Preparation And Immune Efficacy Evaluation Of Inactivated Vaccine Against Avian Metapneumovirus Disease Subtype B

Avian metapneumovirus disease,caused by avian metapneumovirus(aMPV)is an acute upper respiratory tract disease of chicken or turkey,and it is widely prevalent in the world.Many studies have shown that the seropositive rate of aMPV in some chicken populations is as high as 100%.The results of etiological tests show that avian metapneumovirus subtype B is the main viral strain prevalent in chickens in China.When aMPV infects alone,the mortality rate is not high,but it is easy to cause secondary infection with other pathogens.If there is a secondary infection with other bacteria or virus,the mortality rate can be as high as 25%to 40%.In addition,aMPV infection can cause swelling head syndrome of broilers and decrease of egg production and egg quality of layers,causing huge economic losses and seriously affecting the healthy development of poultry industry.Avian metapneumovirus disease is mainly controlled by vaccine immunization in foreign countries,but there is no vaccine for prevention and control of this disease in China at present.In this study,an inactivated vaccine against avian metapneumovirus disease(subtype B)has been developed based on aMPV/B LN16 strain isolated from chickens in our previous work.Besides,its safety and immune efficacy have all been systematically evaluated.aMPV/B LN16 strain was successively cultured in Vero cells for 15 generations.Median tissue infective dose(TCID₅₀),purity test and immunogenicity comparison were conducted among different generations of virus.According to the results of the immunological efficacy experiment,we chose aMPV/B LN16 F11 strain(LN16-I)as the immunogen,and developed avian metapneumovirus disease(subtype B)inactivated vaccine by screening the inactivated conditions and emulsification process.The results of single-dose,single-dose repeated and high-dose immunization experiments showed that the inactivated vaccine had good safety,with no adverse reactions or inflammatory reactions at the injection site observed after immunization.It was showed that the vaccine could induce the production of serum ELISA antibody and neutralizing antibody,and promote the proliferation of B and T lymphocytes in PBMC and the secretion of IL-4 and IFN-?cytokines in spleen.It also provided above 80.0%protection rate against virulent attack of aMPV/B,significantly reduced viral load in nasal swabs and avoided pathological damage to the turbinate.According to the results of the challenge protection experiment,the protection rates of the vaccine were 77.8%and 100.0%in the priming immunization at the age of 3 weeks and in the boosting immunization at the age of 6 weeks,respectively.Therefore,the immunization program was set as priming immunization at 3 weeks of age and boosting immunization at 6 weeks of age.The results of minimum immunization dose experiment showed that boosting immunization with the inactivated vaccine at a dose of no less than 0.25 m L/bird,could provide more than 87.5%protection at 3 weeks after boosting immunization.Therefore,the minimum immunization dose was set at 0.25 m L/bird,and the recommended immunization dose was set as 0.50 m L/bird to ensure the clinical immune protection effect.The results of immunization duration experiment showed that the protection rates of the inactivated vaccine were 100.0%,90.0%,100.0%,90.0%and 88.9%,respectively,at 2,3,4,5 and 6 months after boosting immunization.The immunization duration of the vaccine was initially determined to be 6 months.In addition,the inactivated vaccine could provide 100.0%protection on 17-week-old SPF chickens.The immune protection effect of inactivated vaccine on commercial layers and commercial broilers were further evaluated.The pathogenicity test results showed that aMPV/B LN16 strain had significant pathogenicity to commercial layers and commercial broilers.Infected chickens showed symptoms of nasal fluid and nasal scabs,with incidence of 92.3%and 100.0%,respectively.aMPV can be detected in nasal swab,turbinate,throat,trachea,and Harderian gland.aMPV infection can cause various degrees of pathological damage to the turbinate,trachea and lung of commercial layers and commercial broilers at 9 days post infection.3-week-old commercial layers and commercial broilers were priming immunized and boosting immunized at the age of 6 weeks with the inactivated vaccine and high levels of serum antibody could be induced.3 weeks after boosting immunization,virulent aMPV/B was used to challenge the chickens.The vaccine could significantly reduce the viral load in nasal swabs and alleviate the pathological damage of respiratory organs.The protection rates of commercial laying hens and commercial broilers in the immunization group were 100.0%and 88.9%,respectively.In this study,avian metapneumovirus disease(subtype B)inactivated vaccine was developed,and it was proved to have good safety and immunogenicity,and could provide good immune protection to SPF chickens,commercial layers and commercial broilers.These results provide technical support for the effective prevention and control of avian metapneumovirus disease in China.