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Infectious Disease
Veterinary Pathology
48(2) 338-348
The American College of
Veterinary Pathologists 2011
Reprints and permission:
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DOI: 10.1177/0300985810377182
http://vet.sagepub.com
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
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
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.
necessary requirement is that of initial colonization and establishment in the nasopharynx by adherence to the epithelial surface.17 This binding must provide resistance to the physical
removal by air flow, local innate and adaptive immune mechanism, 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 tracheal cells, this adhesin binds a 165-kDa glycoprotein receptor
onto neutrophils and initiates an oxidative burst.38 Another putative adhesin molecule is present on fimbriae of M haemolytica
and binds to a sialoglycoprotein receptor on respiratory epithelium.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 haemolytica to nasal mucus can be increased by enzymatic degradation of the protein and carbohydrate components of mucus.11
M haemolyticaderived 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 proteins 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
340
Highlander52 proposed that a filamentous hemagglutinin homologue 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 evidence indicates that LPS contributes to the pulmonary lesions
through a variety of complex mechanisms, including the stimulation 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 polysaccharide 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 hypotensive 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 produces clinical signs associated with the hypotension.4 The systemic 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 highmolecular
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 intravascular macrophages and on epithelial cell surfaces.124 LPS
can stimulate alveolar macrophages to produce proinflammatory cytokines, reactive nitrogen intermediates, reactive oxygen 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
Capsule
There are 12 serotypes of M haemolytica (S1, S2, S5S9,
S12S14, S16, S17) based on the differences in capsular polysaccharide antigen typing.103 Log-phase bacteria exhibit good
encapsulation, whereas stationary-phase bacteria exhibit poor
encapsulation.27 The chemical structures of capsular polysaccharides of M haemolytica S1, S2, and S7 have been determined2 and are similar to those from other pathogenic
bacteria. S1 capsular polysaccharide structure is similar to the
widely distributed enterobacterial common antigen; S2 capsular 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 colonization of lung by promoting bacterial adherence to respiratory 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 complementmediated lysis of the bacterium, whereas others have found
that the presence of anticapsular monoclonal antibodies promotes phagocytosis but not complement-mediated killing.127
Singh et al
Czuprynski et al31 found that M haemolytica capsular polysaccharide stimulated release of IL-1 from bovine blood monocytes but not from alveolar macrophages. Vaccination of
cattle with live M haemolytica or capsular polysaccharide promotes production of anticapsular antibodies; however, a positive correlation between capsular antibody and protection has
not been established.26,121
341
leukocyte toxicity because of its ability to form tertiary conformation 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 tolCdependent type I secretion system.28,128 The other members
of this family and their LKTs include Actinobacillus actinomycetemcomitans (LtxA), Actinobacillus pleuropneumoniae cytotoxins (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 downstream.53 The structural gene product of lktA is composed of
953 amino acids, which encodes the structural yet biologically
inactive LKTprotoxin. Gene lktC encodes transacylase that
posttranslationally modifies the inactive LKTprotoxin 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 hydrophobicity 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 binding, 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 aspartaterich repeats, and neutralizing epitopes are present within a
229amino 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
Singh et al
thereby forming transmembrane pores, whereas others have
suggested that LKT-induced regulation of voltage gated channels 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 1dependent pathway,
whereas LKT-associated apoptosis proceeds through a caspase
9dependent pathway.10
Irrespective of the basic process involved in the pore formation, these pores are 0.001 to 0.002 mm in diameter, allow rapid
loss of intracellular K from the host cell cytoplasm, and internalization of Na molecules, causing colloidosmotic imbalances.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 demonstrated 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 parenchyma.33,79,80,131 These by-products cause damage to the pulmonary 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 intracellular 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 cultures 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 LKTinduced 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 completely understood, a role has been suggestednamely, 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 predominates in the upper respiratory tract, whereas immunoglobulin 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 phagocytosis and bacterial killing; it can also selectively degrade host
mucosal sialoglycoproteins.1,72,94 Shewen et al106 used this glycoprotease 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 potentiated 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 cleaving 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 respiratory 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, Mycoplasma 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 virulence 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
Financial Disclosure/Funding
The authors declared that they received no financial support for their
research and/or authorship of this article.
References
Figure 4. Oversimplified hypothetical sequence of events following
pulmonary inhalation of Mannheimia haemolytica. Lipopolysaccharide
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pulmonary macrophages to produce proinflammatory cytokines and
undergo oxidative burst. Various cytokines serve as chemoattractant
molecules for inflammatory cells. Under the influence of LKT,
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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.
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