Adapting Flow Cytometry for Studying Immune Cell Seasonality in Wild Migratory Bats

Identifying the drivers of wildlife immunity is critical for assessing stressor impacts and zoonotic risks. However, such studies are limited by logistical challenges of wildlife research and lack of species-specific reagents. We adapt flow cytometry, typically confined to laboratory settings, to field settings to profile cellular immunity with small blood volumes and extended sample holding times. We then apply these methods to a preliminary assessment of immune cell seasonality in migratory Mexican free-tailed bats (Tadarida brasiliensis). We confirmed four antibodies recognizing CD3, CD79a, MHCII, and CD11b originally validated in Egyptian fruit bats (Rousettus aegyptiacus), enabling quantification of T and B cells, macrophages, and neutrophils, respectively. Flow cytometry outperformed hematology in quantifying leukocyte profiles and revealed variable immune cell seasonality. Adaptive cells increased between spring and fall migration. Neutrophils were most abundant during the reproductive period and decreased during migrations, whereas B cells were most abundant after reproduction and before fall migration; granulocytes as a whole, macrophages, and T cells had no seasonality. Females had more B cells than males but did not differ in other cells. Our findings lay the groundwork for applying flow cytometry to field studies of wildlife and provide initial insights into the seasonality of bat immunity.