Optimisation of bronchoalveolar lavage fluid preparation for mononuclear cell isolation and cytologic evaluation in free-ranging African elephants (Loxodonta africana).

Understanding immune responses to infectious diseases, such as tuberculosis (TB), is essential for developing diagnostic tests and studying disease progression. Although TB affects African savanna elephants (Loxodonta africana), few studies have investigated immune cells and function in this species, especially in the respiratory tract. Techniques for isolating immune cells from elephant bronchoalveolar lavage (BAL) samples have not been previously reported. Therefore, this study aimed to develop and optimise a protocol to isolate and characterise alveolar cell types in BAL fluid collected from free-ranging, African savanna elephants. The optimised protocol incorporated a mucin digestion step, filtration, Ficoll gradient separation and wash steps to remove contaminants and successfully isolate viable populations of alveolar mononuclear cells. The isolated cells were stained with Rapi-Diff and microscopically examined to differentiate and characterise each cell type present. Cells isolated from healthy African elephant BAL samples, using this method, were predominantly alveolar macrophages (92.5 - 100.0 %) followed by lymphocytes (0.0 - 6.0 %), neutrophils (0.0 - 3.0 %) and eosinophils (0.0 - 1.0 %). This study provides the first optimised protocol for the isolation of alveolar mononuclear cells for future investigations into local immune responses to respiratory diseases such as tuberculosis.