| Newcastle disease(ND),infectious bronchitis(IB)and H9 subtype avian influenza(AI)are three major diseases that must be prevented and controlled in poultry industry.Newcastle disease is caused by the Newcastle disease virus(NDV).In recent years,the prevalentNDVstrains with high virulencein China is the genotype VII.Chicken infectious bronchitis is an acute,contagious disease caused by the infectious bronchitis virus(IBV).H9N2 subtype avian influenza is widely present in birds,with high transmssion efficiency.In order to control these three diseases and to reduce the side effects caused by frequent immunization,it is necessary to develope a triplex inactivated vaccine,which has excellent immunity and cross-protection.Four recombinant H9 subtype AIV strains were successfully obtained through reverse genetic technology.These strains were made into inactivated vaccines individually.By comparing the titers of antibody after immunization,we found that AH515-PR8 strain had betterimmunogenicity thanother strains.The recombinant H9 subtype AIV(AH515-PR8 strain),NDV(A-VII strain)and IBV(QXL87 strain)were used to make the triplex inactivated vaccine,then we compared the immune efficacy of this vaccine with a similar product from the market1.Construction of recombinant H9 subtype AIV strainsIn this study,we selected 4 prevalent strains of H9N2 AIV to obtain their envelope glycoprotein(HA,NA)genes.By using reverse genetic technology,four recombinant viruses named A3-PR8,AH515-PR8,TM343-PR8 and XZ349-PR8were constructed,with six internal gene of H1N1 influenza virus A/Texas/05/2009×Puerto Rico/8/1934 as the backbone.Four recombinantstrains was propagatedin 10-day-old embryonated SPF chicken eggscontinuously.Among them,A3-PR8 strain,AH515-PR8 strain and TM343-PR8 strain could grow stably in chicken eggs,and their erythrocyte agglutination price can reached 29.The EID50 of the strain’s sixth generation were determined separately.Compared them with the EID50 of the original strains,the EID50 of the recombinant strain can increase by 0.5-0.8 titers.2.Evaluation of the immunogenicity of recombinant H9 subtype AIV strainsThe test was divided into A3-PR8 group,AH515-PR8 group,TM343-PR8 group,WJ57-PR8 vaccine strain group and challenge control group.The immunization group had 1021-day-old SPF chickens,and the challenge control group had5 SPF chickens.Each immunization group was immunized at a dose of 0.2mLper chicken by subcutaneous injection,which contains 2×108.5 EID50 virus;the challenge control group was inoculated with the same amount of PBS.Blood was collected at the 7,14,21 and 28 days post inoculation(dpi),and the hemagglutination inhibition(HI)antibody titer of H9 subtype AIV in serum was measured.The results showed that the antibody titer of the AH515-PR8 group continued to rise after immunization,and it was always higher than the antibody titer of the other immunized groups.At28 dpi,the antibody titer was 28.9,which was 0.4-0.7 titer higher than that of the remaining groups.After the challenge,AH515-PR8 group and TM343-PR8 group had significantly reduced virus shedding compared with the vaccine strain WJ57-PR8.Among them,the recombinant virus AH515-PR8 group had the lowest virus shedding rate,and the isolation rate of virus was 0%at 5 dpi.3.Evaluation of the immune efficacy of the triplex inactivated vaccineFifty SPF chickens were randomly divided into immunization groups A1,B1,A2 and B2,with 10 chickens in each group;challenge control groups A3 and B3,with 5 chickens in each group.Al and B1 groups were immunized with triplex inactivated vaccine at the age of 28 days,which ocontains 3.5×108EID50 NDV,4×106EID50 IBV,5.8×108EID50 AIV;A2 and B2 groupswere immunized with similar products from the immune market.Blood was collected to separate serum at 7,14,21,28 dpi.The antibody titer of NDV and AIV was determined by using the HI test;the antibody level of IBV was determined by using indirect ELISA method.The results showed that the average value of NDV antibody reached 29.3 at 28 dpi,which was 1 titer higher than that of similar product;the average value of AIV antibody was 210.2,which was 0.7 titer higher than that of similar product;IBV antibody were all negative at 7dpi.At 14 dpi,the positive rate of antibody in Al group was 80%,and it was 10%higher than that in B1 group.The rate of positive antibody at 21 dpi and 28 dpi was 100%in two groups.The JS02/06 strain was used to attack the Al,A2,and A3 groups by nasal drip,and the WJ57 strain was used to attack the B1,B2,and B3 groups by wing vein injection.Throat and cloaca swabs were collected to determine virus shedding rate at 3,5,7 dpi.In the NDV part,the clinical survival rate of the Al group was 100%,and it was 10%higher than that of the A2 group.The virus shedding rate was greatly reduced,the virus isolation rate was 7%at 7 dpi.In the AIV part,the virus shedding rate of the B1 group was 3%on the 3rd day,and it reached 0%on the 5th day,which was lower than that of the B2 group.In summary,four recombinant viruses named A3-PR8,AH515-PR8,TM343-PR8 and XZ349-PR8 were constructed.Among them,the AH515-PR8 strain had the best immunogenicity.It was made into a triplex inactivated vaccine with recombinant NDV(A-Ⅶ strain)and IBV(QXL87 strain).The vaccine had shown good immunoprotective effects in animal experiments,which lays the foundation for the development of a new commercial vaccine. |
PDF Full Text Request