METHODOLOGICAL ASPECTS OF ASSESSING PHAGOCYTOSIS OF VIBRIO-ANGUILLARUM BY LEUKOCYTES OF GILTHEAD SEABREAM (SPARUS-AURATA L.) BY FLOW-CYTOMETRY AND ELECTRON-MICROSCOPY

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the ba cterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes w ere incubated with the heat-killed fluorescein isothiocyanate (FITC)-l abelled bacteria for different periods, during which the kinetics of p hagocytosis was studied. Attached and interiorized bacteria were disti nguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of inges ted bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phag ocytosis. Monocyte-macrophages and acidophilic granulocytes showed pha gocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousan ds of cells, and individual cell evaluation, by quantifying interioriz ed particles in fish phagocytes. Our ultrastructural results demonstra te that V. anguillarum is actively phagocytized by seabream macrophage s and acidophilic granulocytes.