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

Sarah K. Highlander From the Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030.

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 PhD

Abstract

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. (Am J Vet Res 1997;58:749–754)

Abstract

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. (Am J Vet Res 1997;58:749–754)

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