Unmasking Budgerigar Splenic Leukocyte Populations With Single-Cell Transcriptomics and Multiplex RNA In Situ Hybridization.

The current understanding of the avian immune system primarily stems from research conducted in chickens, given their economic significance as a food source. Extending the research to other avian species like Psittaciformes requires the use of label-free techniques. Therefore, budgerigar (Melopsittacus undulatus) splenic leukocytes were characterized in this study, with the help of an immunological toolbox, integrating single-cell transcriptomics and multiplex RNA in situ hybridization (ISH). Twenty-four distinct psittacine splenic leukocyte populations were identified and characterized, including, amongst others, germinal center (GC) B cells and regulatory T cells (Tregs). For each of these 24 populations, markers were defined for subsequent use in immunological assays. To further examine splenic organization, multiplex RNA ISH was applied, successfully characterizing six out of the nine selected markers. This study showed that the psittacine immune system closely mirrors that of chickens. However, a detailed, comprehensive examination was hindered by the lack of a complete sequenced and annotated budgerigar genome and the limited number of replicates. Consequently, further investigation is imperative to advance our understanding of the avian immune system.