Pasteurella haemolytica leukotoxin: physicochemical characteristics and susceptibility of leukotoxin to enzymatic treatment
Chang, Y.F.; Renshaw, H.W.; Richards, A.B.
American Journal of Veterinary Research 47(4): 716-723
1986
ISSN/ISBN: 0002-9645 PMID: 3963575 Document Number: 279555
Sterile, concentration culture supernatant from Pasteurella haemolytica (biotype A, serotype 1) strain 630 was subjected to physical, chemical, and immunologic treatments to determine their influence on leukotoxin (cytotoxin) activity contained in the supernatant. Each treated sample contained approximately 8 chemiluminescence inhibitory units of leukotoxin. Treatment effects were evaluated for their ability to inactivate leukotoxin activity. Leukotoxin activity in treated samples was determined by inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. Optimal leukotoxin synthesis by P. haemolytica occurred when the bacteria were at the logarithmic growth phase, whereas stationary phase cultures contained minimal amounts of leukotoxin activity in their culture supernatant. Leukotoxin activity was heat labile; activity was substantially decreased when concentrated culture supernatant samples containing leukotoxin activity were incubated at 37.degree. C for several hours. When concentrated culture supernatant was incubated at progressively decreasing temperatures, there was a progressive increase in the length of time that the leukotoxin retained its biologic activity. Samples stored at -70.degree. C retained activity for at least 2 months. Leukotoxin activity was nondialyzable and was able to withstand considerable extremes in hydrogen ion concentration. Leukotoxin activity could not be pelleted when subjected to forces of 100,000 .times. g for 1 hour. Chemical and enzymatic studies suggested that P. haemolytica leukotoxin contained carbohydrate and protein moieties. Chemical treatment with 0.2% sodium lauryl sulfate, 0.5% sodium deoxycholate, 7.5 mM EDTA and 8M urea with 8 mM 2-mercaptoethanol and enzymatic treatment with lipase, ribonuclease, and deoxyribonuclease had no discernible effect on leukotoxin activity. However, chemical treatment with appropriate concentrations of potassium metaperiodate and enzymatic treatment with chymotrypsin, trypsin, protease, and amylase completely inactivated leukotoxin activity. The leukotoxin preparation was without hemolytic activity against erythrocyte suspensions prepared from 12 different non-human animals and from human beings with each of the 4 ABO blood groups (A, B, O, and AB). Bovine fetal serum was without neutralizing activity against leukotoxin. Neutralizing activity was found in a serum pool from young cattle and in a convalescent phase serum collected from a cow that had recovered from spontaneously occurring pneumonia caused by P. haemolytica. Collectively, results from this study and previous studies suggest that metabolically active P. haemolytica synthesize and secrete into the culture supernatant a heat-labile, oxygen-stable, pH-stable, nondialyzable, nonhemolytic, and water-soluble antigenic glycoprotein that is toxic to ruminant leukocytes and that may be an important virulence factor of the microorganism.