Isolation And Identification Of Goose Stellate Virus And Preparation And Quality Evaluation Of Its Yolk Antibody

Goose Astrovirus(Gastv)has been identified as the pathogenic cause of viral Goose gout in recent years.Since 2017,outbreaks of Gastv have occurred in many areas in China.Gastv mainly infects goslings aged from 5 to 20 days and causes a large amount of urate deposition in joint cavity and viscera,causing great economic losses to goose raising industry in China with a high mortality rate of 10%to 30%.In this study,the liver and kidney tissues of suspected gout dead geese in a goose farm in Liaoning Province were collected.After positive astrovirus detection by RT-PCR,the tissue suspension was inoculated into susceptible goose embryos for virus isolation.The isolated virus was identified by RT-PCR identification,ORF1b gene sequencing,phylogenetic analysis,determination of half lethal dose of goose embryo,hemagglutination test,purification test and animal regression test.The results showed that after the goose embryos were inoculated into the allantoic cavity for 4 generations,the goose embryos began to die stably,and the time of death was mostly concentrated in 72-120 hours after inoculation,and the mortality rate reached 90%-100%.The allantoic membrane of the dead embryo thickened,edema and bleeding all over the body,and the half lethal dose(ELD₅₀)of goose embryos was?10^5.0ELD₅₀/0.3m L.The isolated virus bacteria,mycoplasma and exogenous virus were all negative and had no hemagglutination characteristics,so the isolated virus was named goose astrovirus DT strain.The ORF1b gene sequence of DT strain was compared with that of other Ast V isolates collected in NCBI and phylogenetic analysis showed that DT strain had high homology with goose astrovirus GD strain,SD01 strain and SDPY strain,with 99.1%-99.8%nucleotide homology and 100%amino acid homology,while only58.8%-67.6%nucleotide homology with turkey,chicken and goose ASt V FLX strains.The homology of amino acid is only 55.6%-68.7%.The results of genetic evolution analysis showed that DT strain was in the same clade with goose astrovirus strain GD,SD01 and SDPY,but different from turkey origin,chicken origin and goose origin ASt V FLX strain.The DT strain was inoculated into 1-day-old healthy goslings for animal regression test.The goslings began to detoxify from the 3rd day after infection,and the peak was from 6 to 10 days after infection.On the 5th day,the goose began to die,and the mortality rate was 30%.The body mass of the infected geese was reduced,and autopsy showed that there were serious urate deposits on the surface of internal organs such as heart,liver and gallbladder,and kidney swelling.The isolated and identified GAst V DT strain was used as virus seed,the susceptible goose embryo was inoculated,and the goose embryo liquid was harvested as the virus solution to prepare GAst V inactivated vaccine.The layers were immunized with GAst V inactivated vaccine according to different immunization procedures,and the best immunization procedure was selected by comparing the amount of immunogen and the titer of antibody in yolk after immunization,and the optimum extraction methods and parameters of yolk antibody were screened and optimized by acidified water dilution method,caprylic acid method,sodium alginate method and acidified water dilution-caprylic acid method.The inactivation process of yolk antibody was screened by pasteurization,filtration and formaldehyde inactivation,and the concentration process of yolk antibody was screened by hollow fiber filter column and tangential flow ultrafiltration membrane stack.Three batches of goose astrovirus yolk antibodies were prepared according to the optimized production process,and the contents of character,asepsis,mycoplasma,exogenous virus,caprylic acid and formaldehyde were tested.The safety of egg yolk antibody was evaluated by single dose,single dose repeated injection,overdose injection and non-target animal mouse injection,and the efficacy of egg yolk antibody was evaluated by the minimum preventive dose of antibody,the relationship between antibody titer and challenge protection,the duration of passive immunity,the time of immune protection and so on.The results showed that the best immunization procedure was as follows:subcutaneous injection of GAst V inactivated vaccine in healthy laying hens,basic immunization of 1.0m L/goslings,booster immunization of 1.5m L/goslings,intensive immunization of 2.0m L/goslings at an interval of 21 days,14 days after enhanced immunization,the neutralization titer of yolk fluid was not less than 1:1024,which could be used to extract antibodies and maintain immunity when the neutralization titer was less than 1:1024.The best preparation method of egg yolk antibody is acidified water-caprylic acid method,the best dilution of acidified water is 1:8,the p H value is 5.2,the best precipitation time is 8-15 hours,the best concentration of caprylic acid is 1.0%,and the best action time is 4 hours.The indicator microorganisms added in egg yolk can be completely inactivated by pasteurization,0.2%formaldehyde and 0.22?m filtration.Three batches of goose astrovirus yolk antibodies were prepared according to the determined production process of egg yolk antibodies,and all the test results were qualified.The results of single-dose,single-dose repeated,over-dose injection on target animals and safety tests on non-target mice were all safe,and the neutralization titer of three batches of egg yolk antibodies against goose astrovirus was positively correlated with challenge protection,and the protection rate of goslings inoculated with antibodies with neutralization titer not less than 1:256 was more than 80.0%.The minimum preventive dose of 1-3-day-old goslings was 0.3m L/goslings,and that of 4-day-old goslings was 0.5m L/goslings.The earliest time for antibody to produce immune protection was 24 hours after injection,and the passive immune protection period was 7 days.The purpose of this study is to develop a safe and effective egg yolk antibody against Goose Stellus Virus,and to provide technical support for strengthening the comprehensive prevention and control of Goose Gout in China.