Biochemical characterization of the leukotoxins of three bovine strains of Fusobacterium necrophorum.
The biochemical characteristics of the leukotoxins of 3 bovine isolates of Fusobacterium necrophorum which represent biotypes A, AB, and B were compared. Two methods were used for the production of the leukotoxins: medium M-1 continuous dialysis sac cultures and brain-heart infusion agar plate cultures. The supernatant cultural fluids were fractionated sequentially by membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. The ultrafiltrates (less than 500 mol wt) were fractionated by gel-permeation chromatography, using G-10 Sephadex. The leukotoxins of the 3 F necrophorum strains were estimated to have a molecular weight between 350 and 450. The leukotoxins in the ultrafiltrates (less than 500 mol wt) were stable at 60 C for 4 hours and at 100 C for 30 minutes, stable to extremes of pH (3 to 11), and stable to degradative enzymes including trypsin, protease, alpha-amylase, lipase, deoxyribonuclease, and ribonuclease. Significant differences were not observed in the biochemical characteristics of the leukotoxins produced in vitro by the 3 F necrophorum biotypes. These assays were done, using monolayers of mouse peritoneal macrophages. The monolayers were exposed to the 4 ultrafiltrates of both the continuous dialysis sac and brain-heart infusion agar cultures (pH 7.2) for 4 hours at 4 C, 25 C, and 37 C. Maximal cytotoxic activity in the assays was at 37 C.
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D005098:Exotoxins / Q000302:isolation & purification / Q000633:toxicity*; D005674:Fusobacterium Infections / Q000382:microbiology / Q000662:veterinary*; D005675:Fusobacterium necrophorum / Q000032:analysis* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D013696:Temperature
D003470:Culture Media; D005098:Exotoxins; C018579:leukotoxin