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CHAPTER SEVEN Immunophenotyping of Avian Lymphocytes IMPLICATIONS AND FUTURE FOR UNDERSTANDING DISEASE IN BIRDS Jeanne M. Fair, Kirsten J. Taylor-McCabe, Yulin Shou, and Babetta L. Marrone Abstract. Chicken (Gallus gallus domesticus) T-cell populations can be delineated into subsets based on their expression of cell-surface proteins such as cluster of differentiation (CD) cell surface markers. However, immunophenotyping using flow cytometry in birds has focused on cell characterization in the thymus and spleen during development in chickens. West Nile virus (WNV) causes differential infections in birds, ranging the entire spectrum of pathogenesis. In order to accurately assess immunocompetence to diseases such as WNV in birds, more efficient methodology to access natural variability in avian immune function must be devised and understood . Previously, lymphocyte subpopulations CD4⫹ and CD8⫹ have been found to be critical for clearing infection of WNV in mammals. Focusing on chickens, a species that is susceptible but not infective for WNV, our objectives were to: (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations, and (3) estimate repeatability and application to other avian species susceptible to WNV. Immunophenotyping of CD3⫹, CD4⫹, CD8⫹, and CD45⫹ was successfully completed for chicken peripheral blood but not for the Common Raven (Corvus corax) or Black-billed Magpie (Pica hudsonia). Future studies include immunophenotyping during infection studies of WNV in chickens and further development of flow cytometry for other bird species. Key Words: chicken, flow cytometry, Gallus gallus domesticus, host range, immunophenotyping, lymphocytes, West Nile virus. Caracterización Inmunofenotípica de Linfocitos de Aves: Las Implicaciones y el Futuro Para el Entendimiento de la Enfermedad en Las Aves Resumen. Las poblaciones de las células T del pollo (Gallus gallus domesticus) pueden ser caracterizadas en base a la expresión celular de sus proteínas de superficie, tales como los marcadores de antígenos de diferenciación leucocitaria (CD). Sin embargo, la caracterización inmunofenot ípica usando citometría de flujo en aves se ha enfocado a la caracterización celular en el timo y el bazo durante el desarrollo de pollos. El virus del Nilo Occidental (VNO) causa diferentes formas de infección en las aves, y cubre todo el espectro patológico. Para poder evaluar de manera precisa la inmunocompetencia de las aves a las enfermedades como el VNO es necesario desarrollar metodologías más eficientes, que permitan accesar la variabilidad natural de la función Fair, J. M., K. J. Taylor-McCabe, Y. Shou, and B. L. Marrone. 2012. Immunophenotyping of avian lymphocytes: implications and future for understanding disease in birds. Pp. 81–90 in E. Paul (editor). Emerging avian disease. Studies in Avian Biology (vol. 42), University of California Press, Berkeley, CA. 81 STUDIES IN AVIAN BIOLOGY NO. 42 Paul 82 de CD3⫹, CD4⫹, CD8⫹ y CD45⫹ fue exitosa para la sangre periférica de pollos, pero no fue exitosa para el Cuervo Común (Corvus corax) y para la Urraca (Pica hudsonia). Los estudios para el futuro deberán incluír la caracterización inmunofenotípica de los pollos durante la infecci ón con el VNO y el desarrollo de la citometría de flujo para otras especies de aves. Palabras Clave: caracterización inmunofenotípica, citometría de flujo, Gallus gallus domesticus, linfocitos , pollo, rango de hospedero, virus del Nilo Occidental. as probes to proteins expressed by cells. Lymphocytes express distinct assortments of molecules on their cell surfaces, many of which reflect either different stages of their lineage-specific differentiation or different states of activation or inactivation. Lymphocyte cell surface molecules are routinely detected with anti-leukocyte monoclonal antibodies (mAbs). Using different combinations of mAbs, it is possible to profile the cell surface immunophenotypes of different leukocyte subpopulations, including the functionally distinct mature lymphocyte subpopulations of B cells, helper T cells, cytotoxic T cells, and natural killer cells, as well as estimate the whole leukocyte count. The cluster of differentiation (CD) applies to the subpopulations of lymphocytes that describe the antibody directed to the antigen on the cell, and not to an epitope of that antigen (Table 7.1). For example, in humans, Stewart and Nicholson (2000) noted that CD11a refers to all antibodies that bind to any epitopes on the alpha chain of LFA1. Conventions in nomenclature arose out of the need to identify the antigen, rather than the epitope. The CD molecules most commonly referred to are CD4⫹ and CD8⫹, which are markers for...

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