Internalization of Vibrio salmonicida in isolatedmacrophages from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) evaluated by a paired immunofluorescence technique

A procedure was developed to culture isolated head kidney macrophages fromsalmonid fish and to study their phagocytic activity. The method was used to compare in vitro macrophage phagocytic activity against Vibrio salmonicida in rainbow trout and Atlantic salmon. These two species of salmonid fish differ in their natural resistance to V. salmonicida, with rainbow trout being more resistant. In both species, the isolated cell population from head kidney had morphological and functional characteristics typical of macrophages. The cellsʹ functional activities were evaluated by studying their ability to grow and adhere to glass, to phagocytise uncoated monodispersed polymer beads, their acid phosphatase activity, and their uptake and degradation of bacteria in a phagocytic assay. To distinguish between extra cellular/cell-adherent bacteria and intracellular bacteria, a paired immunofluorescence technique was developed, based on monoclonal antibodies identifying epitopes on the lipopolysaccharide component of the bacterium. A unique identification of bacterial localisation was achieved by sequential incubation steps with intermittent cell membrane permeabilisation with absolute ethanol. At initial time points macrophages from rainbow trout exhibited a significantly higher phagocytosis of unopsonised V. salmonicida, compared to Atlantic salmon macrophages. Phagocytosed bacteria were easily degraded intracellularly, but fragments of the bacterium were identified by the two monoclonal antibodies at 72 h after inoculation.