Biological and biochemical characterization of Fusobacterium necrophorum leukotoxin

Z. L. Tan From the Department of Animal Sciences and Industry (Tan, Nagaraja, Smith) and the Department of Pathology and Microbiology, College of Veterina Medicine (Chengappa), Kansas State University, Manhattan, KS 6506.

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 BVSc, PhD
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T. G. Nagaraja From the Department of Animal Sciences and Industry (Tan, Nagaraja, Smith) and the Department of Pathology and Microbiology, College of Veterina Medicine (Chengappa), Kansas State University, Manhattan, KS 6506.

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 MVSc, PhD
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M. M. Chengappa From the Department of Animal Sciences and Industry (Tan, Nagaraja, Smith) and the Department of Pathology and Microbiology, College of Veterina Medicine (Chengappa), Kansas State University, Manhattan, KS 6506.

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 DVM, PhD
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J. S. Smith From the Department of Animal Sciences and Industry (Tan, Nagaraja, Smith) and the Department of Pathology and Microbiology, College of Veterina Medicine (Chengappa), Kansas State University, Manhattan, KS 6506.

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 PhD

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Summary

Biological and biochemical characteristics of the leukotoxin of Fusobacterium necrophorum were determined. Culture supernatant of F necrophorum was toxic to polymorphonuclear neutrophilic, leukocytes from cattle and sheep, but not to those from pigs and rabbits. Culture supernatant and sonicated bacterial cell fractions had low hemolytic activity and did not cause dermonecrosis in a guinea pig. Supernatant derived leukotoxin was inactivated at 56 C for 5 minutes and became unstable at pH > 7.8 or < 6.6. Chemical treatment with 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 5.2% sodium sulfide, or 0.25 mM titanium (III) citrate markedly decreased leukotoxicity. Enzymatic treatment with protease, trypsin, and chymotrypsin inactivated the toxin completely, whereas amylase had no effect. Use of protease inhibitors failed to prevent loss of leukotoxin activity. Using membrane partition chromatography and gel filtration, the estimated molecular weight of the toxin was > 300,000. On reduction and denaturation, the toxin dissociated into several components by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Summary

Biological and biochemical characteristics of the leukotoxin of Fusobacterium necrophorum were determined. Culture supernatant of F necrophorum was toxic to polymorphonuclear neutrophilic, leukocytes from cattle and sheep, but not to those from pigs and rabbits. Culture supernatant and sonicated bacterial cell fractions had low hemolytic activity and did not cause dermonecrosis in a guinea pig. Supernatant derived leukotoxin was inactivated at 56 C for 5 minutes and became unstable at pH > 7.8 or < 6.6. Chemical treatment with 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 5.2% sodium sulfide, or 0.25 mM titanium (III) citrate markedly decreased leukotoxicity. Enzymatic treatment with protease, trypsin, and chymotrypsin inactivated the toxin completely, whereas amylase had no effect. Use of protease inhibitors failed to prevent loss of leukotoxin activity. Using membrane partition chromatography and gel filtration, the estimated molecular weight of the toxin was > 300,000. On reduction and denaturation, the toxin dissociated into several components by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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