Immune response and distribution of antigen in chickens after infection LPAIV (H4N6)
Authors: Muzyka Denys , Lillehoj Hyun , Rula Olexandr , Stegniy Borys 
Published: May 2017

ObjectiveTo study the immune response in chicken on the administration ofLPAIV isolated from the natural reservoir.IntroductionInfluenza is a serious problem for the health of people, animals andbirds. Therefore, comprehensive study of influenza virus, its naturalreservoir, pathogenesis and immune response will provide furtheropportunity to ensure protection for animals, birds and people fromthis infection.MethodsFour-week-old commercial chickens were intranasally inoculatedwith a H4N6 LPAIV A/Garganey/Chervonooskilske/4-11/2009(H4N6), isolated from the cloacal swab of clinically healthy garganeyin 2009 in Ukraine. Cecum, spleen, lung, and trachea samples werecollected from infected chickens on 1 - 14 dpi and examined byimmunohistochemical and virology techniques. On these days,we collected blood samples for serological analysis. Detection ofantibodies to avian influenza virus subtype H4 was performed withchicken serum samples by HI test and ELISA. The studies were doneaccording to IACUC.ResultsUpon intravenous and intranasal infection with this virus(A/Garganey/Chervonooskilske/4-11/2009), no clinical signs wereobserved in chickens and no pathological changes were found atnecropsy. Infection of poultry with this virus provoked an antibodyresponse at 10 days after intranasal inoculation which ranged from1:8 to 1:32 serum antibody titers. Only 2 of 5 chickens were positiveby the HI test and 3 of 5 were positive by ELISA at intranasalinoculation. All 10 chickens were positive both by HI test and byELISA after intravenous inoculation. Specific antibodies (HI test) toinfluenza virus H4 were detected in titer ranges of 1:128 to 1:1024.In immunohistological studies, the respiratory tract organs (lungsand trachea) showed higher level of humoral immunity (IgM, IgG,IgA-expressing cells) in the lung compared to the trachea. Also,indicators of cell mediated immunity as measured by the CD4 andmacrophage markers were higher in LPAIV-infected chickens inthe lungs at 14 days post infection compared to uninfected chickens.Lymphocytes expressing CD8 were increased starting 7dpi.The chickens in the infected group showed 2 times higher levels ofCD8 cells compared to the control chickens. IFN-γtranscripts wereobserved in the AI-infected chickens starting at 7dpi that coincideswith the increasing level ofCD4 cells. The number of lymphocytes which secrete IL-2 andIL-15 in AI-infected chickens were in general 1.5 to 2 times highercompared to the uninfected chickens. In AIV-infected chickens, thelevel of cells expressing IFN-γ, IL-2, and IL-15 increased at 7-daysafter infection. The peak time coincided with a period of increasingCD8 cells. However, there was no significant difference in thesecytokine levels between the AIV-infected and uninfected groups.In the cecum, lower levels of CD4 cells were seen on 5 dpi butlevels slowly increased from 7 dpi to 14 dpi following AIV infection.In the ceca, a significant increase in the number of cells expressingIgM and IgG was found.LPAIV infection induced an increase in macrophages andlymphocytes expressing CD4 and CD8 in the spleen throughout theperiod examined in this study indicating their role in host responseto viral infection. The levels of macrophages in chickens of AIV-infected group were 2 times higher than the control after 1 dpi.ConclusionsAlthough infection with a LPAIV did not cause obvious clinicaldisease, viral replication was detected in the trachea and spleen andboth local and systemic cellular and humoral immune responses wereelicited in these LPAIV-infected chickens. Our results indicate thepotential possibility for infection of poultry with viruses isolated fromwild birds. But currently it is not completely known why some virusesfrom wild birds can cause infection in poultry, while others can not.Further study of the immune response will enable us to determine thefeatures of the pathogenesis of low pathogenic avian influenza.