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.
10 P haemolytica A1 strains and 1 P multocida strain.
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.
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.
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.
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)
Objective—To characterize ultrastructural changes of
bovine lymphocytes exposed to Pasteurella haemolytica
Sample Population—Partially purified LKT from a wild
type P haemolytica A1 strain and inactive pro-LKT from
an isogeneic mutant P haemolytica strain. Isolated bovine
lymphocytes were obtained from 2 healthy calves.
Procedure—Isolated bovine lymphocytes were incubated
with various concentrations of LKT and pro-LKT
for 3 hours at 37 C and examined by use of transmission
electron microscopy. A cytochemical Klenow
DNA fragmentation assay was used to examine lymphocytes
for DNA fragmentation.
Results—Lymphocytes incubated with LKT at a high
concentration (1.0 toxic U/ml) had ultrastructural evidence
of cytoplasmic and nuclear membrane rupture
and swelling or lysis of mitochondria. Low concentrations
of leukotoxin (0.1 toxic U/ml) induced DNA
fragmentation in 80% of lymphocytes. Ultrastructurally,
these cells had nuclear membrane blebbing,
cytoplasmic vaculation, chromatin condensation,
nuclear fragmentation, and membrane-bound
apoptotic bodies. Incubation of lymphocytes with
LKT at extremely low concentrations (0.001 toxic
U/ml) or with pro-LKT did not alter their ultrastructure.
Inclusion of 0.5 mM ZnCl2
in the medium
blocked leukotoxin-induced ultrastructural changes in
Conclusions and Clinical Relevance—Low concentrations
of LKT induce apoptosis and high concentrations
induce oncotic cell lysis in bovine lymphocytes.
The ability of low LKT concentrations to induce apoptosis
in host leukocytes may allow bacteria to escape
host immune surveillance and colonize the host.
(Am J Vet Res 2000;61:51–56)