ELECTRON-MICROSCOPE STUDIES OF THE IN-VITRO PHAGOCYTOSIS OF MYCOBACTERIUM SPP. BY RAINBOW-TROUT ONCORHYNCHUS-MYKISS HEAD KIDNEY MACROPHAGES

The cytological response of rainbow trout Oncorhynchus mykiss head kid ney macrophages to ingested Mycobacterium spp, was examined in vitro. Mycobacterium marinum or Mycobacterium sp. TB267 isolated from snakehe ad fish Channa striata Bloch were opsonised with either fresh rainbow trout serum, serum which had been heat-inactivated, or rainbow trout a ntiserum against the extracellular products (ECP) of the 2 Mycobacteri um spp. A monoclonal antibody against the ECP was also used as an opso nin. Suspensions of macrophages were prepared (1 ml of 1 x 10(7) cells ml(-1)), mixed with the opsonised bacteria (100 mu l of 2 x 10(9) ml( -1)), and incubated at 18 degrees C for 0.5, 1, 2, 4 or 6 h to allow p hagocytosis to occur. A quantitative evaluation of the phagocytosis of the mycobacteria by the macrophages was carried out by electron micro scopy. Macrophage phagosomes and their contents were examined and numb ers of intact and partially degraded bacteria determined. Pre-labellin g dense granules (secondary lysosomes) with ferritin enabled phagosome lysosome fusion to be identified and their frequency determined. Opso nisation of the mycobacteria was found to greatly enhance the phagocyt ic and killing activity of the rainbow trout macrophages.