Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity [30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml)]. Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

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.

Alterations in the size and functions of porcine alveolar macrophages exposed to sublytic amounts of heat-labile, hemolytic cytotoxin produced by Actinobacillus pleuropneumoniae (App) serotype 1, strain HS54 into the culture medium were studied in vitro. Alveolar macrophages were sensitive to the cytotoxin; treatment of the macrophages with low concentrations of cytotoxin (0.016 hemolytic unit) resulted in severe, irreversible cell swelling. However, high doses of cytotoxin (2.0 hemolytic units) were required to cause substantial cell death, as indicated by the influx of propidium iodide into and release of lactate dehydrogenase from cells. Macrophages exposed to low, sublytic doses of cytotoxin failed to migrate toward chemoattractant, were unable to attach to glass, and failed to phagocytize optimally opsonized erythrocytes. Macrophages already attached to glass surfaces detached when exposed to sublytic doses of cytotoxin. The swelling and impairment of functions of alveolar macrophages observed in this study could not be attributed to endotoxic effects, because heat treatment of the cytotoxin preparation for 60 minutes at 60 C resulted in complete loss of cytotoxicity. We conclude that sublytic doses of heat-labile, hemolytic cytotoxic substances produced by App depress alveolar macrophage function at concentrations likely to develop in association with acute pulmonary infection with App. The Apx (A pleuropneumoniae Rtx toxins) exotoxins secreted by the bacteria into culture medium were considered responsible for the toxic activity of the cytotoxin preparation. The Apx of the App field strain used in this study were likely to be similar to those of serotype-1 reference strain (S4707). Analysis by use of DNA-DNA hybridization indicated that genomic DNA of the field strain contained sequences similar to those encoding structural protein of ApxI (apxIA) and ApxII (apxIIA) of the serotype-1 reference strain. Therefore, Apx produced by the field strain of App used in this study are likely to be of similar pathogenic importance worldwide.