| Duck growth retardation disease is a new infectious disease in China in recent years,which is characterized by lethargy and growth retardation.The susceptible animal of the new disease is cherry valley ducks,and the age of onset of ducks is mainly concentrated in 10 to 25 days,some ducks aged from 5 to 14 days also have symptoms.The disease can occur all year round but the overall incidence is below 15%.An unknown virus was isolated from the diseased ducks,which has a capsule and the diameter ranged from 70 to 100 nm.The virus was identified as a new RNA virus,and named duck growth retardation virus(Anhui2).The virus does not kill ducks by the artificial infection,but it can spread horizontally across the ducks,to cause the duck grows slowly.In this study,duck growth retardation virus(Anhui2)was used to immunize 6-week-old BALB/c mice.After 3 times immunization,the antibody titers of mouse sera reached 1:10000 or higher,and the mouse spleen cells with highest antibody titerse after strengthen immunity were fused with SP2/0 myeloma cells.After indirect ELISA screening and three consecutive subcloning,two hybridoma cells that stably secrete anti-Anhui2 antibodies were obtained,named 1-5 and 3-6G,respectively.The antibody titers of both supernatants of two hybridoma cells were 1:1000,and their titers of induced ascites was 1:10000 and 1:1000 respectively.After neutralization test,the two hybridoma cells had specific neutralization activity.Anhui2 virus was used as coating antigen and 1-5 monoclonal antibody was screened for use.To optimize the conditions of ELISA,the optimum dilution of the antigen was 1.5μg/mL,the optimum dilution of the serum was determined as 1:10,and the reaction time was 1h at 37°C,the optimal dilution of monoclonal antibody was determined as 1:20 and the reaction time was 37°C for 1 h.The optimum dilution of the Goat-anti-mouse secondary antibody was determined as 1:3000,and the duration of action is 37°C 1h.A blocking ELISA method was established for the detection of Duck growth retardation virus antibody.Statistical analysis was performed based on the test result of 316 negative sera,The mean inhibition rate(?X)of serum samples was calculated as 1.9%,standard deviation(SD)was 8.0%,It was positive when the percent inhibition(PI)value was more than 25.9%,it was negative when PI was less than 17.9%,repeated analyses were performed on the sera whose PI values were between 17.9% and 25.9%,and the sera were considered negative when the values were less than 25.9%.Positive sera against other pathogens of duck(such as duck hepatitis virus typeⅠ,duck hepatitis virus typeⅢ,influenza virus,duck enteritis,duck reovirus,new duck reovirus,muscovy duck reovirus)were tested by the blocking ELISA,the results showed that the blocking ELISA has good specificity.In addition,the antibody sensitivity experiments showed the method has goog sensitivity.The Anhui2 virus was adopted to pass serially by inoculating into the allantonic cavity of 10-day-old specific pathogen free(SPF)embryonated chicken eggs.To observe the pathogenicity of passage virus to the ducklings through animal experiments.Results show that the body weight of ducks was not serious affected by 100-passage viruses compared with the parental virus.After immunized with 111-passage virus,ducks were protected totally from Anhui2 virus attacks.It was suggested that 111-passage virus has been attenuated and has the simillar immunogenicity as its parental virus.It is suggested that passage attenuation virus is a fessible method for vaccine research. |
PDF Full Text Request