The Synergistic Pathogenesis Of Infectious Bronchitis Virus And Newcastle Disease Virus In The Chickens Immunized With Newcastle Disease Vaccine

Infectious bronchitis virus(IBV)and Newcastle disease(ND)virus are epidemic in chicken flocks in China.There are increasing evidences of NDV co-infection with IBV,however,studies describing the pathogenesis of the co-infection of NDV and IBV are not available.Due to high pathogenicity of NDV to chickens,we decided to develop the co-infection model of NDV and IBV in NDV vaccinated chicken chickens.We check the clinical symptom,death rate,histopathological change,antibody level,virus titers and expression patterns of innate immune related genes,by the means of clinical observation,histopathological analysis,serologic test and quantitative Real-time PCR.The study is as follows:During the epidemiological investigation,we isolate a new infectious bronchitis virus(IBV)and named CK/CH/HD/171018.The entire genome of the virus obtained(Gen Bank: MH020185).Two recombinations were observed in Gene 1 of CK/CH/HD/171018 virus by the QX-like and CK/CH/LSC/99 I like strains.One hundred and fifty-two specific pathogen free(SPF)chickens were divided into 8 groups.Chickens in the group A to F were vaccinated with La Sota inactivated vaccine at the age of 50 days,and the birds in the G and H group were non-vaccinated and served as the positive controls.Chickens in the groups A to F were challenged with PBS,ZJ1,QX,QX+ZJ1(+24h),ZJ1+QX(+24h),QX+ZJ1,and at the same time group G and H were challenged with ZJ1 and QX at 64 days of age,via the eye and choana.All the chickens were observed for the clinical signs and mortality upto 20 days post infection(dpi).After the infection birds were sacrificed at 2dpi,3dpi,5dpi,7dpi,and collected the trachea,lung,spleen,kidney,caecum and bursa to examine the histopathological alteration,virus loads,and expression level of cytokines.The result showed that all the non-immunized chickens(group G,ZJ1 positive control),inoculated with ZJ1 died on 5dpi.Interestingly,there was not any mortality in the birds inoculated with PBS,ZJ1,QX(Group A to C)and group H(QX positive control).The mortality rates of QX+ZJ1(+24h),ZJ1+QX(+24h)and QX+ZJ1 co-infection group were 30%,20% and 30% respectively,which was obviously higher than group B and C.In addition,co-infection of QX with ZJ1 increased the severity of clinical signs and pathological changes.Therefore,there is a pathogenic synergism between IBV and NDV in the chickens immunized with Newcastle disease vaccine.Antibody levels of NDV and IBV were checked by heamagglutination inhibition(HI),and the results showed that the NDV antibody levels of all the groups increased on the second day after the challenge,but the antibodies of IBV until the 7th day after the challenged.Absolute quantification Real-time PCR methods of NDV and IBV were developed respectively to check the virus shedding in cloaca and the virus titers in trachea,lung,spleen,kidney,intestines and bursa.While,relative quantification Real-time PCR method was used to check the expression level of IFN α,IFN β,1L-1β,1L-6,MX and PKR in every organ.Results showed that,significantly increased virus shedding,as well as duration was observed for the ZJ1 and QX strains in co-infected birds,compared to birds infected with one virus.Similarly,virus loads were higher in co-infected chickens.In the early phase of infection,results of cytokine expression revealed that it was significantly higher in the chickens infected with one virus,ZJ1 or QX compared to co-infected groups.But,the expression pattern of these cytokines were reversed during late phase,and were higher for co-infected groups.In conclusion,we developed co-infection model of NDV and IBV,and systematically checked the dynamic changes of NDV and IBV,host innate immune response after co-infection,which provided assertive evidence for the enhancement of pathogenicity after NDV and IBV co-infection.It will also provide the information to design the control programme of NDV and IBV co-infection.