Setup of a Simple and Cost-Effective pH-Sensitive Assay to Evaluate Phagocytosis in Rainbow Trout (Oncorhynchus mykiss) Peripheral Blood Leukocytes

Phagocytosis in vitro assays are widely used to assess the health status of fish, since it may be affected by diseases and environmental factors. To date, studies have evaluated phagocytosis in fish by observing fluorescent beads or bacteria engulfed by phagocytes. However, particles adhering to the external surface of the phagocyte membrane cannot be easily distinguished from those that are truly internalized. This work aimed to: (1) test whether a pHrodo™ (pHR)-based method improves the accuracy of phagocytosis detection compared to traditional assays; (2) evaluate the influence of opsonization, incubation time, and temperature on the phagocytosis ability of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs). Opsonization increased the PBLs’ phagocytic activity. To evaluate the effects of incubation time and temperature on PBL phagocytosis ability, 30% autologous plasma was used. Leukocytes incubated at 16 °C for 2 h showed a significantly higher (p < 0.05; H = 45.43) percentage of phagocytic cells. This incubation setting also resulted in increased pHR-related fluorescence intensity (p < 0.05; F = 58.39), directly proportional to the number of phagocytosed particles. These findings indicate that opsonization enhances phagocytic ability and capacity, and that a 2 h incubation at 16 °C may be recommended for future large-scale studies aimed at evaluating innate immune function in rainbow trout.