Masamitsu Kanoe

Effects of Fusobacterium necrophorum on the mesenteric microcirculation of guinea pigs

Thrombi formation was demonstrated in mesenteric microcirculation of guinea pigs inoculated with Fusobacterium necrophorum and the bacterial hemagglutinin (HA). The thrombi were initially observed in venules and later, in arterioles. Immunofluorescence study revealed that the HA bound to the thrombi in the microcirculation. These results indicate that thrombosis is an early step in the pathogenesis of necrosis.

Dermonecrotic activity of a cell wall preparation from Fusobacterium necrophorum.

A cell wall preparation of Fusobacterium necrophorum induced haemorrhagic necrosis in the skins of guinea pigs and rabbits. Effects in mice and rats were weak or absent. The toxic activity of the cell wall preparation was not reduced by heat treatment. A dermonecrotic toxin was isolated from the cell wall preparation with sodium dodecylsulphate and concentrated by precipitation with ethanol. A preparation of the bacterial cytoplasm from Fus. necrophorum induced mainly erythema.

Purification and partial characterization of Fusobacterium necrophorum hemagglutinin

Hemagglutinin (HAin) of Fusobacterium necrophorum was separated from the bacterial cells by trypsinization-sonication, and purified by the gel filtration on Sephadex G-100 column. The final product obtained from gel filtration gave one precipitin line in the immunodiffusion gel and produced a single band in polyacrylamide gel electrophoresis. The molecular weight of the HAin was estimated to be about 19000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was heat labile and comparatively rich in alanine, glutamine and histidine. Electron microscopy observation revealed that the HAin was a filamentous rod with 0.5-1.0 nm width or frequently showed a cluster form. The hemagglutinability was inhibited by addition of albumins but not by sugars and lipopolysaccharide. Anti HAin rabbit serum inhibited hemagglutination.

Partial characterization of leukocidin from fusobacterium necrophorum

Leukocidin from Fusobacterium necrophorum was produced in the diffusate of a dialysis culture. It was free from deoxyribonuclease, fibrinolysin, gelatinase, haemolysin, lipase, caseinase and endotoxin. The leukocidin had a molecular weight between 10,000 and 5,000 as estimated by membrane partition chromatography. It formed precipitin lines with anti-leukocidin-serum in double immunodiffusion tests. Mouse peritoneal cells were characteristically damaged by the leukocidin, as revealed by scanning electron microscopy. The damaged cells lost microvilli and suffered partial destruction of their cell membranes.

Adherence of Fusobacterium necrophorum to Vero cells

The adherence of the anaerobic species Fusobacterium necrophorum to the surface of Vero cells was studied. Adherence between the bacterium and the tissue culture cells was paralleled by the hemagglutinability of F. necrophorum. Treatment of the bacterial cells with lactoalbumin hydrolysate or anti-F. necrophorum-hemagglutinin serum reduced the intensity of the attachment. The purified hemagglutinin bound to the membranes of Vero cells. It lost its adherent property when mixed with the homologous anti-hemagglutinin serum. These observations suggest that the adherence of F. necrophorum to the surface of Vero cells is mediated by the bacterial hemagglutinin.

Cytotoxic effects of a leukocidin from Fusobacterium necrophorum on bovine hepatic cells

Cytotoxic effects of a leukocidin from Fusobacterium necrophorum were demonstrated on bovine hepatic cells. The cytotoxic response was dose-dependent and could be inhibited by homologous antiserum. Scanning electron microscopy revealed damaged hepatic cell surface. These findings indicated a pathogenic role of the leukocidin in F. necrophorum infections.

Effects of leukocidin from fusobacterium necrophorum on bovine peripheral leukocytes in vitro

Partially purified leukocidin from Fusobacterium necrophorum damaged bovine peripheral leukocytes as demonstrated by trypan blue staining. Granulocytes were most and T-lymphocytes least sensitive to the leukocidin. Heating for 30 min at 60 degrees C completely inactivated the leukocidin. The cytotoxicity of the leukocidin could be neutralized by homologous anti-leukocidin. Scanning electron microscopy of the exposed cells revealed an apparent destruction of the cell membranes, loss of the microvilli and smoothing of the cell surfaces.