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Abstract
Avian species are unique in that they lack a conventional lymphatic system, denoted by the absence of lymph nodes. As a result, they have an elaborate mucosal immune network, multiple paired thymi and a discrete B cell producing organ, the bursa of Fabricius. Characterizing the phenotypes of immune cells in the head associated lymphoid tissues (HALT) during juvenile maturation is critical for understanding the immunological responses to avian respiratory diseases and vaccine development. Thus, the focus of this study was to optimize techniques to effectively enumerate and phenotype select mucosal leukocytes of the conjunctiva-associated lymphoid tissue (CALT), Harderian gland (HG), nasal associated lymphoid tissue (NALT), and trachea leukocytes from White Leghorn chickens at 4, 6, 8 and 10 weeks-of-age. HG, NALT, and trachea cellularity increased with bird age whereas the CALT had minimal to no change in cellularity. Cell viability varied by tissue and was likely linked to enrichment technique. Still, the leukocyte enrichment processes used in this study resulted in a consistent yield of predominantly lymphocytes and macrophages in all four tissues. As predicted, all mucosal tissues by week 10 post hatch yielded increased CD4+ and CD8+ cell numbers. In summary, a standardized leukocyte enrichment protocol was established for the mucosal tissues of the HALT and the trachea, which allowed for cell enumeration, viability and phenotypic analysis. Further, the results of this study showed that the T cell subset numbers, but not B cell numbers, increased in these tissues with bird age.