Infectious Disease

Veterinary Pathology
48(2) 338-348
Mannheimia haemolytica: Bacterial–Host ª The American College of
Veterinary Pathologists 2011
Reprints and permission:
Interactions in Bovine Pneumonia sagepub.com/journalsPermissions.nav
DOI: 10.1177/0300985810377182
http://vet.sagepub.com

K. Singh1, J. W. Ritchey2, and A. W. Confer2

Abstract
Mannheimia haemolytica serotype S1 is considered the predominant cause of bovine pneumonic pasteurellosis, or shipping fever.
Various virulence factors allow M haemolytica to colonize the lungs and establish infection. These virulence factors include
leukotoxin (LKT), lipopolysaccharide, adhesins, capsule, outer membrane proteins, and various proteases. The effects of LKT
are species specific for ruminants, which stem from its unique interaction with the bovine b2 integrin receptor present on
leukocytes. At low concentration, LKT can activate bovine leukocytes to undergo respiratory burst and degranulation and
stimulate cytokine release from macrophages and histamine release from mast cells. At higher concentration, LKT induces
formation of transmembrane pores and subsequent oncotic cell necrosis. The interaction of LKT with leukocytes is followed
by activation of these leukocytes to undergo oxidative burst and release proinflammatory cytokines such as interleukins 1, 6,
and 8 and tumor necrosis factor a. Tumor necrosis factor a and other proinflammatory cytokines contribute to the
accumulation of leukocytes in the lung. Formation of transmembrane pores and subsequent cytolysis of activated leukocytes
possibly cause leakage of products of respiratory burst and other inflammatory mediators into the surrounding pulmonary
parenchyma and so give rise to fibrinous and necrotizing lobar pneumonia. The effects of LKT are enhanced by
lipopolysaccharide, which is associated with the release of proinflammatory cytokines from the leukocytes, activation of
complement and coagulation cascade, and cell cytolysis. Similarly, adhesins, capsule, outer membrane proteins, and proteases
assist in pulmonary colonization, evasion of immune response, and establishment of the infection. This review focuses on the
roles of these virulence factors in the pathogenesis of shipping fever.

Keywords
bronchopneumonia, cytokine, leukotoxin, Mannheimia haemolytica, shipping fever, virulence factors

Mannheimia haemolytica (formerly Pasteurella haemolytica)
is the major cause of fibrinous and necrotizing lobar pneumo-
nia and pleuropneumonia of cattle which is often called ship-
ping fever (SF) (Fig. 1 and 2). SF has a marked economic
significance worldwide and costs approximately $1 billion per
year to the US cattle industry.125 The term shipping fever
emphasizes circumstances (stress) under which the disease pre-
dominantly occurs. Various combinations of environmental
stress can serve as cofactors in the pathogenesis of SF—such
as inclement weather, shipment, weaning, overcrowding, and
complex interactions among several infectious agents, includ-
ing bovine herpesvirus 1, bovine parainfluenza virus 3, bovine
respiratory syncytial virus, bovine viral diarrhea virus, Myco-
plasma sp, and other bacteria. The underlying processes by
which various environmental factors alone or in combination
with microorganisms predispose cattle to SF are not fully
understood. Alterations of local innate and adaptive immune
responses presumably contribute to the development of the dis-
ease process.45,64,107,109,110,111 Although SF is a multifactorial
disease, M haemolytica serotype S1 is considered the major
cause of the most severe form of SF pneumonia, and it is
generally believed that control of M haemolytica S1 infection
would markedly reduce the prevalence and severity of SF.
M haemolytica is an opportunistic pathogen; it is a normal
inhabitant of the nasopharynx and tonsils of cattle and
sheep.46,118 In healthy cattle, the relatively nonpathogenic
M haemolytica serotypes S2 and S4 predominate, whereas
pathogenic serotype S1 is present in low numbers.42,43
Although the exact mechanisms are not known, stress or
concurrent viral infections result in microenvironmental
1
Veterinary Diagnostic Laboratory and Department of Pathobiology, College
of Veterinary Medicine, University of Illinois, Urbana, Illinois
2
Department of Veterinary Pathobiology, Center for Veterinary Health
Sciences, Oklahoma State University, Stillwater, Oklahoma
Corresponding Author:
Kuldeep Singh, DVM, MS, PhD, Diplomate ACVP, Assistant Professor,
Anatomic Pathology, Veterinary Diagnostic Laboratory and Department of
Pathobiology, College of Veterinary Medicine, University of Illinois, 2001
South Lincoln, Urbana, IL 61802
Email: ksingh08@uiuc.edu

338
Singh et al 339

Figure 1. Lung; bovine. Fibrinous bronchopneumonia. The ventral regions of cranial, middle, and caudal lobes are markedly dark purple,
consolidated, regionally collapsed, and sharply demarcated. Thick mats of fibrin strands are adhered to the visceral pleura, and the interlobular
septa are expanded by edema. Figure 2. Cut section of lung; bovine. Necrotizing bronchopneumonia. The lobular pattern is accentuated by
severe edema of interlobular septa and necrosis, centered on airways imparting a characteristic marble pattern. Necrotic foci are irregular,
discrete, sharply delineated from the remaining parenchyma, and bordered by a pale white necrotic rim. Note that a thrombus (arrow)
completely occludes the lumen of a blood vessel.

change that favors increased multiplication and colonization of
serotype S1 in the upper respiratory tract.11 The selective
growth and colonization of the nasopharyngeal region by
M haemolytica serotype S1 is a prerequisite for the develop-
ment of SF.61 The resulting colonization of the nasal mucosa
with large numbers of serotype S1 contributes to the inhalation
of aerosol droplets containing bacteria, into the trachea and
lungs.50 Healthy calves can clear the inhaled bacteria; however,
stressed calves develop pneumonia. Although the most
common isolate from the cattle lung with SF is M haemolytica
serotype S1; other reported serotypes include S2, S5, S6, and
S9, and group S6 and S2 constitute approximately 25% and
10% of the isolates, respectively.7,100
Various virulence factors possessed by M haemolytica S1
promote lung colonization and evasion of host immune
response; therefore, the host immune status is critical in counter-
acting these strategies. When the host immune response is not
successful, these interactions contribute to the development of
SF. Multiple review articles have addressed these interactions
in detail.103,113 Several virulence factors of M haemolytica sero-
type S1 promote host–pathogen interaction and influence the
outcome of infection. Of these, leukotoxin (LKT) and lipopoly-
saccharide (LPS) are important, and their roles in pathogenesis
of SF are well documented.23,61,98,113 Other virulence factors
with less-defined pathogenic roles are capsule, adhesins, outer
membrane proteins, and various proteases, such as neuramini-
dase and glycoprotease.23,38,58,68 The following is an overview
of the virulence factors of M haemolytica serotype S1.
Surface Proteins and Carbohydrates
Adhesins
A naı̈ve host encounters M haemolytica from the environment
through air or droplet inhalation. For development of SF, a
necessary requirement is that of initial colonization and estab-
lishment in the nasopharynx by adherence to the epithelial sur-
face.17 This binding must provide resistance to the physical
removal by air flow, local innate and adaptive immune mechan-
ism, and mucociliary clearance. The adhesins of M haemolytica
involved in the nasopharyngeal epithelial binding are not fully
understood.52 De la Mora et al38 demonstrated a 68-kDa adhesin
molecule associated with M haemolytica that is involved in the
adhesion to cultured tracheal cells. In addition to binding tra-
cheal cells, this adhesin binds a 165-kDa glycoprotein receptor
onto neutrophils and initiates an oxidative burst.38 Another puta-
tive adhesin molecule is present on fimbriae of M haemolytica
and binds to a sialoglycoprotein receptor on respiratory epithe-
lium.56,88 However, a locus encoding such an adhesin has not yet
been identified.53 Kisiela and Czuprynski63 recently identified
the heat-modifiable M haemolytica outer membrane protein
OmpA and lipoprotein Lpp1, which are involved in adherence
to the bovine bronchial epithelium.
Alternatively, adhesion molecules are not always necessary
for niche colonization. Nonspecific adherence to epithelial
surfaces via capsular or other bacterial surface proteins can
occur, or bacteria can simply remain within the mucus layer.88
This view is supported by the fact that adherence of M haemo-
lytica to nasal mucus can be increased by enzymatic degrada-
tion of the protein and carbohydrate components of mucus.11
M haemolytica–derived neuraminidase has been shown to exert
a pathogenic role by degrading mucus components and thus
enhancing adherence of M haemolytica to epithelium.115
Retzer et al102 proposed that outer membrane iron-binding pro-
teins transferrin-binding protein A and B (Tbp A and Tbp B)
could serve as adhesin molecules because their homologue in
Neisseria meningitidis is expressed on the bacterial surface.102
Furthermore, based on evaluation of the M haemolytica
genome sequence and in comparison with the sequences from
N meningitidis and Bordetella sp filamentous hemagglutinin,

339
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Highlander52 proposed that a filamentous hemagglutinin homo-
logue of high molecular weight (340 Kd) in M haemolytica is an
adhesin molecule. In addition, several other gene sequences
of putative adhesins were proposed after analysis of the M
haemolytica S1 genome sequence; however, their role as
adhesins has not yet been confirmed.47,52
Lipopolysaccharide
LPS plays a critical role in the pathogenesis of SF. Current evi-
dence indicates that LPS contributes to the pulmonary lesions
through a variety of complex mechanisms, including the stimu-
lation of leukocytes to produce proinflammatory cytokines, the
activation of complement and coagulation cascade, and direct
cell cytolysis.69,83,85 The LPS molecule consists of polysac-
charide side chain (O antigen), lipid A, and inner and outer
cores of oligosaccharides, with the size of the immunogenic
side chain determining whether the LPS type has a rough or
smooth material.8,67 The lipid A moiety of LPS is responsible
for eliciting endotoxic effects, such as pyrexia and hypoten-
sive shock. Although O antigen of LPS is immunogenic and
antibodies exhibit cross-reactivity among different serotypes,
there is unfortunately no correlation between high antibody
response to LPS and protection of the host from development
of pneumonia.23,24
LPS is a potent vasodilator; it stimulates pulmonary
endothelial changes consistent with the vascular leakage.86 In
addition, when administered intravenously in sheep, LPS pro-
duces clinical signs associated with the hypotension.4 The sys-
temic effects of LPS are considerably important in acute
pasteurellosis, which causes septicemia in lambs, resulting in
high mortality. The pathological effects of LPS are mediated
by binding with LPS-binding protein, and further interactions
are most likely mediated by CD14. LPS forms high–molecular
weight complexes with LKT, enhancing the pathogenic effect
of each other.69,76 Additionally, in vitro studies revealed that
bovine alveolar macrophages simultaneously challenged with
LKT and LPS produced more tumor necrosis factor a (TNFa)
and interleukin 8 (IL-8), compared to cells challenged with
each factor individually.69 There is some similarity between the
effects mediated by LKT and LPS. Because LPS and LKT can
physically interact and it is difficult to obtain purified LPS
alone without LKT, it is possible that results of some in vitro
studies using LPS or LKT alone are influenced by the presence
of the other molecule.
In pneumonic calf lungs, LPS can rapidly cross the alveolar
wall and become localized within the cytoplasm of neutrophils,
alveolar macrophages, endothelial cells, and pulmonary intra-
vascular macrophages and on epithelial cell surfaces.124 LPS
can stimulate alveolar macrophages to produce proinflamma-
tory cytokines, reactive nitrogen intermediates, reactive oxy-
gen intermediates, and other mediators that can actively
participate in the inflammatory process; these include IL-1b,
IL-8, leukotriene 4, prostaglandin E2, and TNFa from bovine
leukocytes.55,69,83,129,131 Subsequently, these proinflammatory
cytokines and chemotactic mediators initiate influx of
340
Veterinary Pathology 48(2)
neutrophils.13,69 In vitro challenge of bovine neutrophils with
both LPS and LKT resulted in degranulation, generation of
superoxide and nitric oxide, and lysis.80 Another in vitro study
demonstrated increased expression of elastase, myeloperoxi-
dase, nitric oxide, and alkaline phosphatase in neutrophils.123
The in vitro effect of LPS on bovine alveolar macrophages
demonstrated a dose-dependent increase in iNOS gene expres-
sion, and nitric oxide generated from LPS-stimulated bovine
alveolar macrophages caused cytotoxic injury to pulmonary
endothelial cells in a dose-dependent manner.131 Similarly,
inoculation of M haemolytica in calf lung lobes revealed iNOS
gene expression in leukocytes and epithelial cells.101
LPS can cause pulmonary damage directly through toxic
effects on pulmonary endothelium and indirectly through neu-
trophil recruitment.55,85,97 The toxicity of LPS can be
enhanced by complexing it with the phospholipids in the pul-
monary surfactant, which allow LPS to persist in the lung and
initiate inflammation.12,90 Other systemic effects induced by
LPS include fever and production of acute-phase proteins
by the liver.
Similar to LKT, LPS exhibits a dose-dependent effect on the
bovine peripheral blood leukocytes. At low concentrations,
LPS decreases the phagocytic ability of neutrophils, whereas
at higher concentrations, the phagocytic activity increases.25
Moderate concentration is mitogenic for mononuclear cells,
whereas a high concentration has the opposite effects.25 Emau
et al41 studied the molecular mechanisms underlying these
effects using purified M haemolytica LPS infusion. The authors
concluded that cyclic nucleotides mediated the action of LPS at
the cellular level, as evidenced by an increase in plasma arachi-
donic acid, thromboxane B2, and prostaglandin E levels.
Capsule
There are 12 serotypes of M haemolytica (S1, S2, S5–S9,
S12–S14, S16, S17) based on the differences in capsular poly-
saccharide antigen typing.103 Log-phase bacteria exhibit good
encapsulation, whereas stationary-phase bacteria exhibit poor
encapsulation.27 The chemical structures of capsular polysac-
charides of M haemolytica S1, S2, and S7 have been deter-
mined2 and are similar to those from other pathogenic
bacteria. S1 capsular polysaccharide structure is similar to the
widely distributed enterobacterial common antigen; S2 capsu-
lar polymer is identical with capsular polysaccharides of
N meningitidis B and Escherichia coli K1; and S7 structure
is similar to polysaccharides associated with N meningitidis
group L and Haemophilus influenza type F.2-4
The capsule of M haemolytica S1 may be involved in colo-
nization of lung by promoting bacterial adherence to respira-
tory epithelium.89 Except for lung colonization, the role of
capsule in the pathogenesis is not yet established. Chae et al,
among others,14,31,32 demonstrated that the presence of capsule
reduces leukocyte phagocytosis as well as complement-
mediated lysis of the bacterium, whereas others have found
that the presence of anticapsular monoclonal antibodies pro-
motes phagocytosis but not complement-mediated killing.127
Singh et al
Czuprynski et al31 found that M haemolytica capsular polysac-
charide stimulated release of IL-1 from bovine blood mono-
cytes but not from alveolar macrophages. Vaccination of
cattle with live M haemolytica or capsular polysaccharide pro-
motes production of anticapsular antibodies; however, a posi-
tive correlation between capsular antibody and protection has
not been established.26,121
Outer Membrane Proteins
M haemolytica possess multiple outer membrane proteins.96
Several of these proteins are iron-regulated outer membrane
proteins, such as Tbp 1 and Tbp 2, and are physiologically
and pathologically relevant because they are in involved in
iron acquisition.93 Because M haemolytica does not produce
siderophores, expression of these iron-regulated outer mem-
brane proteins is the main mechanism of iron acquisition.62,93
Furthermore, Iovane et al57 demonstrated that outer membrane
proteins are chemotactic agents for neutrophils and inhibit their
phagocytic and subsequent bacterial-killing mechanisms, thus
favoring bacterial pulmonary colonization.
The protective antigens of M haemolytica are not com-
pletely understood; however, data indicate that protective
immunity against M haemolytica can be acquired through the
production of neutralizing antibodies against outer membrane
proteins and LKT.22,108 One of the immunogenic outer mem-
brane proteins that offer protection following vaccination is a
45-kDa S1 outer membrane lipoprotein designated PlpE.94,95
This outer membrane protein was identified in all serotypes,
but its structural and physiologic function is not known. The
antibodies generated against PlpE offer cross protection against
serotype 6 and promote phagocytosis and complement-
mediated bacterial killing.94
Another outer membrane protein, OmpA, is a highly con-
served protein that is involved in binding to specific host cell
receptors in the upper respiratory tract, thereby playing a role
in adherence and colonization and in generating host specifi-
city.37,63 Other studies have found that outer membrane pro-
teins stimulate the release of nitric oxide, induce the
expression of iNOS in interferon g–activated macrophages,
increase actin polymerization, and modify oxidative burst in
neutrophils.57,84
Toxins and Extracellular Enzymes
Leukotoxin
M haemolytica LKT and LPS are well-characterized virulence
factors with respect to their pathogenic role in the SF.52,60
Since its initial discovery, LKT has been the subject of intense
investigation relative to its role in the pathogenesis of SF. LKT
is an exotoxin and a member of the RTX (Repeats in ToXin)
family of toxins. These toxins are genetically related and share
a common, highly conserved motif consisting of a series of gly-
cine–aspartic acid nonapeptide repeats in the carboxy terminal
third of the LKT protein molecule. This conserved motif is
involved in calcium binding and plays a vital role in inducing
341
leukocyte toxicity because of its ability to form tertiary confor-
mation required for target host cell binding.29 The conserved
motif also contains a recognition site required for transport
of LKT across biological membranes in bacteria by the tolC-
dependent type I secretion system.28,128 The other members
of this family and their LKTs include Actinobacillus actinomy-
cetemcomitans (LtxA), Actinobacillus pleuropneumoniae cyto-
toxins (ApxI, ApxII, ApxIII and ApxIV), E coli alpha
hemolysin, Actinobacillus suis subs haemolyticus toxin (Aqx),
Fusobacterium necrophorum LKT, Bibersteinia trehalosi, and
Bordetella pertussis hemolysin.66,91,105,106 Although these
members belonging to the RTX family of toxins are genetically
related owing to the similarity of mechanisms involved in LKT
activation, secretion, and partially shared amino acid
sequences, they differ markedly in target cell specificity.122
M haemolytica LKT is a 102- to 105-kDa protein produced
by all serotypes during the logarithmic phase of bacterial
growth in vitro.107 The genetic organization of the LKT gene
complex is composed of four genes, with genes lktC and lktA
located upstream and genes lktB and lktD located down-
stream.53 The structural gene product of lktA is composed of
953 amino acids, which encodes the structural yet biologically
inactive LKT–protoxin. Gene lktC encodes transacylase that
posttranslationally modifies the inactive LKT–protoxin by
fatty acid acylation, thereby converting it into a biologically
active LKT (LKT-A). During acylation, the fatty acid groups
are added to the lysine residues located on LKT-A, which is
a critical step in removing charge and increasing the hydropho-
bicity of LKT-A and which allows LKT-A to insert into host
cells and form transmembrane pores. Although acylation is not
required for LKT-A binding to the target cells, it is required for
increasing intracellular calcium ion concentration, generation
of reactive oxygen species, and production of IL-8 and for
causing cytolysis.119 Gene products of lktB and lktD are
required for transport of LKT-A from the bacterial cytoplasm
into the outer environment (Fig. 3).
LKT-A contains domains that are responsible for receptor
binding, pore formation, and calcium binding.29,116 The
N-terminal region is putatively involved in the receptor bind-
ing, whereas the adjacent residues of LKT-A consist of a series
of hydrophobic residues implicated in spanning the host cell
membrane and, possibly, pore formation.91 The carboxy
terminal domain of LKT-A contains glycine and aspartate-
rich repeats, and neutralizing epitopes are present within a
229–amino acid region at the C-terminal end of LKT-A.53
LKT follows a species-specific dose-dependent activation–
inhibition paradox on bovine leukocytes.91 At low
concentrations, LKT can activate neutrophils and macrophages
to stimulate respiratory burst and degranulation, proinflammatory
cytokine (TNFa, IL-1, and IL-8) release from neutrophils and
alveolar macrophages, and histamine release from mast cells, as
well as reduce mitogen-mediated lymphoid proliferation.53,80
At high concentrations, LKT stimulates bovine leukocytes to
undergo apoptosis by engaging extrinsic and intrinsic
mechanisms, whereas at highest concentrations, LKT causes
transmembrane pore formation, cell swelling, and subsequent cell
341
342
Figure 3. The gene complex of Mannheimia haemolytica S1
leukotoxin is composed of genes lktC, lktA, lktB, and lktD. The
structural gene product of lktA is biologically inactive LKT–protoxin
(pro-LKT-A). Gene lktC encodes transacylase that posttranslationally
modifies the pro-LKT-A, thereby converting it into a biologically
active leukotoxin (LKT-A), which allows LKT-A to insert into host
cells and form transmembrane pores. Gene products of lktB and lktD
are required for transport of LKT-A from the bacterial cytoplasm
into the outer environment. bp, base pairs; aa, amino acids.
necrosis (oncotic cell death).10,20,120 The transmembrane pores
in the plasma membrane of activated macrophages and neutro-
phils and their subsequent oncotic cell necrosis cause leakage
of products into the surrounding pulmonary parenchyma,
contributing to the pulmonary damage—specifically, products
of respiratory burst (ie, oxygen free radicals, superoxide
anions, and hydrogen peroxide) and others, such as nitric
oxide, lysosomal enzymes (including myeloperoxidase), and
arachidonic acid metabolites (such as leukotriene B4 and
5-hydroxyeicosatetraenoic acid).31,51,79,80,131 In addition,
M haemolytica LPS can complex with LKT, enhancing
LKT-induced cytotoxicity.69,76
LKT-A is a key virulence factor that contributes to the
pathogenesis of inflammation and pulmonary necrosis in SF,
and it is specific for ruminant leukocytes.54,117 In diseased
lungs, LKT is associated with the cell membranes of degener-
ating inflammatory cells located in alveoli.124 Although most
strains of M haemolytica from cattle and sheep produce LKT,
not all strains are equally pathogenic, because LKT from these
strains do not exhibit similar leukotoxic activity and the
amount of LKT produced is variable.36,103,104 The importance
of LKT in causing pulmonary necrosis was established by
intratracheal inoculation of calves with a LKT-deficient mutant
M haemolytica, which caused lower mortality and decreased
lung lesions, as compared to animals challenged with the parent
strain capable of producing LKT-A.54,117
M haemolytica–induced pneumonia and other diseases are
observed in only ruminants—including cattle, sheep, bighorn
sheep, goats, bison, and exotic ruminants—because LKT-
induced effects are specific for ruminant macrophages,
lymphocytes, neutrophils, and platelets.34,35 This species
specificity stems from the selective interaction of LKT with the
342
Veterinary Pathology 48(2)
b2 integrin LFA-1 (lymphocyte function-associated antigen 1;
CD11a/CD18) on target host cells.35,40,48,59,71,77,78 Integrins
are expressed on most cell types and are involved in cell–cell
interactions and cell–extracellular matrix interactions, whereas
subtype b2 integrins are expressed exclusively on leukocytes,
including T lymphocytes, macrophages, neutrophils, monocytes,
and dendritic cells.6
Despite a consensus that LKT-induced specificity is due to
b2 integrin binding, a complete agreement is lacking on the
specific b2 integrin and subunits of b2 integrin required for
receptor–ligand interaction.34,70,71 Most scientific evidence
supports LKT interaction with the CD18 subunit of LFA-1.36
Furthermore, in vitro incubation of bovine leukocytes with
proinflammatory cytokines (IL-1b and TNFa) results in
increased expression of LFA-1 and a simultaneous increase
in LKT binding, cytotoxicity, and apoptosis.30,73,74 Similarly,
in vivo and in vitro results indicate that bovine herpesvirus 1
infection increases susceptibility of the bovine leukocytes to
LKT binding and cytotoxicity by increasing LFA-1 expression
on neutrophils and peripheral blood mononuclear cells.75
Recent studies using LKT-resistant mouse histiocytoma,
human K562, and other cell lines demonstrated that these cell
lines could be rendered susceptible to LKT effects by expres-
sing cattle CD18 subunits on these cells.
However, other evidence suggests that LKT binds not only
with the CD18 subunit of other b2 integrins but also with the
CD11a subunit.40,120 It appears that binding of LKT to CD18
is critical in eliciting the pathologic effects of LKT, and during
this binding, LKT may interact with CD11a subunit.34,36,40 It
was recently suggested that LKT particularly binds to the integ-
rin epidermal growth factor 3–like domain of bovine CD18.39
Furthermore, interaction not mediated by b2 integrin may
occur, not only with cells from nonruminant species, but with
cells lacking b2 integrins, such as erythrocytes and platelets.116
It is therefore possible that some effects are mediated by non-
specific receptor–ligand interactions.
In addition, in vitro studies have revealed that at lower
doses, LKT can induce apoptosis, activate leukocytes, or inhi-
bit lymphoid proliferation.103 Those latter effects are partially
abrogated by preincubation of lymphocytes with IL-1 and
IL-2.81,82 The mechanism underlying this response is
unknown; however, the authors suggested failure of IL-2
production by lymphocytes or altered IL-2 receptor expres-
sion.81,82 In addition, this effect raises interesting concerns
on whether increased susceptibility to other bacteria or viruses
in SF is due to the underlying lymphoid necrosis. However,
lymphopenia is not a clinical feature of SF, and LKT is
restricted to the lungs of affected animals with no systemic dis-
tribution, suggesting that LKT-induced inhibition of lymphoid
proliferation may be only an in vitro phenomenon.
LKT-induced cytotoxicity of host leukocytes is character-
ized by the formation of transmembrane pores and, eventually,
oncotic cell lysis.19,20 LKT inserts its N-terminus into the host
cell membrane to form a hydrophilic pore, but how these termi-
nal transmembrane pores are formed is controversial. Studies
have indicated that polymerization of LKT occurs on host cells,
Singh et al
thereby forming transmembrane pores, whereas others have
suggested that LKT-induced regulation of voltage gated chan-
nels and, thereafter, formation of transmembrane pores.19,56,87
In addition, Atapattu and Czuprynski9 recently demonstrated
that LKT binds to the cell receptors and relocates to lipid rafts
and clathrin-coated pits, resulting in LKT internalization and
cytotoxicity. LKT-induced cytotoxicity has been shown to
be associated with a caspase 1–dependent pathway,
whereas LKT-associated apoptosis proceeds through a caspase
9–dependent pathway.10
Irrespective of the basic process involved in the pore forma-
tion, these pores are 0.001 to 0.002 mm in diameter, allow rapid
loss of intracellular Kþ from the host cell cytoplasm, and inter-
nalization of Naþ molecules, causing colloid–osmotic imbal-
ances.19 Subsequently, water moves into the cell to correct
the imbalance, resulting in rapid cell swelling.20 Uncontrolled
transmembrane calcium influx occurs and likely activates
membrane phospholipases and proteases, or it may disrupt the
cytoskeletal structure.
The outcome of these alterations is degradation of the cell
membrane and subsequent cytolysis of leukocytes, as demon-
strated by release of cytoplasmic lactate dehydrogenase.20
Similarly, LKT can enhance ruminant platelet adhesion and
activate and induce transmembrane pore formation in platelets
by calcium-dependent mechanisms.15,21,92 The transmembrane
pores in the plasma membrane of activated macrophages and
neutrophils and their subsequent oncotic necrosis may cause
leakage of products of respiratory burst, lysosomal enzymes,
and arachidonic acid metabolites into the pulmonary parench-
yma.33,79,80,131 These by-products cause damage to the pul-
monary parenchyma, giving rise to fibrinous and necrotizing
bronchopneumonia typical of SF (Figs. 1, 2).79 LKT can also
induce release of histamine from isolated bovine mast cells,
causing increased vascular permeability and rapid influx of
serum proteins and more LKT-susceptible neutrophils at the
location of inflammation.5 Because b2 integrins are present
on lymphocytes, LKT-induced damage to these cells may
allow bacteria to evade host adaptive immune responses.
The influence of calcium ions on the cytotoxic activity
of M haemolytica LKT is critical because depletion of intracel-
lular calcium ion by addition of a calcium chelator eliminated
the cytolytic effect of low doses of the toxin. Likewise, addition
of calcium to target bovine leukemia cell line (BL-3) cell cul-
tures depleted of the divalent cations restored the cytolytic
effect of the LKT. Addition of a calcium channel blocker
resulted in dose-dependent protection of BL-3 from LKT-
induced cytolysis. These results suggest that calcium positively
influences the rapid initial phase of cell death resulting from
exposure to the toxin.55,130
Proteases
A number of proteases have been associated with M haemolytica.
Although their physiological and pathological roles are not com-
pletely understood, a role has been suggested—namely, one of
circumventing innate and adaptive immune response, thereby
343
enhancing lung colonization and establishment of the disease. For
instance, in the bovine respiratory tract, immunoglobulin A pre-
dominates in the upper respiratory tract, whereas immunoglobu-
lin G (IgG) is the primary antibody in the lower respiratory
tract.99,126 Both IgG1 and IgG2 are believed to be important in
defense against M haemolytica infection. Glycoprotease obtained
from the culture supernatant of M haemolytica can selectively
hydrolyze IgG1, thereby reducing opsonization-induced phago-
cytosis and bacterial killing; it can also selectively degrade host
mucosal sialoglycoproteins.1,72,94 Shewen et al106 used this gly-
coprotease to demonstrate that vaccination of calves with the
recombinant glycoprotease alone and in combination with M
haemolytica culture supernatant vaccine enhances protection
against experimental disease. The pathogenic function of this
enzyme is unknown; however, adherence of bacteria to host
epithelial cells and aggregation of platelets in the alveoli have
been demonstrated, whereas glycoprotease activity can be poten-
tiated by coincubation with the LKT.92
All strains of M haemolytica produce the enzyme
neuraminidase; however, its function is unknown.44,115 Many
respiratory pathogens, including Hemophilus influenzae,
Streptococcus pneumoniae, and Pseudomonas aeruginosa,
express neuraminidases that can cleave a2,3-linked sialic acids
from glycoconjugates. Because mucosal surfaces are heavily
sialylated, neuraminidases may modify epithelial cells by
exposing potential bacterial receptors, enhance biofilm
formation, and evade local innate immune response by cleav-
ing salivary glycoproteins.49,65,112 Therefore, it is possible that
neuraminidase produced by M haemolytica may play a role in
the initial colonization and evasion of antimicrobial killing and
immune response.
Summary
The pathogenesis of SF is elaborate and complicated.
M haemolytica S1 is a normal inhabitant of the upper respira-
tory tract of cattle. Under the influence of predisposing factors,
which include stress induced by change in the environment
(shipment, inclement weather, cold air) or by microbial agents
(bovine herpesvirus 1, bovine parainfluenza virus 3, bovine
respiratory syncytial virus, bovine viral diarrhea virus, Myco-
plasma sp, and other bacterial infection), M haemolytica S1
rapidly replicates, resulting in inhalation of droplets containing
bacteria in the lungs. In the alveoli, it initially interacts with the
resident pulmonary alveolar macrophages that serve as primary
line of host defense.
By the help of various virulence factors, M haemolytica can
evade the innate and adaptive immune responses, colonize
lungs, and establish the infection. The foremost of these viru-
lence factors are LKT and LPS. In low subcytotoxic dose, LKT
can activate macrophages through its interaction with the b2
integrin receptor, which is present not only on macrophages but
also on other ruminant leukocytes, including neutrophils and
lymphocytes.80,84 This interaction of LKT and LPS with
bovine leukocytes is followed by activation of leukocytes to
undergo oxidative burst and release proinflammatory cytokines
343
344
Figure 4. Oversimplified hypothetical sequence of events following
pulmonary inhalation of Mannheimia haemolytica. Lipopolysaccharide
(LPS) and leukotoxin (LKT) of M haemolytica activate the resident
pulmonary macrophages to produce proinflammatory cytokines and
undergo oxidative burst. Various cytokines serve as chemoattractant
molecules for inflammatory cells. Under the influence of LKT,
transmembrane pores are formed in the infiltrating leukocytes and
platelets, which possibly results in leakage of cellular constituents,
such as reactive oxygen and nitrogen species (ROS and RNS,
respectively), arachidonic acid metabolites, and lysosomal enzymes.
These cellular constituents damage pulmonary parenchyma and give
rise to necrotizing bronchopneumonia. Activation of complement
during this process contributes to the infiltration of susceptible
leukocytes into lungs and subsequently enhances damage induced by
the M haemolytica. IL, interleukin; TNFa, tumor necrosis factor a.
such as IL-1, IL-8, and TNFa, leading to accumulation of
inflammatory cells in the lungs (Fig. 4).69,83,90,126
To overcome host defenses, M haemolytica has evolved a
powerful mechanism of LKT-mediated cytotoxicity of the
leukocytes involved in the innate and adaptive immune
responses.18 LKT forms transmembrane pores in bovine lym-
phocytes, neutrophils, macrophages, mast cells, and platelets,
eventually causing oncotic cell cytotoxicity (Fig. 4).16,18,20
These transmembrane pores and the cell cytolysis of activated
leukocytes possibly cause leakage of oxygen radicals and other
products, such as nitric oxide, lysosomal enzymes, and inflam-
matory mediators, into the surrounding pulmonary parenchyma,
contributing to the pulmonary necrosis.30,79,80 Moreover,
increased expression of LKT receptor b2 integrin was observed
on bovine neutrophils incubated in vitro with LKT, LPS, and
cytokines such as IL-1b and TNFa, thereby increasing LKT
binding to the leukocytes and, subsequently, cytotoxicity.74,114
The biological effects of these inflammatory molecules and
LKT are morphologically evident in the form of necrotizing
bronchopneumonia and formation of neutrophil-derived oat
cells typical of M haemolytica infection.79,80 To circumvent
host-adaptive defense mechanisms of antibody-mediated bacter-
ial phagocytosis, M haemolytica has generated a unique method
of cleaving IgG delivered to the site of inflammation by using its
immunoglobulin proteases.72 In addition, in vitro studies have
revealed that LKT at sublethal doses is capable of inhibiting
mitogen-induced lymphoid proliferation, potentially inhibiting
adaptive immune responses.81,82
344
Veterinary Pathology 48(2)
Acknowledgement
We appreciate the comments by anonymous reviewers. We also
acknowledge Dr W. E. Hoffmann for editing this article.
Declaration of Conflicting Interests
The authors declared that they had no conflicts of interest with respect
to their authorship or the publication of this article.
Financial Disclosure/Funding
The authors declared that they received no financial support for their
research and/or authorship of this article.
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