Preparation And Immunoprotection Of Yolk Antibody Against Novel Duck Reovirus Strain LY20

Since 2017,a new type of duck reovirus disease characterized by severe spleen necrosis and immunosuppression has been prevalent in large-scale duck farms in Shandong Province,whose pathogen is a novel duck reovirus（NDRV）,which can be transmitted vertically.Infected ducklings are easy to cause mixed infection and secondary infection,and the younger the age of the diseased duck is,the higher the morbidity and mortality is.Immunoglobulin of yolk（Ig Y）is a specific polyclonal antibody found in immunized poultry eggs.Compared with serum antibodies,egg yolk antibodies have better stability,acid and alkali resistance and sensitivity to temperature.Because of its safety,high yield,low cost and high specificity,yolk antibody is widely used in poultry industry.In this study,we analyzed and compared two strains characterized by splenic necrosis,selected the virulent strain to prepare oil-emulsion inactivated vaccine against the novel duck reovirus.The vaccine was used to immunize laying hens to produce antibodies against the yolk of the new duck reovirus,which was then tested in clinical trials.The main contents include:1.Screening of virus strains for vaccine preparationTwo strains of NDRV（NDRV-SDYC and NDRV-LY 20）causing spleen necrosis were isolated and preserved in our laboratory as candidate strains for virus purity test and 50%tissue culture infective dose(TCID₅₀)test,and carried out animal regression experiments respectively.The results showed,when 0.5 m L of the NDRV-SDYC(TCID₅₀=10^-7.65/0.1 m L)virus was injected into the leg muscle of ducklings,there is depression in spirit and a decline in feeding.Autopsies of the dead ducks showed that splenomegaly,bleeding and irregular necrosis.When 0.5 m L of the NDRV-LY 20(TCID₅₀=10^-6.66/0.1 m L)virus was injected into the leg muscle of ducklings,the clinical symptoms and autopsy changes were similar to those of the former,but the degree of spleen lesion,necrosis rate,and the inhibition of body weight growth of ducklings in the latter group were more severe.Finally,the LY 20 strain was selected as the virus strain for vaccine production.2.Preparation of oil emulsion inactivated vaccineThe LY 20 strain was used as the inoculum and expanded after inoculating LMH,and then produce oil emulsion inactivated vaccine.Using different concentrations of formaldehyde solution to inactivate virus solution at different temperatures and at different times.The results showed that the optimum inactivation conditions were 0.2%formaldehyde and 24 h at 37℃.The aqueous phase of the vaccine is a mixture of inactivated virus solution and Tween-80 at the ratio of 96:4;The No.10 pharmaceutical white oil was mixed with Span-80 at 94:6,then 3%aluminum stearate was added and stirred evenly;After aseptic treatment,it was used as the oil phase for vaccine preparation.Subsequently,inactivated vaccine of NDRV was prepared according to the ratio of oil to water 2:1,and test the character of the vaccine.The result of inspection reveals that the dosage form of the vaccine is oil-in-water type and has well centrifugal stability,and there is no pathogenic microbial contamination.3.Preparation of immunoglobulin of yolkThe laying hens were immunized with two doses（±0.5 m L）of the above oil emulsion inactivated vaccine,collect eggs with high concentrations of Ig Y after maintenance of immunity,and the yolk antibody was prepared by caprylic acid method;The titer of the prepared antibody was detected by indirect ELISA and VNT.The result of inspection reveals that the titer of yolk antibody in high immune dose group was significantly higher than that in low dose group,the titers of ELISA and VNT were 2^-9（1:512）and 10^-3.82（1:6607）,respectively.Subsequently,physical characteristics and animal safety tests were carried out,the results showed that the antibody was a yellowish transparent clear liquid,and no local redness and swelling and systemic adverse reactions were observed when 1.0 m L was injected into the leg muscle of 1-day-old ducklings.4.Immune protection test of immunoglobulin of yolkThe detection results of yolk antibody in ducklings showed that the antibody level increased rapidly after injection,reached the maximum at about 4 days,and then decreased continuously.The antibody level in negative control group was basically unchanged and continued to be negative;At about the 17th day,the antibody level of ducklings in 0.3 and 0.5 m L antibody group basically decreased to negative,and the serum antibody titer of 0.7 m L yolk antibody group was negative on the 21st day;On the 21st day,the antibody level in 1.0 m L yolk antibody group was still slightly higher than the critical value,so injection of 1.0m L yolk antibody could effectively protect ducklings from NDRV infection.The results of prevention and protection test of ducklings showed that when the ducklings were injected with 1 m L yolk antibody into their legs at the age of1-day-old,no infection was found in the experimental group,indicating that the antibody can effectively prevent the damage of NDRV-LY 20 to ducklings.Compared with the healthy control group,the results showed that the weight gain of ducklings in the positive control group decreased by up to 24.7%,while the weight gain of the experimental group using egg yolk antibodies decreased by a minimum of 7.7%,indicating that the antibody can significantly reduce the weight gain caused by NDRV infection.The duration of passive immune protection test showed,when the ducklings were injected with 1 m L yolk antibody at 1-day-old,and infected with the NDRV 5 days later,the spleen necrosis rate and necrosis degree were the lowest,indicating that the passive immune protection duration of single injection of this yolk antibody was about 5 days.To sum up,he yolk antibody against novel duck reovirus prepared in this study has a good preventive and protective effect,which can resist infection of NDRV significantly.It provides a rapid and effective specific biological product for the prevention and treatment of novel duck reovirus disease in China.