Microscopic identification of active phagocytes in the blood of the European pond turtles (Emys orbicularis)

Upon stimulation with antigens, phagocytes act as key cells of innate immunity. Recognized mammalian blood phagocytes are neutrophils and monocytes, but evolutionary older classes, such as reptiles, have a diversity of blood cells able to fully perform phagocytosis. It was previously shown that in turtles, besides heterophils and monocytes, eosinophils, B lymphocytes, erythrocytes and thrombocytes have a powerful phagocytic capacity as well. The aim of this study was to identify different blood phagocytes in a group of European pond turtles (Emys orbicularis), after in vitro stimulation with zymosan particles. The study included six turtles, residents of the Belgrade Zoo, that initially took part in a larger study, and were proven to be clinically healthy. Each of the residual heparin blood samples (30 μL) was mixed with opsonized zymosan (6 μL, 1 mg/mL), and incubated 15 minutes at 37°C and 15 minutes at room temperature, respectively. Total leukocytes (40×, average number×2000) and differential count (100×) were assessed microscopically, using Romanowsky-stained blood smears. For each sample, the ability of blood cells to perform phagocytosis was confirmed and estimated based upon their zymosan intake. The main phagocytes were heterophils, but zymosan particles were also engulfed by monocytes, eosinophils, basophils, lymphocytes and red blood cells. Due to their possible interference with tube heparin, thrombocytes were dominantly found in clusters, resulting with doubtable phagocytic performance. Our study confirms that different blood cells of European pond turtles have preserved the potential to perform phagocytosis, and identifies for the first time that basophils also possess this function. ; Proceedings