015,D-GPV has caused huge economic losses to the Chinese Cherry Valley duck farmers.Therefore,the systematic study on the infection of D-GPV in ducks and the preparation of high-price vaccines have become an urgent task.In this study,a clinically isolated D-GPV was used to artificially infect the cherry valley ducks to investigate the dynamic distribution of D-GPV in cherry valley ducks and to immunize laying hens to prepare yolk antibodies.The main findings are as follows:1 Isolation and Identification of D-GPV and Analysis of VP1 GeneThe filter-sterilized virus solution was inoculated into the 9-day-old Cherry Valley duck embryo via the allantoic cavity and passed blind for 5 generations.Viral DNA was extracted and the VP1 gene was amplified by PCR.The recovered fragment was ligated with the T vector and transformed into a self-prepared DH5? competent cell.The bacterium was shake cultured and identified as a positive clone by PCR and then sequenced and analyzed.The other allantoic fluid was used to make nine consecutive 10-fold serial dilutions and the ELD50 was calculated.At the same time,animal regression experiments were performed.The results showed that the dead duck embryo embryo edema,systemic scattered bleeding,part of the duck gills hypoplasia,deformity,bending.Sequence analysis of VP1 revealed that the VP1 gene has a size of 2203 bp and encodes a total of 734 amino acid residues.The VP1 gene has a homology of 85.4% to 99.5% with the amino acid encoded by the VP1 gene of the waterfowl parvovirus.The ELD50 is 10-6.54/0.2 m L.The results of animal regression experiments showed that after 14 days,the growth of the infected group was slower than that of the control group,and the length of sputum was shorter than that of the control group.The infected group had slight swelling of the sacrum.Through isolation,identification and animal regression experiments,the virus was identified as a strain of D-GPV and was named QH-L01 strain.2.Dynamic distribution and pathogenic pathological study of D-GPV in vivoThe allantoic fluid was divided into groups by intramuscular injection.The2-day-old Cherry Valley duck was inoculated with a dose of 106.54ELD50 0.2 m L.At the time points of 0.5h,1h,2h,4h,8h,12 h,24h,48 h,72h,144 h,216h,the cherry valley ducks were dissected to obtain organs,tissues and blood,and virus DNA was extracted-80?.Preservation and neutral formaldehyde fixation.Use VP1 sequence to establish fluorescence quantitative standard curve.The extracted D-GPV nucleic acids were quantified quantitatively by fluorescence.Paraformaldehyde-fixed tissues were paraffin sections and HE staining for pathological observation.The results showed that the establishment of a fluorescence quantitative standard curve was Ct =-3.326Ślg [virus copies]+38.902(R2= 0.997).Fluorescent quantitative results showed that D-GPV was detected only in the spleen and blood at 1 h after D-GPV inoculation,and the copy numbers were 102.86 copies/g and 102.62 copies/m L,respectively.D-GPV was detected in all tissues up to 8 h after inoculation.After 12 h,24 h,48 h,and 72 h-216 h,the highest levels of virus were detected in the lung,cecum,ileum,and duodenum,respectively 108.28copies/g,109.23copies/g,and107.1copies/g,109.83copies/g.Histopathological examination revealed 12 h lung atrophy and a significant reduction in the number of pulmonary lobule;48h meningeal inflammatory cell infiltration;48h cecal and ileal mucosal apical necrosis and loss,lymphocytes increased;72h duodenal villous epithelial cell necrosis and other pathological Variety.Visible brain,lung,cecum,ileum,duodenum is the main target organ of D-GPV,and the virus is mainly transmitted through the blood at the site of infection(muscle)through the blood circulation,to reach the initial point of infection(spleen),through the blood The circulation first proliferates to the lungs,then to the cecum,ileum,duodenum and other tissues to proliferate,and then reach the whole body.3.Preparation of anti-D-GPV yolk antibodyThe allantoic fluid was inactivated with 0.2% formaldehyde and mixed with FCA and FIA at a ratio of 1:1 to inactivate the vaccine A and the inactivated vaccine B.Ten SPF laying hens were divided into two groups.Inactivated vaccine A was injected intramuscularly in the leg of the experimental group,1 m L/body,with interval of 7days;the second injection was the intramuscular injection of inactivated vaccine B,1m L/body,interval 7 day.The third injection of inactivated vaccine with the leg muscle was immunized with B,1 m L/body.Eggs were collected after the immunization and Ig Y was extracted by water extraction.Potency was determined by agar diffusion method.The purity of the antibodies was determined by SDS-PAGE electrophoresis.The purified Ig Y was used for animal protection experiments.The results showed that the optimal aqueous extraction yolk antibody conditions were p H 5.2 and water dilution 1:8.After 3 immunizations,the titer of D-GPV yolk antibody was 1:16.The purity of Ig Y was 87.9% after the secondary ammonium sulphate purification.SDS-PAGE results showed that Ig Y showed obvious bands at 67 k Da and 38 k Da,which were Ig Y heavy and light chains,respectively.The results of animal protection experiments showed that Ig Y can effectively protect cherry valley ducks inoculated with D-GPV;the body weight of treated ducks at 9 days of age was increased compared with the infected group,and the difference was extremely significant;histopathological observations showed that the structural of main target organs for damage were restored.In summary,the present study used a D-GPV isolated from the clinic and found a dynamic distribution of D-GPV in the cherry valley duck body from muscle to lung to cecum and duodenum,and to cherry.The main damage target organs of the grain duck are lung,cecum,ileum,duodenum and other organs,and prepare Ig Y with protective effect on BADS caused by D-GPV,supplement the basic research of D-GPV,and provide clinical prevention and treatment D-GPV provides effective technology. |
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