Newcastle Disease, Avian Influenza Inactivated Vaccine (la Sota Strain + Strains N28 + Sd696 Strain) Developed

The study on biological characterization such as virulence, propagation and immunology characterization, specificity of Newcastle disease (ND) virus La Sota strain, H5 subtype Avian Influenza(AI) virus N28 strain and H9 subtype Avian influenza virus SD696 strain are carried out separately. For NDV La Sota strain in this study, the intracerebral pathogenecity index (ICPI) is 0.31, the Mean Death Time in embryonated eggs with minimal lethal dose (MDT/MLD) is 110.4 hours, the lethality rate to 10-day old embryo is 8/10, the erythrocyte HA titer is 1:1024, the virus titer is 10^8.5EID50/0.1ml, 2-7 days old SPF chickens are safe post inoculation with the La Sota strain of NDV, and the virus could be neutralized differentially by the antiserum of NDV; For H5 AIV N28 strain in this study, the intravenous pathogenecity index is 0.98, the lethality rate to 10-day old embryo is 1/10, the positive isolation rate after inoculation of 4-5 weeks old SPF chickens is 10/10, the erythrocyte HA titer is 1:512, the virus titer is 10^7.2EID50/0.2ml, the virus with good immunogenicity could be neutralized differentially by the positive antiserum of H5 AIV; For H9 AIV SD696 strain in this study, the intravenous pathogenecity index is 0.91, the lethality rate to 10-day old embryo is 4/10, the positive isolation rate after inoculation of 4-5 weeks old SPF chickens is 10/10, the erythrocyte HA titer is 1:1024, the virus titer is 10^7.5EID50/0.2ml,the virus with good immunogenicity could be neutralized differentially by the positive antiserum of H9 AIV.10-day-old embryos derived from non-immunized chickens are inoculated separately with NDV La Sota strain, H5 AIV N28 strain and H9 AIV SD696 strain, allantoic fluid are harvested, the final virus titers are 10^8.5 EID50/0.1ml(NDV), 10^7.17EID50/0.2ml(AIV, H5) and 10^8.17EID50/0.2ml(AIV, H9). All the viruses are inactivated by formaldehyde with 0.2% final concentration, and all the allantoic fluid are concentrated 3-4 times by tangential flow with 300 thousand grade molecular, the HA titers of the three kinds of virus reached 1:1024, 1:512 and 1:1024 separately.Three bathes of vaccines are manufactured with the above gained virus fluid according to the following final proportion: 9.6%(NDV), 10.8%(AIV, H5) and 10.8%(AIV, H9).The vaccine proved to be ivory w/o emulsion by physical test; A centrifuging tube with 10ml vaccine sample are centrifuged for 15 minutes,3000r/min, no batches of vaccine appears delamination; 1ml vaccine sample at 25℃are assimilated into an 1ml straw (the upper diameter is 2.7mm, the lower diameter is 1.2mm), then discharged naturally, the time is recorded when 0.4ml sample finish discharging, the time all the samples needed should be within 8 seconds.Ten 4-5 weeks old SPF chickens are vaccinated with the trial-manufactured vaccine via neck subcutaneous injection or intramuscular injection, 2.0ml vaccine is used per chick, the chickens are examined daily for 14 days, all of them are live post vaccination, and no systemic or local side responses related to the vaccine are found.Fifteen 4-5 weeks old SPF chickens are vaccinated with the trial-manufactured vaccine via neck subcutaneous injection or intramuscular injection, the blood sample are collected 21 days post vaccination, and serum are separated. All the HI antibody titers of NDV, H5 AIV and H9 AIV are higher than 1:64, HI antibody titers of serum from the control chickens are negative. The vaccinated chickens are challenged via intramuscular injection with 10⁵ELD50 virulent NDV Beijing strain, the chickens are investigated daily for 14 days, 10/10 vaccinated chickens are protected, and all the chickens in the control group died; The vaccinated chickens are challenged via intravenously injection with 2×10⁶EID50 AIV SD696 strain, the negative rate of virus isolation is 10/10 in the vaccinated group, and the positive rate of virus isolation is 9/10 in the control group.The related trials on duration and storage are carried out, it proved that the immunity duration of the manufactured vaccine is more than 20 weeks and the shelf life is more than 12 months. The results of these trials proved that all the examined indexes are accordant with the related quality standardizations of inactivated ND vaccine, inactivated H9 AI vaccine and inactivated H5 AI vaccine. The trial-manufactured vaccine can be used to prevent ND, H9 AI and H5 AI in chickens.