Isolation And Identification Of New Goose Astrovirus And Preparation And Application Of Yolk Antibody

An outbreak of acute infection with gout as the main feature in goslings in Shandong and Jiangsu in China since February 2017,and it has brought huge economic losses to the goose industry,the isolation and identification of the causative agent of the disease indicates that it is a novel goose astrovirus.The disease mainly affects goslings within 2 weeks of age.The affected goslings are pathological characterized mainly by severe urate deposition on the internal organ surface and joint cavity,with mortality rate reaching more than 50%.The affected goslings are not in good spirits,lying on the ground and moving,difficulty in feeding,slow growth,increased meat ratio,and significantly reduced the rate of meat goose slaughter.Yolk antibodies are a type of Immunoglobulin found in chicken egg yolk,it refers specifically to antibodies against specific antigens extracted from immunised poultry eggs.Compared to serum antibodies,yolk antibodies are more stable,heat and acid resistant and remain active at room temperature.The yolk antibody is safe,efficient,residue-free and environmentally friendly,with a significant therapeutic effect,high specificity and can be suitable for mass production.In the study,N-GAst V was isolated from samples of clinically probable disease,the isolates were identified and analysed,the dominant strain was selected to prepare an inactivated vaccine in oil emulsion,immunised laying hens to produce yolk antibodies,and clinical application studies were conducted with the aim of providing technical support for the prevention and treatment of the new goose stellate virus.1.Isolation and identification of a Novel Goose Astrovirus.Isolation and identification of novel goose stellate virus from some of the suspected infected samples collected in our laboratory from 2019 to 2020,and animal regression experiments on the isolated strains.The results showed that seven novel goose astrovirus was isolated.The nucleotide homology of the seven viruses ranged from 96.1%to 99.9%.Homology analysis of the isolate with N-GAst V showed that it has between 96.4%and 99.0%nucleotide homology with the classic strain SDPY,indicating a relatively high level of homology,and on the same branch of the genetic evolutionary tree.The isolates are in different branches from the classical turkey,duck and chicken origin astroviruses and are more distantly related.The isolated JLCC strain was the closest relative to the SDPY strain and was selected as the vaccine strain for animal regression experiments based on its pathogenicity and immunogenicity analysis.After 1.0 m L(TCID₅₀=10^-3.22/0.1 m L)of viral solution was administered intravenously to 1-day-old fledgling geese,the goslings showed clinical signs such as depression,lying down and moving around,and reduced feeding.Through autopsy,it was found that there were pathological changes such as urate adherence and deposition on the heart,liver surface and inner thigh muscles of the dead geese,swollen and bleeding kidneys in the form of flecks,thickened ureters and urate deposition.2.Preparation of a novel goose astrovirus oil emulsion inactivated vaccine.The vaccine strain was inoculated with chicken embryo liver cells for amplification.The antigen liquid was inactivated by formaldehyde(shaken every 6-8 hours)and mixed with Tween-80 at a ratio of 96:4 to produce an aqueous phase,and the oil phase was emulsified with the water phase at 2:1 to prepare a novel goose astrovirus oil emulsion inactivated vaccine,and the physical properties of the vaccine were checked.The results showed that the best inactivation conditions for the antigen were formaldehyde at a final concentration of1.0‰and kept at 37°C for 24 hours.The physical and chemical properties of the vaccine showed that it was a water-in-oil type with good centrifugal stability and no microbial contamination such as bacteria and mycoplasma.3.Preparation of yolk antibodies to a novel goose astrovirus.Inactivated oil emulsion vaccine used to immunise laying hens.Highly vaccinated eggs were collected 14d after the fourth immunisation.The potency of the yolk antibodies was checked using an i ELISA method and the extracted yolk antibodies were prepared.The extracted yolk antibodies were evaluated for physical property test,safety test,chick protection effect test and chick treatment effect after attacking the poison.The results showed that after the fourth immunization of the hens,the potency of the in-yolk antibody was 1:1024and the potency of the prepared yolk antibody solution was 1:512.The physical and chemical properties of the antibody showed that it was a pale yellow clear liquid containing a small amount of white precipitate over a long period of time,with no microbial contamination such as bacteria or mycoplasma.The safety of the antibody is good.1.0m L was injected subcutaneously into the neck of 1 day old goslings and no local or systemic adverse reactions were observed.The i ELISA results show that the novel goose astrovirus can be effectively prevented when the OD₄₅₀value comes to the 0.8.Antibody monitoring results show that chick-specific antibodies begin to rise at 1dpi,reach a maximum at 3 days of age and then begin to decline,but can still be kept at a high level.The results of the chick attack treatment trials have shown that,in the case of naturally poisoned geese,the shortest detoxification time,high survival rate,rapid weight gain and the best treatment effect were achieved by injecting the yolk antibody once at 1 day of age and once at 7 days of age respectively.In summary,the novel goose astrovirus yolk antibody prepared has good immunoprotective effect in this study,and it has a good effect on the treatment of affected goslings and can be used as a preventive and treatment against N-GAst V.It provides a rapid and effective novel biological product for the prevention and treatment of novel goose astrovirus in China,and provides technical support for the comprehensive prevention and control of gout disease in goslings.