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The objective of this proposal is to characterize the mucosal immune response in the Harderian glands of chicken upon experimental exposure to avian influenza (AI) virus. Mucosal immunity, which provides protection at the portal of entry of viruses , is important for protection of chickens against viruses including AI. The magnitude and the distribution of mucosal and systemic immune responses induced after nasal spray, ocular or drinkwater exposure to low pathogenic AI strains H5N1 and H7N3 will be evaluated. ELISA, ELISPOT assay, immunohistochemistry and FACS analyses will be performed to analyze the mucosal immune response in the Harderian glands to AI virus in chickens. The observed humoral immune response in mucosal secretions (e.g. lacrimal fluids, bile and feces) and plasma will be measured. In addition to analyses of T and B cell responses, crossprotection to AI in mucosal inductive and effector site (Harderian glands) after exposure to AI will be analyzed.
<p> Non-Technical Summary- Increasing the resistance of the poultry population against AI will not only reduce the risk of enormous economic losses due to high mortality in chicken flocks but also reduce the risk for the human population. Presently there are numerous projects oriented towards the design of new vaccines against AI (recombinant vaccines, gene deleted vaccines, vaccines generated by reverse genetics, etc). However, there is little knowledge of the mucosal immune response against AI despite the fact that this is the port of entrance of the virus. This research proposal will not only improve the understanding how to control AI but also provide insight in the avian mucosal immune system and in particular the role of the Harderian gland, which is the site of primary replication of numerous respiratory viruses. Thus, the purpose of this project is to better understand the mucosal immune response induced by avian influenza virus, specifically the Harderian glands, in order to develop better vaccines targeted to the appropriate mucosal sites to induce protection against AI and to better understand crossprotective immunity to different serotypes of AI virus .
In order to address mucosal immunity to avian influenza virus we will collect mucosal secretions, such as tears, feces and bile as well as plasma, to measure induction of avian influenza specific antibodies by ELISA. This will involve the analyses of IgA, IgM and IgG antibodies. Furthermore, IgA antibodies will be analyzed for whether or not it is polymeric or monomeric. This is important to know since only polymeric IgA will be secreted at mucosal surfaces. In addition, avian influenza specific IgA, IgG and IgM secreting B cells will be analyzed in Harderian glands, Bursa of Fabrisius, intestinal lamina propria cells, caecal tonsils and lungs. The lymphocyte composition of Harderian glands, lungs and spleen will be analyzed by FACS and combined with immunohistochemistry will provide a better understanding of the role of these organs in immunity to low pathogenic avian influenza virus (LPAI). Different T cell subsets from these organs will be sorted by flow cytometry and analyzed for cytokine production by RT-PCR. This will involve the analyses of both Th1 and Th2 cytokines. Finally we test the cross-protection between H7N3 and H5N1 serotypes after immunization with one and challenge with the other serotype by measuring viral load in Harderian glands , eye and lungs. Other approaches to identify crossprotective antibodies are ELISA, Westernblot analyses and HAI analyses. This may identify conserved regions of avian influenza that will generate crossprotection against multiple serotypes of AI.