| Duck Tembusu disease is a potent infectious disease in ducks.It is caused by Duck Tembusu Virus?DTMUV?,which causes decreased egg production in laying ducks.It severely affects the reproductive system and 20-day-old ducklings.Head and neck tremors,limb paralysis and other neurological symptoms are the main features of the disease.The disease has seriously affected the development of the duck industry in China.Currently,there are no effective treatment measures.Therapeutic antibodies are passive immunization agents that are safe,specific,efficient,and free of residues.In order to obtain a passive immunobiological agent that can be used for emergency prevention and treatment of duck tembuesu virus disease,the clinical isolate of the duck tembusu virus was used as the basic strain,and was diluted 10-fold after RT-PCR identification.After 10 days of inoculation in the allantoic cavities of 7-day-old duck embryos,the death time was concentrated at 3 to 5days.The dead embryo body was congested,the head and neck muscles were spotted and the liver was necrotic.The dead embryos were significantly smaller than normal duck embryos.The collected allantoic fluid is centrifuged to remove impurities and then concentrated by tangential flow.The?-propylene lactone inactivated virus was added and mixed with the adjuvant in a weight ratio of 3:7 to prepare an inactivated vaccine against duck tembusu virus.The immunized chickens were immunized three times with the inactivated vaccine.The interval between each immunization was 14 days.The first dose was 1 mL/body.The two exemptions and the three exemptions were 0.2 mL/body.The route of immunization was neck back and subcutaneous multi-points.Two weeks after the three exemptions,eggs were removed and egg yolk was extracted.After water dilution extraction,ammonium sulfate salting out,small-scale tangential flow ultrafiltration,dialysis and salt purification,IgY antibody in yolk.In order to determine the biological activity of preparation of yolk antibody,duck embry neutralization test was carried out by using fixed virus to dilute the antibody,and its antibody neutralization titer was determined.To determine the biological activity of the preparation of yolk antibody,the indirect ELISA assay was used to determine its antibody titer.The results showed that the DNA fragment of 153 bp could be amplified by RT-PCR method from the allantoic fluid and was sequenced which confirmed DTMUV The strain had an ELD50 of approximately 102.9/0.2 mL.The inactivated vaccine was milky white and the liquid was homogeneous.Obtained crude antibodies white and the liquid was homogeneous.Indirect ELISA measured yolk antibody titer of 29210.This study laid the foundation for the preparation of inactivated vaccines and yolk antibody starting from the clinical case of duck tabutulose,and provided a theoretical basis and technical means for the effective prevention and treatment of duck tembuesu virus disease.However,there are still many problems to be solved in IgY.Its preparation is influenced by poultry's immunization procedures and methods,age and breed,and its effects evaluation and quality inspection standards have not yet been standardized.These factors will affect the effect of high anti-yolk antibody against duck tembuesu virus disease in actual clinical control applications. |
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