Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish

The phagocytes of fish play an important role in innate host defense against bacterial infection, and participate in various immunoregulatory processes. Here, we investigated the effects of various opsonins in the ingestion and adhesion processes by examining respiratory burst (RB) activity in blood and head kidney (HK) fish phagocytes. RB activity was induced in rainbow trout phagocytes with the bacterium Aeromonas salmonicida (strain MT004) in the presence of various opsonins [purified antibodies (Ab), immune serum (IS), normal serum (NS) and heat-inactivated immune serum (HI-IS)], and measured in terms of luminol-amplified chemiluminescence (CL) emission at 20 °C for 210 min. The RB activity of blood phagocytes was measured directly from highly diluted whole blood and compared to that observed in isolated head kidney (HK) phagocytes measured under similar conditions. In addition, the extracellular RB activity of adhesion (extracellular degranulation) and the intracellular RB activity of ingestion were distinguished through their inhibition by gelatin and cytochalasin D. Our results showed that the first CL peak appeared within 50 min, and decreased or vanished when gelatin was added to the reaction or when the active complement was destroyed by heating. The second CL peak appeared after 50 min, depending on the utilized opsonin, and vanished when cytochalasin D was added to the reaction. Our results indicate that adhesion and ingestion compete for consumption of reactive oxygen intermediates. Specific IgM without an active complement was a relatively inefficient opsonin, whereas specific IgM with an active complement increased the magnitude of ingestion-mediated RB activity and accelerated the ingestion of target bacteria. Taken together, these results indicate that adhesion and ingestion responses competed for limited phagocyte resources and that the bacterial uptake by blood phagocytes can be measured directly from highly diluted blood.