Screening And Immune Efficacy Studies On VFY Attenuated Vaccine Strain Of Duck Virus Hepatitis

Duck Virus Hepatitis (DVH) is an highly fatal infectious disease of the young duck ,which is caused by the Duck Hepatitis Virus (DHV). The main characteristics of the disease are incidence of acute,dissemination of fast, short pathogenesis and high mortality. The disease is caused by three different kinds serotypes of virus which are duck hepatitis type I virus , duck hepatitis type II virus and duck hepatitis type III virus. It is popular with duck hepatitis type I virus in China。It was first reported in our country in 1963. DVH broke out in most areas of China for many times,with serious conditions, affecting a wide range and great economical loss. It is a serious threat to the duck industry of our country。As the duck industry is developing rapidly, the incidence of the disease has been relatively high.Therefore, it is the main measures to control this disease that preventing in timely prior to the onset of the disease.However, there was a phenomenon that using duck virus hepatitis type I attenuated vaccine or Egg Yolk antibodies for the prevention and treatment were nullin recent years.It was doubtful that an variant strain of DHV type I or new type DVH was appeared.For preventing the outbreak of DVH effectively,it is necessary to develope the relative vaccine of type I variant strain of DHV or new type duck hepatitis to prevent the incidence of the disease. The subject was about studying the screening and immune efficacy of the variant strain of DHV type I (VFYstrain) which was separated and preserved by our laboratory.1. The variant strain of DHV type I (VFY strain) was propagated and attenuated with SPF chick embryos. In order to screen the variant strain of DHV (VFYstrain), to compare the immune efficacy by measuring the ELD₅₀ of different generations of the virus mixture and calculating PD₅₀ of the immune ducklings which were challenged with virulent. The best immune generation was designated VFY71 strain by the screening. According to results, VFY71 strain was chosen as the attenuated strain vaccine for researching into immune effectiveness. Virulent (Z1) which was used to efficacy test was prepared, and its LD₅₀ determinated for 2-4.63/0.5mL.2. It was shown that VFY-71 was sterile and single by testing on the purity of the VFY-71 attenuated strain.Through neutralizing with same quantity specific antiserum and inoculating chick embryos, the chick embryos all survived after eight days, VFY-71 attenuated strain and the standard strain vaccine was made cross-protection tests with virulent variant strain 1 (GY) and virulent variant strain 2 (GZ) respectively,indicating VFY-71 attenuated strain had specificity.The sheldrake ducklings that immunized by VFY-71 attenuated vaccine through the skin, leg muscle, oral, nasal and eye five channels were all strongly alive.But the same number of ducklings were injected by virulent(Z1)via the above channels in the control group. Ducklings in each group had different degrees of death.The virulence was not returned strong when VFY-71 strain was inoculated into 1-day-old sheldrake ducklings with 5 times. All indicated that VFY-71 strain was safe. It was diluted into 100 concentration (5.2×104ELD₅₀/0.5mL) to immunize 1-day-old sheldrake ducklings through the skin immunization, leg muscle immunization, oral immunization, nasal immunization and eye immunization.The best immunization way of VFY-71 was determined for leg intramuscular immunization under the same immunization dose.Under the best immunization way, VFY-71 attenuated strain was diluted into 10 concentration,100 concentration,150 concentration, 200 concentration, 300 concentration, 400 concentration,500 concentration, 103 concentration, 104 concentration, 105 concentration and 106 concentration to immunize 1-day-old sheldrake ducklings respectively. The recommend immunization dose range was :5.2×10⁴ELD₅₀/0.5mL～5.2×10³ELD₅₀/0.5mL,and the best immunization dose was 3.5×10⁴ELD₅₀/0.5mL.3. An indirect enzymelinked immunosorbent assay (ELISA) was developed to detect the antibody valence of DHV.The antigen was prepared for indirect ELISA by Freeze-thaw repeatedly,Chloroform extraction and chromatography on Sephadex G-200. The virus protein content was 9.710mg/mL.The healthy muscovy ducks were immunized four times (one time per ten days)by the VFY-71 attenuated strain.The result indicated that the positive price was as much as 1:16.while collecting serum of healthy Muscovy ducks as a negative serum and preparing HRP enzyme-labeled rabbit anti-Muscovy duck antibody.It was shown that the diluting antigen to 10μg/mL,the HRP enzyme-labeled rabbit anti-Muscovy duck antibody to 1:800,the serum sample to 1:25 was the best working concentrations and the critical OD value was calculated as 0.345 between positive and negative .The highest-level extent of dilution of serum was determined to be its terminal point antiboby titer if the diluted serum could produce positive reaction.4. VFY-71 attenuated strain.was diluted into 150 concentration (3.5×10⁴ELD₅₀/0.5mL) to immunize 1-day-old sheldrake ducklings by leg muscle immunization.The serum of immunized sheldrake ducklings were collected and the antibody titer was measured by indirect ELISA test which had been developed.The antibody level reached to the peak in the fifth day ,the average antibody titer was 1:392,and then showed a downward trend.The antibody titer was decreasing to a minimum in the 40th day,it was 1:70. The 1-day-old sheldrake ducklings was immunized by VFY-71 attenuated strain through the leg muscle immunization and skin immunization, the 1-day-old sheldrake ducklings were challenged by efficacy test virulent (Z1)in the third day, the fifth day and the seventh day after immunization. As a result,both two types of immune pathway had the highest rate of protection in the fifth day after immunization.Indicating the protection of animal infection test was consist with antibody titer results that measured by indirect ELISA.VFY-71 attenuated strain was stored at -20℃,sampling one time every three months, the 1-day-old sheldrake ducklings were immunized and challenged by efficacy test virulent (Z1)in the fifth day old.VFY -71 attenuated strain which was stored for 6 months was able to protect all. The protection rate of which was preserved for 9 months and 12 months were 90% and 75% respectively.