Objective—To investigate the concentration-dependent
effects of Mannheimia haemolytica (formerly
Pasteurella haemolytica) leukotoxin (LKT) on apoptosis
and oncosis in bovine neutrophils and to examine the
role of calcium ions (Ca2+) in LKT-induced apoptosis.
Sample Population—Neutrophils isolated from
blood samples obtained from healthy calves.
Procedure—Neutrophil suspensions were exposed
to lytic or sublytic dilutions of LKT and then examined
by use of transmission electron microscopy (TEM) or
gel electrophoresis. Contribution of extracellular Ca2+
to LKT-induced apoptosis was investigated by incubating
neutrophils with LKT or control solutions in
buffer containing 1 mM CaCl2 or in Ca2+-free buffer
containing 1 mM ethylene glycol-bis (b-aminoethyl
ether)- N,N-tetraacetic acid (EGTA) prior to diphenyl
Results—Examination by TEM revealed that bovine
neutrophils exposed to lytic dilutions of LKT had
changes consistent with oncosis, whereas neutrophils
exposed to sublytic dilutions of LKT and staurosporin,
an inducer of apoptosis, had changes consistent
with apoptosis. Effects of sublytic dilutions of
LKT on apoptosis were confirmed by gel electrophoresis.
Replacement of extracellular Ca2+ with
EGTA, a Ca2+ chelator, reduced apoptosis attributable
to the calcium ionophore A23187, but it did not have
significant effects on apoptosis induced by LKT or
Conclusions and Clinical Relevance—The ability of
LKT to cause apoptosis instead of oncosis is concentration-
dependent, suggesting that both processes of
cell death contribute to an ineffective host-defense
response, depending on the LKT concentration in
pneumonic lesions. Furthermore, although Ca2+ promotes
A23187-induced apoptosis, it is apparently not
an essential second messenger for LKT-induced apoptosis.
( Am J Vet Res 2001;62:136–141)
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)