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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.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine the efficacy of leukotoxin-based Fusobacterium necrophorum vaccines and dietary tylosin in providing protection against experimentally induced hepatic abscesses in steers.

Design

30 steers assigned randomly to 6 treatment groups of 5 steers each: 1, phosphate-buffered saline solution (PBSS; control); 2, PBSS control, fed tylosin (100 mg/steer) daily; 3, inactivated whole-cell culture with oil emulsion adjuvant; 4, culture supernatant (crude toxoid) with oil emulsion adjuvant; 5, semipurified leukotoxoid with oil emulsion adjuvant; and 6, semipurified leukotoxoid with saponin adjuvant.

Procedure

Steers were inoculated SC with emulsified antigen or PBSS on days 0 and 21. Blood samples were collected at weekly intervals to monitor serum antileukotoxin antibody titer. On day 42, all steers were challenge exposed intraportally with F necrophorum culture. Three weeks later (day 63), steers were euthanatized and necropsied to examine liver and assess protection.

Results

Antileukotoxin antibody titers of all vaccinated groups markedly increased from baseline values, and mean titers of vaccinated groups were higher than those of the control and tylosin-treated groups. Steers vaccinated with culture supernatant with oil emulsion adjuvant or semipurified leukotoxoid with saponin adjuvant had the highest mean antibody titers. All 5 steers in the control group developed liver abscesses. Tylosin feeding did not protect steers challenge exposed with F necrophorum intraportally.

Conclusions

Culture supernatant was more protective than whole-cell culture or semipurified leukotoxin against experimentally induced hepatic abscesses. Partial purification of leukotoxin appeared to reduce its protective immunity. (Am J Vet Res 1996;57:483–488)

Free access
in American Journal of Veterinary Research