INTERNALIZATION OF VIBRIO-SALMONICIDA IN ISOLATED MACROPHAGES 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 from salmonid fish and to study their phagocytic activity. The method was used to compare in vitro macrophage phagocytic activity against Vi brio salmonicida in rainbow trout and Atlantic salmon. These two speci es of salmonid fish differ in their natural resistance to V. salmonici da, with rainbow trout being more resistant. In both species, the isol ated cell population from head kidney had morphological and functional characteristics typical of macrophages. The cells' functional activit ies were evaluated by studying their ability to grow and adhere to gla ss, to phagocytise uncoated monodispersed polymer beads, their acid ph osphatase activity, and their uptake and degradation of bacteria in a phagocytic assay. To distinguish between extracellular/cell-adherent b acteria and intracellular bacteria, a paired immunofluorescence techni que was developed, based on monoclonal antibodies identifying epitopes on the lipopolysaccharide component of the bacterium. A unique identi fication of bacterial localisation was achieved by sequential incubati on steps with intermittent cell membrane permeabilisation with absolut e ethanol. At initial time points macrophages from rainbow trout exhib ited 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 i dentified by the two monoclonal antibodies at 72 h after inoculation.