Growth of Pasteurella haemolytica and production of its leukotoxin in semi-defined media.

OBJECTIVE: To develop semi-defined media that support growth of the bovine pathogen, Pasteurella haemolytica, and use them to examine production of leukotoxin and an arginine-binding protein by this organism. SAMPLE POPULATION: 10 P haemolytica A1 strains and 1 P multocida strain. PROCEDURE: Bacterial strains were cultivated at 37 C in media containing various amino acids, carbon sources, vitamins, and cofactors, and absorbance (OD600) was measured. Leukotoxin and arginine-binding protein production were assessed by immunoblot analysis. RESULTS: Optimal growth required supplementation with 0.1% fetal bovine serum, gelatin, or purified bovine serum albumin. Calcium pantothenate and thiamine were essential for growth, and a variety of carbon sources could be utilized. In the complete medium, 15 amino acids were included; however, in the minimal medium, no amino acids were required. All strains (except P multocida) grew in the complete medium and 7 grew well in the minimal medium. Leukotoxin was not produced when amino acids were limiting, but could be enhanced by addition of 0.2% NH4SO4. Production of the arginine-binding protein was not affected by nitrogen availability or by presence of L-arginine. CONCLUSIONS: Two media that support good growth of P haemolytica strains were developed. The minimal medium is simple to prepare and manipulate and its use revealed a potential role of nitrogen availability in the regulation of leukotoxin expression. CLINICAL RELEVANCE: Creation of these media will permit continued studies of the response of P haemolytica to environmental conditions that may mimic those encountered in the bovine respiratory tract during shipping.