Preparation Of Fowl Hydropericardium-hepatitis Syndrome IgY Liposome And Chicks Protection Test

Hydropericardium-hepatitis Syndrome(HHS)is an acute and highly contagious infectious disease caused by Fowl Adenovirus Serotype 4(FAV-4).The typical symptoms are sudden death of broilers aged 3~5 weeks and hemorrhagic hepatitis with obvious pericardial effusion can be seen by necropsy.HHS occurred firstly in Ankara of Pakistan,and became popular in many provinces in China,2015.The FAV-4 strain TJ1607 was isolated and identified in our laboratory for the first time in Tianjin on 18 July 2016.Oil-emulsion inactivated vaccine with purified TJ1607 strain as antigen based on the experience of predecessors,the high-yield laying hens were immunized several times to prepare purified high-immunity TJ1607 Ig Y and purified high-immunity TJ1607 Ig Y liposomes,animal protection tests of Ig Y antibody was carried out successively,which showed a certain control effect,at the same time,an indirect ELISA method for detecting TJ1607 antibody was established in the paper.The specific research contents are as follows:1.Preparation of TJ1607 oil-emulsion inactivated vaccine.TJ1607 oil-emulsion inactivated vaccine was prepared by using the proliferated and purified TJ1607 strain as antigen and Freund’s adjuvant as oil emulsion.The vaccine was tested and there was no dispersion in the drip test;no bacterial growth and stratification was observe in the test;the animal test showed no adverse reactions were observed post-inoculation of healthy chicks,and the prepared vaccine could be used in subsequent experimental study.2.Preparation of TJ1607 Ig Y antibody.High-yield laying hens were immunized with TJ1607 oil-emulsion inactivated vaccine for several times,and the titer of antibody reached 1:128 on the 17 th day after the third immunization and remained unchanged,at the same time,eggs were collected and high-titer TJ1607 high-immunity yolk antibody was successfully obtained.The TJ1607 high immunity yolk antibody was purified by octanoic acid-ammonium sulfate method combined with dialysis technology.The purified antibody has the same potency and higher purity,and was encapsulated by liposome.3.Establishment of indirect ELISA for detection of serum antibody in TJ1607.The purified TJ1607 was used as the antigen coated enzyme plate,the TJ1607 serum antibody was the primary antibody,the rabbit-anti-chicken horseradish peroxidase was the secondary antibody,and the best dilution multiples of the antigen,primary antibody and secondary antibody was screened by the checkerboard method,and the standard curve was drawn,the fitting degree of standard curve was checked,and the established detection method was tested by repeatability and specificity test.The results showed that the optimal coating concentration of TJ1607 antigen was 350μg/m L,the optimal dilution of TJ1607 serum was 1:3000,and the optimal dilution of rabbit-anti-chicken horseradish peroxidase was 1:2000;The linear equation of positive serum antibody concentration and OD value was y=21.751x-3.5781,R2=0.9993.The repeatability and specificity test showed that the intra-plate coefficient of variation was 1.68%~4.88%,and the inter-plate variation coefficient was 4.03%~9.85%.It can be used for the detection of subsequent serum samples.4.Animal protection test of TJ1607 high immunity egg yolk antibody.The results of animal protection test showed that the cure rate of refined high immunity TJ1607 Ig Y to HHS in Tianjin was 85%,and the prevention and protection rate was 100%.It could effectively prevent and control the outbreak of HHS in Tianjin.The results of antibody dynamic growth test showed that the purified high immunity TJ1607 Ig Y was absorbed better and produced antibody faster than the crude high immunity TJ1607 Ig Y,and the peak time was faster,but the sustained release effect was not as good as the crude Ig Y,it could be used for emergency epidemic prevention and multiple high-efficiency immunization.The oral test results of Ig Y antibody showed that the purified high immunity TJ1607 Ig Y stock solution was orally ineffective,and it could be used for oral immunization after preparation of purified TJ1607 Ig Y liposome.