Development And Quality Assessment On The Yolk Antibody Against H Strain Gosling Plague Virus

Gosling plague,also known as goose parvovirus disease,is highly contacted and septic infectious disease caused by goose parvovirus,which is the main infectious disease that endangers the goose industry.In recent years,according to the analysis of genetic evolution,the virulence of gosling plague virus presents an increasing tendency,bringing greater challenges to the prevention and control of the disease.At present,the specific prevention and treatment of GP mainly depend on passive immunity with yolk antibody（IgY）,exhibits protective effect and obvious therapeutic effect on infection of GP.Due to the advantages of low price and good control effect,IgY has been accepted and adopted by the majority of goose households.But at present,most of the IgYs in application are crude antibodies,which lack of unified production and test standards,as well as the titer of antibody varies.Therefore,it is imperative to develop a GPV IgY with high purity and titer,good treatment and prevention effect,and a unified production standard.In this study,we collected the sick materials suspected to be GP dead geese from the geese raised in a certain place in Heilongjiang Province,separated and identified GPV.The anti GP IgY was developed based on the separated GPV,and its quality was evaluated.The specific research contents and methods are as follows:（1）Isolation and identification of GPVThe virus was isolated from the liver and spleen tissues of dead geese suspected to GP by chloroform extraction.The virus was isolated through inoculating 3-days goslings and 12-days geese embryos.The comprehensive identification was performed on isolated virus,including specific test（AGP,neutralization test of goose embryo）,ultrastructural observation,toxicity determination,physical and chemical properties test（thermal sensitivity test,ether sensitivity test,chloroform sensitivity test,trypsin sensitivity test,acid resistance tes）,purity test,nucleic acid detection,VP3 gene sequence identification and phylogenetic tree analysis.（2）Development of GPV IgYThe GPV（named as H strain）obtained by isolation and identification,was passed through goose embryo to F10 generation.After diluted for 1000 times,it was inoculated to 12-day goose embryo and the harvesting goose embryo fluid was taken as the antigen of GPV inactivated vaccine.When antigen sterility test and virulence test are qualified,concentration and inactivation were performed.Inactivation test was conducted by inoculating 12-days goose embryos.Preparation and emulsification of GPV inactivated vaccine were carried out after inactivation qualification test was passed.The physical character,sterility test and safety test were performed on the obtained inactivated GPV vaccine.The laying hens were immunized with qualified GPV inactivated vaccine according to the classical immune procedure,when the titer of GPV IgY antibody in high immunity egg yolk fluid was≥1:64,it is degreased,extracted and inactivated to prepare anti-H strain GPV IgY.（3）Quality evaluation of anti-H strain GPV IgYThe physical and chemical properties（temperature,p H,pepsin action time,freeze-thaw times）of GPV IgY were evaluated by monitoring the antibody titer of GPV IgY.Safety test was performed by single dose,repeated single dose and single large dose subcutaneous injection of GPV IgY in 3-day-old goslings and mice to observe the health status and local and systemic reactions of goslings and mice so as to evaluate the safety of GPV IgY.The treatment experiment of GPV IgY with different antibody titers and doses on goslings with GPV virulent attack for 24hours,experimental study on the treatment of goslings with different time of GPV infection and different age were implemented to evaluate the therapeutic effe ct of GPV IgY according to the cure rate of gosling.Subcutaneous inoculation of goslings with GPV IgY with different antibody titers and immune doses for 24 hours was conducted,followed by the use GPV to attack the virus,to evaluate of prevention effect based on gosling protection rate.The difference between GPV IgY and similar products was evaluated through immunizing 3-day-old goslings with similar products,according to the protection rate and cure rate of goslings.Through physical character,steril ity test,mycoplasma test,GPV IgY antibody titer monitoring and the protection rate of goslings against the virus,the storage temperature and time of GPV IgY were determined.Test results showed that:（1）Isolation and identification results of GPVGoslings inoculated with the isolated virus,displayed the same clinical symptoms and pathological changes as those of GP.Inoculation in goose embryo can proliferate in goose embryo,and it is a non hemagglutinating infection virus.AGP showed that there was a pre cipitation line between the virus fluid isolated from goslings and geese embryos and the GP positive serum.The neutralization test of goose embryo suggested that the virus fluid could be neutralized by GP positive serum,and inoculating goose embryo with neutralized virus solution did not cause death.Under electron microscope,H strain virus particles have typical characteristics of parvovirus.When the H strain was passaged to F6-F10 generation in goose embryo and Gosling,the highest and stable poison price was achieved with the ELD₅₀ and LD₅₀ of virus higher than 10^-5.0/0.2 m L and 10^-4.0/0.2 m L,respectively.The isolated H strain was inoculated to 3-day-old,10-day-old and20-day-old goslings respectively and it was found that the pathogenicity decreas ed with the increase of gosling age.The infectivity of the isolated virus could be maintained at 56℃for 3hours,and was resistant to ether,chloroform,trypsin and acidic conditions（p H3.0）.The bacteria,mycoplasma and exogenous virus were all negative in the virus fluid,proving the purity of H strain virus.The specific DNA bands were observed at 500 bp by PCR and DNA size was consistent with that of GPV.The above results showed that the isolated virus accorded with the biological characteristics of GP,and therefore the virus belongs to GPV（named H strain）.VP3 gene amplified by PCR was compared with the VP3 gene sequence of the reference strain,and the homology of nucleotides is between 95.9%and 99.1%with the homology of amino acids between97.9%and 99.6%.Phylogenetic tree analysis showed that the relationship between H strain and SP,ZZ,ZD,DQ,JX strains isolated in Heilongjiang Province was closer to that in GD（Guangdong）strains,YG（Yangzhou）strains.（2）Results of GPV IgY preparationThe antigen sterility test of GPV H strain inactivated vaccine was negative and ELD₅₀ of goose embryo is 10^-5.22/0.2 m L.The inactivation test found that all goose embryos were alive and healthy.The physical character,sterility test and safety test of GPV in activated vaccine were all qualified.The laying hens were immunized with the GPV inactivated vaccine to collect yolk fluid of high immune eggs with AGP≥1:64.The high immune yolk fluid was diluted with acidified water at a ratio of 1:8 for 15 h at p H 5.2 and 4℃,followed by centrifugation at 1900 g for 20 min to remove grease.Acidified water-1.5%octanoic acid-33%ammonium sulfate was employed for extraction.The final concentration of 0.05%formaldehyde solution after inactivation at 20～25℃for 24 hours presented no significant effect on the antibody titer,protein concentration and protein purity of GPV IgY.It was confirmed that the above conditions were the optimum for degreasing,extraction and inactivation.（3）Quality evaluation results of GPV IgYAfter the above-mentioned anti-H strain GPV IgY acted for 2 hours under 60℃,the titer of GPV IgY antibody did not decrease compared with the control.When the temperature reached above 70℃,antibody titer decreased significantly and when the temperatur e achieved 75℃for 30min,the antibody titer became negative.When the p H is ranged 4.0～11.0,there is no significant difference between the GPV IgY antibody titer and the control.At p H 3.0 and 12.0,the titer of GPV IgY antibody was significantly lower than that of the control group.When pepsin acts for 6hours,the antibody titer does not change,and significant decrease was observed after action for more than 8 hours.There was no significant difference in antibody titer between GPV IgY and control group after repeated freezing and thawing for 5 times.GPV IgY immunized goslings and mice showed no local and systemic adverse reactions,with good mental state and normal food intake.The therapeutic effect of GPV IgY on goslings suggested that the highe r the antibody titer and the higher the immune dose,the higher the cure rate of goslings.The longer the virus infects goslings and the older the goslings are,the lower the cure rate is.The preventive effect of GPV IgY showed that at the same dose,the higher the antibody titer,the higher the protection rate of goslings.With the same antibody titer,the protection rate of goslings increased along with the increase in immune dose.The protection rate of GPV IgY to goslings was over 88.00%,after immunization for 12 h-10 days,which could effectively resist virus infection.The difference comparison test showed that the protection rate and cure rate of GPV IgY were higher than those of similar products.（4）Storage conditions of GPV IgYAfter 24-month storage at 2-8℃,the physical character,purity and antibody titer of GPV IgY were stable,and the protection rate of gosling infected with GPV was over 88.00%.（5）Recommended dosage and method of GPV IgYFor goslings infected with GPV,the titer of GPV IgY agar antibody was above 1:16,1.0m L/only;Emergency immunization of uninfected goslings,0.5 m L/only,subcutaneous injection,the cure rate and protection rate of GPV infection were 80.00%.