Preliminary Study On The Mechanism Of Different Protection Efficacy Of H120 And 4/91 Vaccines Against IBV QX Genotype Strain

Infectious bronchitis virus(IBV)is an acute and highly contagious respiratoryinfectious disease,causing great economic loss.Currently,the most effective prevention and treatment measures are still vaccine immunization for IBV.But new variantion,serotypes and genotypes of strains result in differences in the immunoprotective effects of vaccines for different strains.Previous studies showed that these two attenuated vaccines of 4/91 and H120 had different protective efficacy for the prevalent genotype QX strain,but the mechanism is still unclear.To systematically reveal the mechanism of different protective efficacy bwteen 4/91 and H120 attenuated vaccines,the 4/91 and H120 vaccine were used to immunize for chickens,and the cellular,humoral and mucosal immune response were detected.Then chickens were challenged with QX-type strain A/CK/MeiShan/17(hereafter referred as MS strain).Finally,the results were collected for the morbidity,mortality,the tissue pathological changes,the expression of cellular immune related genes in tracheal mucosa and the viral loads of MS strain in trachea and kidney.In this study,the standard curve of ACTB,PFP,GAMA,IFN-?and MS1a was established,using a real-time quantitative RT-PCR.The results showed that amplification efficiency(E)of standard curves were 1.95,2.00,1.97,2.01 and 2.03 for ACTB,PFP,GAMA,IFN-?and MS1a,respectively.The correlation coefficient(R~2)of were 1.00 for five standard curves.The dissolution peaks of these five amplified products were specific single-peaks.The real-time fluorescent quantitative RT-PCR of sensitivity were 100 times for ordinary PCR.And,this RT-PCR with a good reproducibility within the range of template concentration 10~2-10~7 copies/uL.Therefore,these five standard curves can be used for the relative quantification and absolute quantification in this study.One-day-old SPF chickens were randomly grouped into 4 groups(I~IV).On 7 days old and 21 days old,groups I and II with 35 chickens were immunized using 4/91 and H120,respectively.Group III with 14 chicks,7 days old and 21 days old did had PBS.However,chickens were challenge with MS strain at 35 days old with group III.Group IV,25 chickens,used as the blank control,did do had PBS at7 days old,21 days old and 35 days old.The chickens in each group were held in separate biosafety level 2(BSL2)isolators under negative pressure.On 21,28 and 35 days,5 chickens in group I,II and IV were randomly selected,and blood samples were collected from the isolation of lymphocytes and serum.The percentage of T lymphocytes in peripheral blood was detected by flow cytometry test,the specific antibody titer in serum was detected by neutralization test.The chickens were euthanized.Then the collected trachea was flushed with PBS and the suspension was collected,and the level of SIgA antibody in the tracheal flushing fluid was detected by ELISA.The results showed that 4/91 could significantly increase the number of CD3~+and CD4~+T lymphocytes(P<0.05)at 28 days.H120 could not significantly increase the number of T lymphocytes,but increase the CD4~+:CD8~+ratio significantly at 28days(P<0.05),breaking the original balance of the body.Both 4/91 and H120 could effectively induce humoral immune response,and the specific antibody titer in serum were significantly higher than that of group IV(P<0.01).At 28 days,the antibodies titers induced by 4/91 was significantly higher than H120(P<0.05).The 4/91 and H120 could effectively inducetracheal mucosal immune response,and the level of SIgA antibody was significantly higher than group IV(P<0.01).Although there was no significant difference between the immunized groups,the 4/91 induced mucosal immune response were higher than that of H120.At 35 days old,groups I,II and III were challenged with QX strain MS strain and observed for 10days.The clinical symptoms were recorded and the morbidity and mortality were calculated.On the 5th and 10th day after challenge,the anatomical lesion were recorded and the lesions were scored for tracheal hemorrhage and renal enlargement.The relative expression of CMI gene in the tracheal mucosa and the viral load of MS strain in the tracheal mucosa and kidney were also detected by the established real-time quantitative RT-PCR method.The results showed that the protection efficacy among those three groups were group I>group II>group III,the pathological damage in the trachea and kidney,the relative expression of CMI gene in tracheal mucosa,and the viral load of MS strain in tracheal mucosa and kidney among those three groups were group I<group II<group III.In conclusion,the results of this study showed that,compared with the H120 vaccine,the 4/91vaccine strain could induce higher levels of cellular,humoral and mucosal immune response,and also reduce the tissue damage and viral load after the challenge of the prevalent QX genotype strain.