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.

Poly(methacrylic acid) hydrogels were tested for oral delivery of a vaccine against Pasteurella haemolytica infection in cattle. Culture supernatants of P haemolytica, the most common bacterium associated with pneumonia in cattle, were used as the antigens in the vaccine. Hydrogels containing culture supernatants were administered orally to calves. Calves were then challenge-exposed with virulent P haemolytica. Calves were euthanatized 3 days after challenge exposure. The lungs of each calf were scored for severity and size of pneumonic lesions. Results indicated that vaccinated calves had smaller, less severe pneumonic lesions and lived longer than nonvaccinated calves. These results indicated that hydrogels can be used to deliver vaccines orally to calves to enhance resistance to pneumonia caused by P haemolytica.

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples. However, similar analysis of data on GAG release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of LPS derived from different bacterial sources made a significant difference in the response of explants to incubation with LPS.