Application of Multiparametric Flow Cytometry Panels to Study Lymphocyte Subpopulations in Tuberculin-Positive Cattle

SIMPLE SUMMARY: Flow cytometry is a technique that allows identifying different cell populations based on their morphological characteristics and the presence of proteins in the membrane or inside the cell. It is widely used in biomedical research and clinical diagnosis; however, in the veterinary area, its use has been limited due to the reduced availability of fluorochrome-conjugated antibodies that recognize specific proteins. In order to implement multiparametric analyses in the study of bovine tuberculosis, this work designed two panels composed of different conjugates and tested them to identify helper, cytotoxic, activated, and memory T cells in heifers positively and negatively tested for tuberculin (diagnostic test for bovine tuberculosis). Both panels allowed the identification of the cells of interest; in addition, a higher number of activated and memory cells were observed in tuberculin-positive heifers. These panels can also be used in immunopathogenesis studies, as well as in the evaluation of vaccines in cattle. ABSTRACT: Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25(+) and CD45RO(+) in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis (M. bovis). We identified subpopulations of T cells from peripheral blood mononuclear cells using two multicolor panels. These panels could be used to analyze total bovine blood in immunopathogenic studies and vaccine development. The same strategy could be implemented in other species of veterinary interest.