Serum-free culture of Pasteurella haemolytica optimized for leukotoxin production.

Objective: To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification.

Sample Population: One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf.

Procedure: Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated.

Results: Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1 M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1% glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium.

Conclusions: Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT.