The Clinical Immune Experimental Study On Attenuated Living Vaccine Of Duck Viral Hepatitis

Abstract:Duck viral hepatitis (DVH) is an acute, high mortality disease which is caused by duck hepatitis virus (DHV). The ducklings under 30-day-old especially those under 2-week-old are the easyest to be infected by DHV, the mortality may reach 90%, which produces significant economic losses in the duck cultivation. Vaccination for breeder ducks or ducklings with DHV vaccine is the best way to prevent this fatal disease, but high quality vaccines are needed. A serial studies were developed around the detection on immune effects of breeder ducks and ducklings which were vaccinated by the tested attenuated living vaccine of DHV:①The indirect enzyme linked immunosorbent assay (ELISA) and chicken embryos virus neutralization test were developed and used to detect humoral immune,②The MTT was performed to detect cell mediated immunity,③The immune protection effect of ducklings was tested by virus-attacking,④The Indirect Immunoperoxidase Staining(IIS) test was developed to detect the location and distribution of attenuated DHV in different tissues at different time points, which can be used to interpret the immune mechanism of DHV attenuated vaccine. The results were as follows:1. The developement of indirect ELISA which was used to detect antibodies of DVHAn indirect ELISA was developed using virus of duck hepatitis virus serotype 1(DHV-Ⅰ) which was purified by chloroform defattening and ultra-centrifugation as coating antigen. This method had high sensitivity and specificity and could be used to detect antibodies of DHV. The protein quantity of purified antigen was 1.9242 mg/ml; the best dilution of second antibody was 1:2000;the best dilution of coating antigens and sera were 1:200 (the protein quantity of antigens were 9.621μg/ml)and 1:100,respectively; the threshold of negative was 0.2439. Other four kinds sera were detected by ELISA and the results were all negative, which showed the developed ELISA had high specificity. The sensitivity tests between ELISA and Virus Neutralization were performed and showed the ELISA had much higher sensitivity than Virus Neutralization. From the specificity and sensitivity tests, we could infer that the developed ELISA could be used to detect antibodies of DVH.2. The immune experiments of breeder ducksThe breeder ducks were vaccinated with attenuated vaccine of DHV at 1 immune dose per duck, the bloods were collected and used to perform MTT and extract sera, the eggs laid by breeder ducks were collected to hatch ducklings and extract yolk antibodies. The sera and yolk antibodies were detected by ELISA and Virus Neutralization, the ducklings were infected by virulent DHV. The antibodies in sera and yolk could be tested by ELISA at 3 days post inoculation (PI), it reached the peak at 21d,28d PI respectively and decreased slowly afterwards, but it still kept positive at 56d. The increasing and decreasing state of sera and yolk detected by ELISA showed the same state detected by Virus Neutralization, however, the results of control group were all negative. The lymphocyte proliferation of external bloods test was performed. From 3d on, there was a significantly difference between vaccination group and control group (P<0.05), it reached peak at 21d post inoculation. Ducklings hatched from the eggs laid by vaccinated breeder ducks were attacked by 10000 LD50 virulent DHV-Ⅰat 0.1ml per duckling. The survival rate of 5d was 10%, it increased from 5d to 21d, the peak was 100% and reached at 21d, afterwords it kept at 100% until 56d, whereas the survival rates of control group were all zero. Breeder ducks in the third group were injected by attenuated vaccine for 2 times at a interval of 14d,1 immune dose every time. The sera were collected at 30,60,90,120,150,180d post the first inoculation and detected by ELISA and Virus Neutralization, the results showed the titers of antibodies were much higher than that of the first group and it kept at a high lever within 6 months.3. The immune experiments of ducklingsOne-day-old ducklings were vaccinated with attenuated vaccine of DHV at 1 immune dose per duckling, the bloods were collected and used to perform MTT and extract sera at 3,5,7,10,15,30d. The sera were detected by ELISA and Virus Neutralization. The antibodies in sera could be tested by the two methods at 3 days post inoculation, it reached the peak at 10d and decreased from 10d to 30d, the antibodies were still positive at 30d. The increase and decrease state of sera detected by two methods showed the same state, however, the results of control group were all negative. The lymphocyte proliferation of external bloods test was performed. From 3d on, there was a highly significantly difference between vaccination group and control group (P<0.01), it reached peak at 10d post inoculation. Ducklings vaccinated by vaccine were attacked by 10000 LD50 virulent DHV-Ⅰat 0.1ml per duckling. The protection rate of 3d was 40%, it increased from 3d to 10d, the peak was 90% and reached at 10d, whereas the protection rates of control group were all zero.4. The location and distribution of attenuated antigens detected by IISThe IIS test was developed to detect the location and distribution of DHV-Ⅰattenuated antigens in the paraffin wax tissue splices using rabbit anti DHV-Ⅰantibodies. Fourteen tissues from ducklings vaccinated by the tested vaccine were collected at different time points and detected by the IIS test. From 12h post inoculation on, the attenuated virus could be tested in liver, kidney, spleen, bursa of fabricius, thymus, muscular, intestines, the virus antigens were location in the cytoplasm. The sequence of the attenuated virus detection in the injection group was as follows: liver, spleen, kidney>thymus, bursa of fabricius, harderian gland, cecum, duodenum, pancreas, muscle> rectum, cardiac muscle, lung> cerebrum. The attenuated virus of 7d post inoculation mainly distributed at liver, spleen, kidney, thymus, bursa of fabricius, harderian gland and intestines, the strong positive signals showed in nearly all tissues at 14d, it decreased afterwards.