| Newcastle disease(ND),caused by virulent Newcastle disease virus(NDV),is an acute and highly contagious disease that causes serious damage to poultry industry worldwide.Since 1980s,for prevention and control of ND,intensive vaccination with inactivated and attenuated live ND vaccines has been adopted in China,and the ND was controlled effectively.Inactivated ND vaccine can induce high level antibodies which can sustain for long time,while live ND vaccines can induce antibodies fast and also stimulate the mucous to generate effective mucosal antibody immunoglobulins A(IgA),which plays an important role in protection of chickens from NDV infection.However,at present,the evaluation of ND vaccine immunopotency in China is mainly to measure the level of HI antibody in chicken serum post-immunization(p.i).In this study,to comprehensively evaluate the immune efficacy of live ND vaccines,we established an indirect ELISA method for detection of NDV specific IgA antibodies.Currently,genotype Ⅶ NDV is the dominant genotype in China since the late 1990s.In order to control this genotype NDV infection,genotype VII ND inactivated vaccine was developed in 2014 and widely used in China thereafter.Besides,the live attenuated genotype VII ND vaccine is also ready for clinic trial.However,the current ND immunization procedure was formulated based on genotype Ⅱ vaccines such as La Sota and VG/GA.Considering the different immunogenicity and genetic variation between genotype Ⅱ and Ⅶ ND vaccines,determination of the immunization procedure for genotype VII ND vaccine is in urgent need presently.To this end,this novel immunization procedure was determined in this paper,based on the detection and analysis of the HI titer and the specific IgA antibody induced by genotype Ⅶ ND vaccines.1.Establishment of an indirect ELISA for detection of NDV specific IgAantibodiesIn order to analyze the mucosal immunity in chickens immunized with live ND vaccines,in this study,we established an indirect ELISA method for detecting NDV specific IgA using the attenuated live vaccine candidate strain A-NDV-LX/I4 as coated antigen.The indirect ELISA assay exhibited the optimum reaction ability,when the antigen coated concentration is 4.8μg/mL with 1%BSA as blocking reagent,the working concentration of the enzyme-labeled antibody is 1:20000,and the incubation time of TMB substrate is 15 minutes at 37℃.Subsequently,4-week-old SPF chickens was vaccinated with A-NDV-LX/I4 with 106EID50 per bird via intranasal and eye drop routes.Samples of tracheal,duodenal and caecal washing fluids plus bile were collected from immunized chickens at different time points for IgA detection using the established ELISA as well as the commercial chicken IgA detection kit.The results showed that both the established indirect ELISA and the commercialized kit could detect IgA antibodies in the above tissues well,and the dynamic changes of IgA antibody levels were almost consistent.After immunization,higher levels of NDV specific IgA can be detected in chicken bile,duodenal washing fluid and caecal washing fluid.Compared with the commercial assay kits,ELISA method established in this study showed lower OD values for negative samples and displayed the better specificity.2.Construction of a virulent NDV marked with green fluorescent protein(GFP)and determination of the immunization procedure for genotype Ⅶ ND vaccinesUsing NDV virulent strain JS5/05/Go as the backbone,the GFP gene was inserted between NDV P and M genes as well as between the HN and L genes,respectively.The recombinant viruses were named as rNDV/vI4GFP-P/M and rNDV/vI4GFP-HN/L correspondingly.The recombinant virus rNDV/vI4GFP-HN/L possessed the similar biological characteristics and pathogenicity to the parental virus JS5/05/Go,and could be used as an alternative challenge virus.Based on the genotype Ⅶ ND attenuated vaccine candidate strain A-NDV-LX-I4 and the recombinant inactivated ND vaccine strain A-Ⅶ,the ND immunization procedure for commercial chickens was studied.Six and four different immunization methods were developed for commercial layer and commercial broiler chickens,respectively.At different time points p.i,blood serum was collected for HI antibody detection,and tracheal,duodenal and caecal washing fluids together with bile were collected for IgA detection.To evaluate the immune efficacy of different immunization methods,the experimental chickens were challenged with recombinant virus rNDV/vI4GFP-HN/L at 32-day-old.Tracheal and cloacal swabs were collected on day 3,5 and 7 post-challenge for virus isolation.The immune efficacy in commercial layers showed that the first immunization in chickens with live and inactivated ND vaccines should be conducted at 7-day-old and 7 to 14-day-old,respectively,which can induce relative higher HI antibodies p.i.In addition,the NDV-specific IgA antibody levels in the experimental groups immunized with live ND vaccine twice within 21-day-old were generally higher and lasted longer than those in the groups immunized only once.The results of the challenge test showed that no virus shedding was detected in each immunization group post-challenge.The immune efficacy from the commercial broiler showed that the HI antibody was above 5log2 in all the immunized chicken groups before 42-day-old,which indicated the strong immunogenicity of genotype VII ND vaccine.Based on the HI and IgA detection data,the chickens vaccinated with live ND vaccine at 7-day-old and boosted at 20-day-old can obtain higher NDV-specific IgA antibodies for long time.As to the inactivated ND vaccine,the first optimal immunization in broiler chickens should be at 7-day-old,so that the high titer of HI antibodies from the immunized chickens could be generated earlier and sustain for over 5 weeks p.i.Additionally,the experimental broiler chicken group with lower IgA level could not provide effectively protection in terms of the virus shedding post-challenge,which confirmed the importance of mucosal antibodies induced by the live ND vaccine. |
PDF Full Text Request