Download Immune response to fungal infections

Available online at www.sciencedirect.com
Veterinary Immunology and Immunopathology 125 (2008) 47–70
www.elsevier.com/locate/vetimm
Immune response to fungal infections
Jose L. Blanco *, Marta E. Garcia
Departamento Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
Received 7 November 2007; received in revised form 21 April 2008; accepted 25 April 2008
Abstract
The immune mechanisms of defence against fungal infections are numerous, and range from protective mechanisms that were
present early in evolution (innate immunity) to sophisticated adaptive mechanisms that are induced specifically during infection
and disease (adaptive immunity). The first-line innate mechanism is the presence of physical barriers in the form of skin and
mucous membranes, which is complemented by cell membranes, cellular receptors and humoral factors. There has been a debate
about the relative contribution of humoral and cellular immunity to host defence against fungal infections. For a long time it was
considered that cell-mediated immunity (CMI) was important, but humoral immunity had little or no role. However, it is accepted
now that CMI is the main mechanism of defence, but that certain types of antibody response are protective. In general, Th1-type
CMI is required for clearance of a fungal infection, while Th2 immunity usually results in susceptibility to infection.
Aspergillosis, which is a disease caused by the fungus Aspergillus, has been the subject of many studies, including details of
the immune response. Attempts to relate aspergillosis to some form of immunosuppression in animals, as is the case with humans,
have not been successful to date. The defence against Aspergillus is based on recognition of the pathogen, a rapidly deployed and
highly effective innate effector phase, and a delayed but robust adaptive effector phase. Candida albicans, part of the normal
microbial flora associated with mucous surfaces, can be present as congenital candidiasis or as acquired defects of cell-mediated
immunity. Resistance to this yeast is associated with Th1 CMI, whereas Th2 immunity is associated with susceptibility to systemic
infection. Dermatophytes produce skin alterations in humans and other animals, and the essential role of the CMI response is to
destroy the fungi and produce an immunoprotective status against re-infection. The resolution of the disease is associated with a
delayed hypersensitive response. There are many effective veterinary vaccines against dermatophytoses. Malassezia pachydermatis is an opportunistic yeast that needs predisposing factors to cause disease, often related to an atopic status in the animal.
Two species can be differentiated within the genus Cryptococcus with immunologic consequences: C. neoformans infects
predominantly immunocompromised hosts, and C. gattii infects non-immunocompromised hosts. Pneumocystis is a fungus that
infects only immunosupressed individuals, inducing a host defence mechanism similar to that induced by other fungal pathogens,
such as Aspergillus.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Immunity; Aspergillus; Candida; Dermatophyte; Cryptococcus; Pneumocystis; Malassezia; Pathogenicity
1. Mycoses
* Corresponding author. Tel.: +34 91 394 3717;
fax: +34 91 394 3908.
E-mail address: [email protected] (J.L. Blanco).
0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2008.04.020
Mycoses, conditions in which fungi pass the
resistance barriers of animals and establish infections,
are a group of diseases with very varied clinical
manifestations. Mycoses are of growing importance for
the following reasons (Garcia and Blanco, 2000):
48
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
(1) They are produced by fungi that are widely
distributed in the environment and, therefore, very
difficult to eradicate.
(2) The clinical manifestation of disease caused by
fungal infection can be highly variable. For
example, in the case of aspergillosis, with effects
on very diverse organs, there is a variety of
responses, such as local (aspergilloma), systemic
(renal, lung, nervous central system, etc.) or even
allergic (allergic bronchopulmonary aspergillosis in
human).
(3) Diagnosis of these diseases can be problematic
because of the difficulty of interpreting the very
different clinical pictures in individuals in the
presence of colonization, infection and/or disease.
(4) There is a few varieties of vaccines available against
these diseases, which are therefore difficult to
prevent. At this time, vaccines are limited to a few
animal species, to only a few processes and with
variable effectiveness.
(5) Treatment is problematic: compared to the antibacterials, the number of antifungal drugs available
at present is very small, with much greater difficulty
in production, with many side-effects, and with the
possibility of the appearance of resistance, as has
happened with antibiotics in the treatment of
bacterial infection.
In human medicine, the appearance of AIDS and the
evolution of immunosuppressive treatments essential
for the success of organ transplants, have highlighted
the importance of fungal diseases, and efforts have been
made to understand these diseases and to develop means
for their prevention and control. This evolution has been
much slower in veterinary medicine; on many occasions
the fungal diseases have been relegated to post-mortem
discoveries.
At present, the two main foci of attention in the study
of fungal diseases are as follows:
(1) The mechanisms of pathogenicity that cause usually
saprophytic fungal species to transform into an
aggressor toward an animal host causing disease or
even death.
(2) Mechanisms of the host resistance to infection and
disease.
In this review, we discuss this second point in detail
in an attempt to identify the main mechanisms used by
the host immune system to counter fungal infection. A
disease outcome is simply a result of the clash between
the mechanisms of pathogenicity of the fungi and the
mechanisms of resistance of the host, leading to the
removal of the infection or its progression according to
the imbalance of these mechanisms.
At present, much of what is known about fungal
infections is limited to what happens in man. There are
many preliminary results obtained with laboratory
animals, mainly with murine models. Less is known in
the case of domestic animals concerning fungal
diseases, especially by species that produce disseminated processes. These are considered to be characteristic of immunocompromised individuals in humans,
while in many cases this immunosuppression is not
detected in animals. The question is raised of whether
immunosuppression exists or have we not been able to
detect it.
2. Immune response to fungi: innate and
adaptive immunity
Host defence mechanisms against fungi are numerous, and range from protective mechanisms that were
present early in the evolution of multicellular organisms
(innate immunity) to sophisticated adaptive mechanisms that are induced specifically during infection and
disease (adaptive immunity) (Romani, 2004).
Traditionally, innate immunity has been considered
as simply a first line of defence. Nevertheless, innate
immunity has recently received attention because,
despite a certain lack of specificity, it effectively
distinguishes self from non-self and activates adaptive
immune mechanisms by the provision of specific
signals (Medzhitov and Janeway, 1997; Romani,
2004). The innate immune system confers rapid
recognition of microbial infection through a limited
repertoire of germ line-encoded receptors that recognize a group of conserved molecular patterns common
to broad groups of microbial species (Janeway and
Medzhitov, 2002; Roeder et al., 2004).
The first of the defensive innate mechanisms is the
physical barriers that separate the organism from the
environment: i.e. skin and the mucous membranes of
the respiratory, gastrointestinal and genito-urinary
tracts. The skin and mucous membranes are physical
barriers, and they have antimicrobial substances on their
surface, some of them synthesized by the epithelial and
endothelial cells. Also, they have a commensal
microflora of saprophytic microorganisms that impede
colonization by pathogenic microorganisms (Romani,
2004).
Once the fungi have passed the physical barriers,
they are met with a series of innate mechanisms of
defence, including cellular membranes, cellular recep-
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
tors and several humoral factors. In the tissues,
phagocytes, consisting of neutrophils, mononuclear
leukocytes (monocytes and macrophages) and dendritic
cells, have an essential role, and natural killer cells, gdT-cells and non-hematopoietic cells, such as epithelial
and endothelial cells are involved in the host defence.
The host defence mechanism can adapt to different
kinds of fungal infection; For example, macrophages
are the primary cells involved in fungal killing during
infection by Cryptococcus and Pneumocystisi, whereas
neutrophils are the primary effector cells in preventing
infection by Candida albicans and Aspergillus fumigatus (Traynor and Huffnagle, 2001).
A critical point in the defence is the production of
chemotactic factors at the site of fungal infection for
effective recruitment of leukocytes to that site. These
chemotactic factors are very varied, and include
peptides derived from activation of the complement
pathway, leukotrienes and products synthesized by the
fungi; certain cytokines have secondary chemotactic
activity. Another important group of chemotactic
factors is the chemokines, a supergene family of small
inducible peptides with potent chemotactic activity for
leukocyte subpopulations. These chemokines are
produced by a variety of cells, including leukocytes,
epithelial cells, endothelial cells, fibroblasts and smooth
muscle cells following stimulation by cytokines or
microbial products. Chemokines regulate an array of
biological activities in addition to chemotaxis, such as
hematopoiesis, angiogenesis, cytokine induction, antigen presentation and Th cell differentiation. All of these
activities are important in both acute and chronic fungal
infections (O’Garra et al., 1998; Murphy et al., 2000;
Rossi and Zlotnik, 2000; Zlotnik and Yoshie, 2000;
Traynor and Huffnagle, 2001).
The synthesis of these chemotactic factors is
activated by invariant molecular structures shared by
large groups of pathogens (also known as pathogenassociated molecular patterns, [PAMPs]) that are
recognized by germ line-encoded proteins (pattern
recognition receptors [PRRs]) present in different cells
of the organism, mainly monocytes, macrophages,
dendritic cells, B-cells, T-cells and endothelial cells.
PRRs include the Toll-like receptors (TLRs), a protein
family of cellular receptors that mediate recognition of
microbial pathogens and subsequent inflammatory
response in vertebrates (Janeway and Medzhitov,
2002; Roeder et al., 2004; Romani, 2004). TLRs and
other PRRs confer PAMP recognition and their
signalling triggers synthesis followed by release of
proinflammatory cytokines, and induces expression of
co-stimulatory molecules for promoting activation of
49
adaptive immunity during antigen presentation. The
simultaneous activation of multiple PRRs by one fungal
pathogen endows the immune system with a broad
range of possibilities for a specific and effective
immune response (Roeder et al., 2004).
Bacterial and viral infections have been the major
focus of research, and less is known about the function
of TLRs against fungal pathogens and fungal PAMPs,
though its participation in the defence against C.
albicans, A. fumigatus, C. neoformans, Pneumocystis
and Coccidioides has been reported (Wang et al., 2001;
Netea et al., 2002, 2006; Meier et al., 2003). In contrast,
several studies have demonstrated that pathogens are
able to manipulate or escape innate immune recognition. Then, certain fungal pathogens use PRR-based
strategies to evade host defence (down-modulate the
microbicidal functions of leukocytes, or escape immune
recognition); in certain circumstances, fungal pathogens induce a strong anti-inflammatory cytokine profile,
which may be a mechanism to escape the host defence
(Netea et al., 2006).
C. albicans induce immunosuppression through
TLR2-mediated IL-10 release, and this leads to
generation of CD4+CD25+ T-regulatory cells with
immunosuppressive potential (Netea et al., 2004). A.
fumigatus evades immune recognition by germination
into hyphae with subsequent loss of TLR-4 recognition,
whereas the TLR2-mediated IL-10 pathways remain
intact, thus shifting the balance towards a permissive
Th2-type profile (Netea et al., 2003). Emphasized is the
fact that dectin-1 recognizes the b-glucans at the level
of budding scars in the yeasts, but it cannot recognize
the b-glucans in the hyphae, where they are shielded by
a layer of mannans (Gantner et al., 2005).
Also are important the host defence peptides, also
known as antimicrobial peptides. They are effector
molecules of the innate immune system that show broad
antimicrobial action against gram-positive and -negative bacteria, and also against fungi. Furthermore, they
likely play a key role in activating and mediating the
innate as well as adaptive immune response in infection
and inflammation (Aerts et al., 2008; Steinstraesser
et al., 2008). Then, they are antimicrobial and
immunomodulating agents that present an important
link between the innate and adaptive immune response
(Zasloff, 2002). One major function of these antimicrobials peptids is to inactivate microbes, including
fungi, through multiple direct effects on their membranes. They have the ability to attack specific external
targets simultaneously (Ganz, 2003; Aerts et al., 2008;
Steinstraesser et al., 2008). Another major function is
their active role in the transition to the adaptive immune
50
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
response by being chemotactic for monocytes, neutrophils, and T-cells and by exhibiting adjuvant and
polarizing effects in influencing dendritic cell development (Steinstraesser et al., 2008). They have a direct
influence on the adaptive immune responses by
activation of different immune factors such as tumour
necrosis factor-alpha (TNF-a), interleukin (IL)-1, and
gamma-interferon (IFN-g) (Ganz, 2002).
Although much progress has been made in recent
years, the complete molecular basis of the mode of
antimicrobial action for most of these antimicrobial
peptides still needs to be unravelled (Aerts et al., 2008).
3. Immune response to fungi: humoral and
cellular immunity
The relative contribution of humoral and cellular
immunity to host defence against fungal infections has
been controversial in the field of medical mycology. For
a long time, only two main observations were
considered, which had been made repeatedly for many
fungal pathogens. First, cell-mediated immunity (CMI)
had consistently been shown to mediate protection
against many fungi, as demonstrated by adoptive
lymphocyte transfer studies, enhanced susceptibility
for hosts with CMI deficiencies and the finding that
granulomatous inflammation is often essential for
control of infection in tissue. Second, humoral
immunity (HI) had been difficult to demonstrate by
either transferring immune sera or correlating antibody
titres with protection (Polonelli et al., 2000). Although a
few studies suggested that antibody might have a role in
protection, the role of HI was uncertain because of
inconsistent results (Casadevall, 1995). The conclusion
was that the CMI was important, whereas HI had little
or no role.
Nevertheless, in the last decade it has been shown
that HI can protect against fungal infection if certain
types of protective antibodies are available in sufficient
quantity. The main recognized functions of antibodies
in fungal infections include prevention of adherence,
toxin neutralization, antibody opsonization and antibody-dependent cellular cytotoxicity (Polonelli et al.,
2000). These findings have been supported by experiment; for example, the identification of protective and
non-protective antibodies for both C. neoformans and
C. albicans, indicating that HI response to fungi could
elicit antibodies of variable efficacy (Casadevall, 1995).
Explanations for the negative historical results in
passive protection experiments with transfer of polyclonal immune sera include inadequate amounts of
protective antibody, the presence of antibody of a
non-protective specificity, and/or the presence of
antibody of a non-protective isotype (Polonelli et al.,
2000). The relative composition and proportion of
protective and non-protective antibodies against fungi
might vary greatly. Also, the amount, specificity,
isotype and idiotype of antibodies have marked effects
on protective efficacy (Casadevall et al., 2002).
Research is in progress to identify antibodies that are
protective, the peptide mimetics that elicit them
specifically and putative candidate vaccines that elicit
protective immunity.
The long debate between the relative merits of
humoral and cellular immunity in medical mycology
has given way to a new consensus, that CMI remains the
main mechanism for defence against fungal infections
but that certain types of antibody responses can also
protect (Polonelli et al., 2000). In conclusion, it is clear
that the immune system works as a whole, and that very
diverse components contribute to the defence of the host
organism. Some parts contribute more than others under
certain circumstances, but they are all important for the
overall protection against infection.
The type of CMI induced is critical in determining
resistance or susceptibility to fungal infection. In
general, Th1-type CMI is required for clearance of a
fungal infection, while Th2 immunity usually results in
susceptibility to infection or allergic responses (Traynor
and Huffnagle, 2001). Th1 cells produce predominantly
cytokines such as IFN-g, and promote cell-mediated
immunity and phagocyte activation. In contrast, Th2
cells produce predominantly cytokines such as interleukins 3 and 4 (IL-3 and IL-4) and tend to promote
antibody production (Traynor and Huffnagle, 2001;
Crameri and Blaser, 2002; Bellocchio et al., 2005).
4. Practical applications of the immune
response: diagnosis and vaccination
The detection of an effective HI response against
fungal infections has driven the research in two
practical aspects of great interest: the development of
immunologic methods of diagnosis and the application
of vaccines.
4.1. Immune diagnosis
The development of an HI response as a consequence
of fungal infection, and the fact that in domestic animals
these diseases generally take place in immunocompetent individuals, at least as concerns the synthesis of
antibodies, open the possibility of immune diagnoses as
a screening method for the detection of fungal diseases.
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
The results obtained using this kind of test would be
useful for early diagnosis, and have the advantage of
requiring only simple and routine collection of blood
samples (Garcia et al., 2001b).
Nevertheless, determination of the level of antifungal antibodies in serum highlights a series of
problems that should be kept in mind (Blanco and
Garcia, 2000):
(1) Some fungi, as Aspergillus, are ubiquitous microorganisms that are in continuous contact with man
and animals, with colonization even in healthy
individuals. This stimulates an immune response in
the host, so that all the animals routinely have a
certain level of antifungal antibodies, which makes
precise high dilutions of serum necessary for an
immunologic diagnosis to differentiate between
infected and non-infected individuals.
(2) Different fungal genera have many common
antigens, thus giving rise to cross-reactive immune
reactions, and it is difficult to make an exact
diagnosis.
Besides diagnostic procedures, we have used these
methodologies to monitor the evolution of treatment
against disease in such a way that antifungal treatment
should not be discontinued before the titre of antifungal
antibodies subsides to negative values that allow us to
guarantee an effective cure (Garcia et al., 2001a,b,
2004a,b).
4.2. Fungal vaccines
A clearer understanding of the mechanisms involved
in the immune response to fungal infection is needed to
aid the development of effective vaccines. Besides the
traditional concept of vaccines that protect against
fungal diseases, it is important to consider the
administration of immunomodulators and the use of
the so-called therapeutic vaccines.
Research has involved both cellular and humoral
immunity, but recent discoveries related to the activity
of the HI have reactivated the search for vaccines, once
it was recognised that protective antibodies to fungal
antigens would be potentially useful for adjunctive
therapy of fungal infections. Encouraging results have
been obtained with certain antigen preparations and it is
considered that a vaccine can be developed in the near
future against diseases like aspergillosis or candidiasis
(Polonelli et al., 2000).
Unlike the fungi for which virulence factors have
been defined, such as C. neoformans, Histoplasma
51
capsulatum, and Blastomyces dermatitidis, identification of similar properties for A. fumigatus has been
difficult (Feldmesser, 2005). Such knowledge has
allowed for the development of conjugate vaccines
aimed at eliciting effective immunity to these targets,
such as the glucuronoxylomannan conjugate for C.
neoformans, or for the development of attenuated
strains by targeted disruption of these molecules that
can function as live attenuated vaccines, such as the B.
dermatitidis BAD-1-deleted strain (Wuthrich et al.,
2000; Casadevall et al., 2002; Feldmesser, 2005).
An additional approach toward vaccine development
has stemmed from finding a broad-spectrum protective
action of killer antiidiotypic monoclonal antibodies.
Antiidiotypes to a monoclonal antibody specifically
reacting with killer toxins from yeasts and are lethal to
pathogenic microorganisms expressing specific cell
wall receptors. Because the target of this protective
antibody is involved in b-glucan synthesis, the
development of host response to this compound could
be beneficial. b-Glucans are important constituents of
the cell wall of many fungi, including Aspergillus spp.,
which, like many carbohydrates, are themselves poorly
immunogenic (Cenci et al., 2002).
On the basis of the observations of antibodymediated protection against fungi, the following criteria
need to be taken into account when considering the
possibility of protective antibodies against fungal
diseases (Casadevall, 1995):
(1) Specificity: The experience with C. neoformans and
C. albicans indicates that specificity is a critical
variable in determining antibody efficacy. For
example, C. neoformans protective antibodies
against cryptococcosis are specific for glucuronoxylomannan; however, not all antibodies to glucuronoxylomannan are protective, suggesting that
individual epitopes in this polysaccharide antigen
differ in their ability to elicit protective and nonprotective monoclonal antibodies.
(2) Ig isotype: The experience with C. neoformans
suggests that antibody isotype is an important
variable in determining the efficacy of antibodies
against fungi. However, the relative efficacy of the
various isotypes may differ, depending on the fungal
pathogen. IgM and IgG1 are effective against
cryptococcosis, but IgG3 is not. IgM and IgG3 are
protective against candidiasis.
(3) Titre: Antibody-mediated protection against fungal
pathogens is likely to depend on an optimal amount
of serum antibody. For C. neoformans, the passive
antibody is effective only at certain doses.
52
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
An ideal vaccine formulation would result in
reproducible and predictable fulfilment of each of the
three criteria described above.
At the moment, no standardized vaccine exists for
the prevention of any of the fungal infections in humans,
a situation that is attributable to both the complexity of
the pathogens and their sophisticated strategies for
survival in the host (Romani, 2004). In contrast, there
are useful fungal vaccines in veterinary medicine,
especially in the field of dermatophytosis, as will be
discussed below, and for protection against other
groups. For example, the fungus Pythia insidiosum
causes a debilitating cutaneous, subcutaneous or
systemic disease in horses and other mammals. Two
vaccines have proved to be of use to both prevent the
infection and to treat horses infected with P. insidiosum.
The former is composed of sonicated hyphal antigens,
and the latter is prepared from culture filtrate antigens
(Deep, 1997).
5. Aspergillus
Of all the fungal diseases, aspergillosis has been the
subject of the most intensive study, including the
immune response to the disease. It has been described in
many species of mammals and birds, where cellular
characteristics have been described that might predispose to respiratory aspergillosis (Tell, 2005).
Human invasive aspergillosis is a disease affecting
highly immunocompromised subjects. It is a rapidly
progressive, often fatal, disease characterized by tissue
destruction associated with abundant hyphae in necrotic
tissue (Bozza et al., 2002). Host defects that result in
predisposition to invasive aspergillosis include neutropenia, defective neutrophil function (such as that seen in
patients with chronic granulomatous disease), receipt of
corticosteroid therapy, immunosuppresive agents for
solid organ transplant and late-stage human immunodeficiency virus (HIV) infection (Feldmesser, 2005).
In humans, the lungs are the most common location
of Aspergillus infections. In domestic animals, the
situation is different and varies between species. For
example, in cows, the forestomachs, especially the
omasum, are mostly affected, from which the infection
may spread to other organs, including the placenta
(Garcia et al., 2001a). A few cases of pulmonary
involvement and later dissemination have been reported
in dogs (Clercx et al., 1996).
Various authors have tried to relate these processes to
some form of immunosuppression in animals, as is the
case with humans, but until now it has not been possible
to establish this relationship with neutrophils, T
lymphocytes or macrophage function (Garcia et al.,
2001a).
5.1. Pulmonary aspergillosis
In humans, aspergillosis is fundamentally a disease
of pulmonary origin, acquired by inhalation of conidia,
that affects immunocompromised individuals. There
have been numerous studies carried out with humans
and laboratory animals, mainly rats and mice, that
reveal a lung disease in which the immune system
behaves as it does against other fungal genera. It is
likely that pulmonary aspergillosis in other animals
follows the same pattern.
On the basis of these studies, host defence against
Aspergillus comprises the following sequence of events
(Phadke and Mehrad, 2005): (1) recognition of the
pathogen; (2) a rapidly deployed and highly effective
innate effector phase (non-specific or innate immunity);
and (3) a delayed but robust adaptive effector phase
characterized by immunologic memory (adaptive
immunity).
There are three different mechanisms of defence: (a)
physical barriers; (b) phagocytosis; and (c) humoral
compounds.
5.1.1. Physical barriers
Include mucous membranes, mucociliary clearance
and local secretion of inflammatory mediators by innate
immunity cells (Crameri and Blaser, 2002).
Most of the A. fumigatus conidia, as well as most of the
inspired particles, are eliminated by the action of the cilia
of the pseudoestratified ciliated cylindrical epithelium of
the upper part of the tracheobronchial tree. A. fumigatus
synthesizes toxins, such as gliotoxin, fumagillin and
helvolic acid, that are able to inhibit this ciliary
movement (Amitani et al., 1995). Also, the endothelial
and epithelial cells are capable of internalizing conidia,
which can facilitate the infection (Paris et al., 1997).
The airway mucus constitutes a physical, chemical
and biological barrier of secretory products from the
mucous membrane that facilitates the elimination of
inspired particles, including fungal conidia. This fluid
contains glycoproteins, proteoglycans, lipids, lysozyme
and surfactants (McCormack and Whitsett, 2002).
Host proteins that participate in mediating the
interaction between innate and adaptive immunity are
very relevant for the host defence against Aspergillus.
There are various PRRs such as lung surfactant proteins
A and D (SP-A and SP-D), mannan-binding lectin
(MBL) and TLRs, which strengthen the innate immune
response. SP-A, SP-D and MBL belong to a group of
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
mammalian lectins called collectins, which contain
collagenous regions (McCormack and Whitsett, 2002;
Madan et al., 2005). It is known that they interact with
carbohydrate structures on the surfaces of a wide range of
pathogens, such as viruses, bacteria and fungi, and thus
enhance phagocytosis and killing by neutrophils and
alveolar macrophages. SP-A and SP-D are known to
interact with phagocytic cells and enhance their
chemotactic, phagocytic and oxidative properties
(Wright, 1997; Van de Wetering et al., 2004). Any
defects in these innate immune molecules might
contribute to increased susceptibility to Aspergillus
infections. The collectins are not primitive, innate
molecules but are highly specialized and able to modulate
responses to foreign agents (Madan et al., 2005).
5.1.2. Phagocytes
The role of phagocytes in the defence against
Aspergillus is essential to avoid the development of the
disease.
The first defensive cells that inhaled conidia meet are
macrophages. The ability of the macrophages to
prevent germination of the Aspergillus conidia
depends on the anatomic location; human alveolar
macrophages are competent in this activity, while
those in the peritoneum are not (Latge, 1999). This is
of importance in cases when the entry pathway of
fungi is not pulmonary.
Attachment of the conidia with macrophages is via
non-specific receptors, such as mannosylfucosyl, and
does not rely on the presence of opsonization factors
such as complement or immunoglobulin. The conidia
are internalized by the macrophages and prevented
from growth for several hours until the macrophage
begins to destroy them. At 24 h after internalization,
90% of the conidia are killed. In spite of the
enormous capacity of the macrophages to kill conidia,
their effectiveness is not 100% (Latge, 1999). This
first defensive mechanism greatly reduces pathogenicity by impeding germination and development of the
fungus (Romani, 2004).
Polymorphonuclear neutrophils are responsible for
the destruction of the hyphae of A. fumigatus, and
they are able to kill the conidia that escape destruction
by the macrophages (Duong et al., 1998; Romani,
2004). The hyphae are too large to be engulfed and the
neutrophils binding the surface without the need for
complement or immunoglobulin, although they may
help such action. The contact between neutrophils and
hyphae triggers secretion of reactive oxidative
intermediary agents that rapidly damage the hyphae;
50% of hyphae are destroyed in 2 h (Latge, 1999).
53
Resting conidia are relatively resistant to killing by
either reactive oxygen intermediates or neutrophil
cationic peptides and their ingestion triggers neutrophil degranulation and the respiratory burst only
weakly. Once conidial swelling has occurred, the
response of neutrophils is enhanced, such that
phagocytosis, degranulation and reactive oxygen
production are increased and conidia are killed more
readily (Latge, 1999).
Natural killer cells, an additional component of innate
responses, function in host defence against invasive
aspergillosis, with a very important role in neutropenic patients (Morrison et al., 2003). In this sense has
been showed the important role of NK cells as an
important effector cell in aspergillosis (Walsh et al.,
2005).
The monocyte chemotactic protein-1 (MCP-1) is
required for host defence against Aspergillus in
neutropenic hosts; this effect is mediated by the early
recruitment of NK cells to the lungs. It was no found
induction of MCP-1 in the lungs of immunocompetent animals challenged with Aspergillus conidia.
This observation suggests than in normal hosts, other
components of innate immunity are sufficient to clear
the pathogen. Then, NK cells have a very important
role in neutropenic patients (Morrison et al., 2003).
It has been described that human platelets can have a
role in the protection against aspergillosis, since they
are able to binding to the wall of invasive hyphae of A.
fumigatus and to be activated (Christin et al., 1998).
The thrombocytopenia that takes place in patients
suffering long periods of neutropenia could increase
the risk of aspergillosis. Platelets, like neutrophils,
attach to cell walls of the invasive hyphal form of A.
fumigatus, that induce its activation. Organisms were
damaged as shown by loss of cell wall integrity and
release of defined hyphal surface glycoproteins
(Christin et al., 1998).
Dendritic cells can phagocytose conidia and hyphae
of A. fumigatus via two different phagocytic
mechanisms. Similarly, the fate of each fungal form
is different once it is engulfed by a denditric cell.
While hyphae were degraded progressively, conidia
were still alive 3 h after phagocytosis, a finding that
confirmed the more resistant nature of this morphological form of the organism (Bozza et al., 2002).
Pulmonary dendritic cells ingest conidia and hyphae,
migrate to draining lymph nodes and induce local
disparate T helper-cell response (Romani, 2004).
Sometimes, in spite of these defensive mechanisms, the fungus is able to germinate, develop and
form invasive hyphae. In this case, phagocytes in
54
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
different parts of the host are able to present the same
defence. The hyphae are too large for the phagocytes
to internalize for destruction; however, monocytes
and macrophages have extracellular antifungal
activity that is able to damage the fungi in an
important way (Cox, 1989).
Activated lymphocytes did not prevent growth of
A. fumigatus and did not damage the fungus, but did
affect the adherence of hyphae to plastic surfaces.
Since adherence is one of the first steps in microbial
virulence, this effect might interfere with the
pathogenicity of A. fumigatus (Clemons et al., 2000).
In later steps of the disease, T cells have an
important role in defence in conjunction with
macrophages. Immunohistochemistry studies of
visceral lesions in dogs demonstrated a correlation
between the number of T cells and the number of
macrophages, which suggests a very important role of
T cells in the local proliferation and activation of
macrophages (Perez et al., 1996).
5.1.3. Humoral compounds
If the two defensive mechanisms described above are
negotiated, the fungus spreads from the lung towards
the bloodstream.
Joining together of A. fumigatus and components of
the serum has been observed, but the role of this joining
in the course of the disease is not known. For example, it
is known that the levels of fibrinogen are increased
during invasive aspergillosis, and that fibrinogen is able
to join to A. fumigatus. C-reactive protein, which
activates the complement cascade, can join to certain
fractions of Aspergillus. The direct activation of the
alternative complement pathway by A. fumigatus has
been demonstrated, although the mechanism that begins
the cascade of the complement resting conidia,
germinated conidia and hyphae seems to be different
(Feldmesser, 2005). Also, the capacity of the C3
component of complement to join to the fungus depends
on changes in the biochemical composition of the
surface of each of those three forms (Kozel, 1996).
Cytokines are important tools that have helped us to
understand the control, immunoregulation and activation of pulmonary host defences. Understanding these
properties provides a rational foundation for developing
strategies for immune augmentation in the prevention
and treatment of invasive pulmonary aspergillosis
(Walsh et al., 2005).
Granulocyte colony-stimulating factor (G-CSF)
enhances the antimicrobial activity of neutrophils
against a wide variety of bacteria and fungi, including
A. fumigatus (Roilides et al., 1993, 1995), and
accelerates recovery from neutropenia and thus shortens
the duration of the risk period for development of
invasive pulmonary aspergillosis (Walsh et al., 2005).
Granulocyte-macrophage colony-stimulating factor
(GM-CSF) primes macrophages to release pro-inflammatory mediators, including IL-1 and TNF-a (Rodriguez-Adrian et al., 1998), and enhances the antifungal
activities of intact neutrophils and/or mononuclear cells
against A. fumigatus (Walsh et al., 2005).
Macrophage colony-stimulating factor (M-CSF)
promotes the differentiation, proliferation and activation of mononuclear cells and macrophages (Gonzalez
et al., 2001; Walsh et al., 2005).
TNF-a stimulates neutrophils to damage Aspergillus
hyphae, enhances phagocytosis of conidia, and augments neutrophil oxidative respiratory burst and
degranulation induced by opsonized fungi (Roilides
et al., 1998; Phadke and Mehrad, 2005).
IL-6 is produced rapidly after intranasal infection
with A. fumigatus, and appears to have a protective
proinflammatory and immunomodulatory role in
pulmonary host defence in response to organismmediated pulmonary injury (Cenci et al., 1999).
IL-10 suppresses the antifungal activity of mononuclear cells against Aspergillus hyphae, while increasing their phagocytic activity (Walsh et al., 2005).
IFN-g is a highly potent activator of phagocytes
against opportunistic fungal pathogens in humans. IL12 augments the oxidative antifungal activity of
mononuclear cells against A. fumigatus via an IFN-g
independent route (Roilides et al., 1999).
The immune response against an A. fumigatus
infection is usually a mixed response that is as much
humoral as cellular, but it is effective only if it is
associated with a cellular answer, with increase of CD4
lymphocytes, and elevation of the levels of IL-2, IFN-g
and IL-12. If the response is largely humoral, with an
increase in the production of antibodies, IL-4, IL-5 and
IL-10, it is usually associated with progression of the
disease (Mehrad and Standiford, 1999; Roilides et al.,
1998, 1999).
It can be assumed that this pattern of pulmonary
aspergillosis, perfectly defined in experimental murine
models and in man, happens in pulmonary aspergillosis
in domestic animals.
5.2. Disseminated aspergillosis
The pulmonary entrance pathway in animals seems
not to be predominant. What happens in these cases
from the point of view of the immune system? The
response of the immune system is related strongly to the
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
pathogenicity of the disease, and especially the pathway
of entrance of the fungus.
The dog is the domestic animal species for which
most is known about the working of the immune system
in aspergillosis. In the dog, however, there is a
symptomathology that is not usually accompanied by
pulmonary affectation, unlike the pulmonary clinical
picture seen in humans. Therefore, they differ from the
conventional disease pattern that appears in human
medicine (Guerin et al., 1993; Clercx et al., 1996).
With the increase in knowledge about canine
aspergillosis, it is clear that the clinical picture in the
literature does not apply to all cases or species; for
example, lesions can occur in very different sites and
can be accompanied by very varied symptoms. Until the
1980s it seemed clear that the fungus involved in these
processes was Aspergillus terreus (Day and Penhale,
1991; Berry and Leisewitz, 1996). However, an
increasing number of studies were reported in which
a different fungus was identified as the cause of the
disease, including Aspergillus deflectus, Aspergillus
flavus, and Aspergillus flavipes, as well as members of
other genera, such as Acremonium, Penicillium,
Paecilomyces, etc. (Jang et al., 1986; Watt et al.,
1995; Gene et al., 2003; Zanatta et al., 2006). For this
reason, years ago we proposed that these cases should
not be called aspergillosis but should be described as
systemic mycosis or disseminated mycosis, at least until
the causal agent is identified. However, the actions of
the immune system against all those fungal species are
probably very similar.
The entrance pathway of fungi into the host organism
is not still clear, although it has been suggested as
possibly through old wounds, medium or inner chronic
otitis, or a urinary pathway (Mullanery et al., 1983;
Patterson et al., 1983; Littman and Goldschmidt, 1987;
Starkey and McLoughlin, 1996; Garcia and Blanco,
2000). A digestive pathway of entrance has been
proposed, since on some occasions lesions have been
observed as mesenteric lymphadenitis with fungal
hyphae (Pastor et al., 1993). Recently has been
described the transuterine transmission (Elad et al.,
2008). Once the fungus has gained entry in the dog, it
can spread to different locations, mainly the rachis,
where the disease can develop slowly, with dissemination to the brain, liver, spleen and abdominal lymph
nodes. A clinical disease would be detected only after
several years.
Theories about the primary spread of the fungal
agent in the host suggest that it is intimately related to
the ability of members of the genus Aspergillus to
synthesize substances, such as elastase, that are able to
55
invade non-necrotic tissues and penetrate into the
bloodstream (Markaryan et al., 1994; Blanco et al.,
2002). This capacity is associated with the production
of certain types of lateral spores, named aleuriospores,
which are usually observed when the fungus grows in
vivo (Gelatt et al., 1991; Dallman et al., 1992; Wilson
and Odeon, 1992; Kauffman et al., 1994; Butterworth
et al., 1995; Perez et al., 1996). Once the fungus has
penetrated into the bloodstream, spread to the organs.
Development begins upon arrival at the capillary
vessels, causing an inflammatory local reaction by
the host, in which neutrophils and monocytes participate (Wilson and Odeon, 1992; Butterworth et al., 1995;
Clercx et al., 1996; Starkey and McLoughlin, 1996).
Therefore, because the entrance is not through mucous
membranes, but directly into the bloodstream, the first
line of defence that is so effective in immunocompetent
individuals with lung infection is eluded, and it raises
the possibility that the disease occurs in immunocompetent individuals.
In humans, local infection attributable to contamination of a wound should not develop into disseminated
disease if the cell-mediated immunological system is
intact (Garcia et al., 2000). However, an apparent
predisposing immunosuppressive disease was not found
in cases of canine systemic mycoses, at least with
respect to the synthesis of antibodies, the function of
neutrophils, T lymphocytes and macrophages, the levels
of serum proteins, the levels of complement compounds, or skin test reactivity (Day et al., 1985; Day and
Penhale, 1998).
At first, little importance was assigned to the HI
response in the defence of the host in canine
aspergillosis. It appears that the humoral factors by
themselves are unable to prevent fungal development
and that they are not important in the first stage of the
infection, given the fact that disgammaglobulinemia or
hypogammaglobulinemia is not translated into risk of
aspergillosis. In spite of this, it is obvious that
antibodies have a role in the immune defence of the
host, limiting the growth of fungal hyphae in conjunction with phagocytes. The level of antibodies in the
blood of dogs with invasive aspergillosis shows a
significant increase in the levels of IgG (Gelatt et al.,
1991; Dallman et al., 1992; Wilson and Odeon, 1992;
Guerin et al., 1993; Watt et al., 1995; Clercx et al.,
1996). Some of these studies have shown low levels of
IgA and IgM. Several authors have described the
predisposition of German Shepherd dogs to suffering
aspergillosis (Day et al., 1985; Berry and Leisewitz,
1996), probably due to reduced levels of IgA. However,
in perhaps the most complete study so far, low levels of
56
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
IgA are reported for only 3 of 12 affected dogs.
Nonetheless, the authors suggested a predisposition of
the breed related to some genetically determined
immunodeficiency, but none has been detected so far
(Day and Penhale, 1998). The same thing was described
for the Beagle dog, suggesting that the deficiency of IgA
is due to defective synthesis or secretion, because the
cells producing IgA are present in normal numbers
(Guerin et al., 1993).
The results obtained in immunohistochemistry
studies of the lesions found in different organs of
animals with invasive aspergillosis revealed a high level
of IgA in the lesions (Day and Penhale, 1991; Perez
et al., 1996), with an intense inmunoreactivity of IgA on
the hyphae and in the cytoplasm of cells in the periphery
of the mycotic granuloma. These discoveries demonstrate an unusual systemic distribution of IgA that
suggests some defect in their production or distribution.
In the same studies, a seemingly disproportionate local
cellular response was described surrounding a small
necrotic focus that included fungal hyphae. Very similar
lesions were observed in laboratory animals with the
experimental reproduction of the disease. Such lesions
were compared with those present in equally infected
animals, but they revealed an immunosuppression state
caused by administration of cortisone, and they had a
different appearance, with a large quantity of hyphae
that sometimes invaded the adjacent vessels, accompanied by a limited inflammatory response. These
results suggest that the lesions present in the
immunocompetent animals are effective in stopping
the advance and dispersion of the fungus. However,
although the pattern of lesions in the dogs was similar to
that in the immunocompetent mice, in any case they
were indications that suggest resolution of these lesions
(Day and Penhale, 1991).
In conclusion, the combination of the epidemiological
studies with the results for the immune and inflammatory
local responses allows speculation about the possibility
of the existence of an immunologic deficit of genetic
origin that would have an important role in the pathology
of canine aspergillosis (Day et al., 1985).
In canine nasal aspergillosis, the immune response in
the nasal cavity is mixed, involving antigen-presenting
cells (macrophages and dendritic cells), T lymphocytes
and plasma cells. Mast cells and eosinophils were not a
significant component of the inflammatory response.
IgG+ plasma cells were the predominant type within the
lesions, whereas IgA+ plasma cells were predominant in
the nasal mucosa of healthy dogs (Peeters et al., 2005).
These data are in contrast with findings in dogs with
disseminated aspergillosis, where IgA+ plasma cells
predominated in the granuloma throughout the body,
and IgA dysregulation was thought to be a predisposing
factor to the disease (Day and Penhale, 1991; Perez
et al., 1996). The predominance of major histocompatibility complex (MHC) class II+ antigen-presenting
cells, and IgG+ plasma cells, together with the presence
of numerous CD4+ and CD8+ T cells may indicate a Th1
immune response that is effective in preventing
systemic dissemination of the fungus but ineffective
in clearing the infection from the nasal cavity and
frontal sinus (Peeters et al., 2005, 2007).
Clinical pictures of disseminated aspergillosis
similar to those described in the dog have been reported
in other animal species. In bovine mycotic abortion and
in ovine Aspergillus mastitis an immune response
similar to that described in the dog can be assumed: the
rupture of the defensive barrier of skin and mucous
membrane allows the fungus to enter directly into the
bloodstream, where the immune system is unable to
stop its development (Jensen et al., 1989; Las Heras
et al., 2000; Garcia et al., 2004a).
Aspergillus spp. have been recognized since the early
1800s as an important cause of pulmonary disease in
birds. In contrast to mammals, birds are highly
susceptible to invasive aspergillosis (Feldmesser, 2005;
Garcia et al., 2007). A study of avian aspergillosis in
chickens found that the appearance of serum antibody
reactive with hyphal fragments is correlated with the
development of resistance to infection, and the appearance of precipitins coincides with clearance of hyphae
from the air sac and lungs, suggesting a role for antibody
in acquired resistance. The increased incidence accompanying captivity, some farming practices and/or mating
has been noted repeatedly, leading to the hypothesis that
susceptibility is stress-induced. A number of features
have been proposed to have a role, such as anatomic
differences from mammals and differences in innate
immune cells, including a lack of surface macrophages in
the lung and dependence upon heterophils, which, unlike
mammalian neutrophils, do not have myeloperoxidase or
any other oxidative mechanism for killing fungal hyphae
(Tell, 2005).
Mould-infected feedstuff can have a dual role in the
process; by dissemination of the fungus, and by
containing small quantities of mycotoxins that would
lead to immunodepression and, consequently, to favour
the appearance of the disease.
5.3. Vaccine against Aspergillus
Studies of vaccines that use whole cells, either in
the form of conidia or disrupted whole germlings or
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
mycelia, support the view that protective immunity
against pulmonary or systemic infection can be induced.
This suggests the possibility of the development of an
effective vaccine against aspergillosis (Cenci et al.,
2000).
A vaccine that is not fully protective but improves the
chances of survival with antifungal therapy would still
be extremely useful as a means of augmenting the host
response to an established disease, providing additional
time for immune reconstitution. Since 1939, a wide
spectrum of vaccination strategies against aspergillosis,
involving combination of a variety of proteins,
glycoproteins, live or inactivated conidia used as
immunogens or the adoptive transfer of immunity cells
have been attempted (Bellocchio et al., 2005; Feldmesser, 2005). For example, an endotoxin isolated from
A. fumigatus gave limited protection to hyperimmunized rabbits subsequently inoculated with conidia.
Some protection was seen in ducks and mice using live
conidia injected subcutaneously. It has been shown
clearly in a murine model that vaccination with A.
fumigatus extracts can confer protection against
subsequent challenges with conidia (Bellocchio et al.,
2005).
One attempt at using a live attenuated conidial
vaccine has been reported. Vaccination of mice with an
avirulent p-aminobenzoic acid auxotrophic mutant
produced by chemical mutagenesis protected against
systemic infection with the virulent parental strain
(Sandhu et al., 1976). Recent experimental evidence
suggests that vaccination against Aspergillus through
the use of fungus-pulsed dendritic cells is feasible
(Bozza et al., 2003; Bellocchio et al., 2005).
Recent studies have proceeded in three important
directions: (1) toward a focus on manipulation of the
immune response with different adjuvants; (2) identification of specific antigens capable of inducing
protective responses; and (3) the development of a
vaccine with efficacy against a broad range of
pathogens, including A. fumigatus.
6. C. albicans
C. albicans is part of the normal microbial flora in
human beings and domestic animals, and is associated
with the mucous surfaces of the oral cavity, gastrointestinal tract and vagina. Immune dysfunction can
allow C. albicans to switch from a commensal to a
pathogenic organism capable of infecting a variety of
tissues and causing a possibly fatal systemic disease
(Stevens et al., 1998; Romani, 1999; Traynor and
Huffnagle, 2001). Mucosal infection is the most usual
57
form of the disease but cutaneous lesions are seen on
occasion (Lehmann, 1985).
In cattle, as a consequence of the abundant use, and
occasional abuse, of antibiotics in the treatment of
mastitis, there is a selection of flora, mainly members of
the genus Candida, that are new etiological agents of
these processes, which are initially difficult to diagnose
because their presence is not expected. Candidiasis in
birds is related to malnutrition and stress, generally
produced by the same strains that are found naturally on
the food plants of these animals. Arthritis caused by
yeasts in horses is relatively frequent as a consequence
of contamination of wounds or after surgical treatment.
In pigs, candidiasis usually takes the form of digestive
alterations in young animals, and is usually related to
problems that predispose to the disease, like treatment
with antibiotics (Garcia and Blanco, 2000). C. albicans
is a common causative agent of stomatitis in the dog
(Jadhav and Pal, 2006).
Although fungi need predisponent factors to produce
the disease, it is known that saprophytic colonization of
the mucous membrane by C. albicans does not need the
host to be immunocompromised, since it is detected in
immunocompetent individuals (Hostetter, 1994; Garcia
and Blanco, 2000).
The clinical spectrum of C. albicans infections
ranges from mucocutaneous to systemic life-threatening infections. The main risk factors that predispose to
severe Candida infections are congenital or acquired
defects of CMI, including quantitative and qualitative
defects in neutrophils and dysregulated Th-cell reactivity (Romani, 2004). Candida infections in dogs have
been described in cases with no clinical evidence of an
underlying disease or immunosuppression (Brown
et al., 2005; Kuwamura et al., 2006).
Resistance to infection by C. albicans is associated
with Th1 CMI, whereas Th2 immunity is associated with
susceptibility to symptomatic infection. Neutrophils are
considered to be the primary effector cells for C. albicans
killing in vivo, although macrophages are involved in
CMI to control infection. In addition to their role as
effector cells, neutrophils may have an immunoregulatory role in development of the Th response. The fact that
neutrophils are (i) abundant at the sites of C. albicans
infection and (ii) capable of selectively producing the
directive cytokines IL-12 and IL-10, suggests that these
cells may be important in determining the type of antiCandida Th cell response (Lehmann, 1985; Stevens et al.,
1998; Traynor and Huffnagle, 2001).
Keratinocytes, neutrophils, macrophages, eosinophils and basophils are the first line of host defence
against mucosal C. albicans infections (Oliveira et al.,
58
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
2007). Neutrophils, monocytes, and monocyte-derived
macrophages, as well as committed tissue macrophages
(Kupffer, splenic macrophages, and pulmonary alveolar
macrophages) are the key effector cells in host defence
against deeply invasive candidiasis (Roilides and
Walsh, 2004).
Cytokines such as TNF-a, IL-6, G-CSF and GMCSF have been determined to be important for
neutrophil recruitment during candidiasis (Kullberg
et al., 1999). GM-CSF accelerates hematopoiesis at
early and late steps of differentiation of myeloid cells,
resulting in increased production of neutrophils,
metamyelocytes and eosinophils. This cytokine also
augments the antimicrobial activities of terminally
differentiated neutrophils and monocytes (Roilides and
Walsh, 2004). GM-CSF increases the fungicidal activity
of human neutrophils against Candida pseudohyphae
and of human monocytes against blastoconidia of C.
albicans (Smith et al., 1990; Roilides and Walsh, 2004).
M-CSF accelerates proliferation and differentiation of
mononuclear/macrophage progenitors, recruits monocytes to sites of infection and activates mature
macrophages. M-CSF is a potent immunomodulator
of antifungal activity of monocytes and tissue macrophages against a variety of fungi, including Candida
spp. (Roilides et al., 2000; Roilides and Walsh, 2004).
The efficacy of HI mechanisms against C. albicans has
been a controversial subject for decades. In the last
decade, evidence has accumulated that antibodies
specific for certain cell surface epitopes may be
beneficial for the host (Han and Cutler, 1995; Cassone
et al., 1997; Polonelli et al., 2000).
The finding of protective and non-protective antibodies, and the observation that these have different
epitope specificity suggest an explanation of why some
investigators have reported evidence for protective
antibodies and others have not. Specifically, when
presented with an enormously complex antigenic
stimulus, such as a yeast cell, the immune system will
respond in a variable manner to a finite number of the
displayed determinants. Predicting the resulting antibody specificities and titres of each antibody may be
impossible, and would likely vary from animal to
animal. Thus, even though the final total antibody titres
may be somewhat similar quantitatively amongst
individual experimental animals immunized against
whole fungal cells, their sera will be expected to differ
qualitatively. This same line of reasoning might be
invoked to explain why the presence of fungal-specific
antibodies in severely ill candidiasis patients is not
necessarily evidence against protective antibodies
(Polonelli et al., 2000).
In animal fungal diseases, it has been shown that the
levels of IgM, IgG and IgA are increased. This fact has
been considered of great importance with diagnostic
and prognostic value, although not necessarily involve a
protective status against the disease (Bromuro et al.,
1994).
It is of interest that many authors have reported that
saprophytic colonization of certain mucocutaneous
locations triggers the production of antibodies and
sometimes it is possible to detect systemic fungal
antigens. This can occur in the absence of clinical
disease and even without systemic colonizations (which
always entails spread of the agent by the lymphohematogenous pathway) (Hernando et al., 1993). There
exists a CMI response characterized by the appearance
of reactive T cells that have a vigilance role in avoiding
active infection (Bromuro et al., 1994).
C. albicans can cause invasive disease in immunocompromised animals. In contrast to the resistance seen
at the skin or mucosal surface, T cell-mediated
immunity seems to be unimportant in resistance to this
form of the disease, in which neutrophils have the
central role (Lehmann, 1985).
In the invasive process, C. albicans reaches the
bloodstream. These blood-borne organisms then adhere
to the endothelial cell lining of the vasculature (Klotz,
1992; Clemons et al., 2000). Next, they invade
endothelial cells by inducing their own endocytosis
(Filler et al., 1995; Clemons et al., 2000). Once inside
the endothelial cells, C. albicans injures and eventually
kills these cells (Fratti et al., 1998; Clemons et al.,
2000). Injury of the endothelial cells leads to loss of
integrity of the vascular lining, enabling the pathogen to
invade the deep tissues. Moreover, the loss of
endothelial cells results in the exposure of the subendothelial cell basement membrane, which can be
bound by additional organisms (Clemons et al., 2000).
Endothelial cells are not passive in this process, they
actively respond to Candida infection by synthesizing a
variety of pro-inflammatory cytokines and expressing
leukocyte adhesion molecules (Orozco et al., 2000;
Clemons et al., 2000). These pro-inflammatory mediators recruit activated leukocytes to the site of vascular
invasion to aid the host defence. If a sufficient number
of functional leukocytes are present, the invading
organisms can be killed and the infection can be aborted
(Clemons et al., 2000).
It has been shown in dogs that CMI is an important
determinant of the systemic spread of Candida spp.,
where circulating neutrophils appear to be the major
source of defence. Disseminated candidiasis has been
associated with parvoviral infection, and with treatment
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
with antimicrobials and corticosteroids, administration
of cyclophosphamide and with a defect in the
immunological system (Brown et al., 2005).
The disease can be controlled in poultry by
immunization via intradermal or intramuscular injection of ethanol-killed C. albicans (Lehmann, 1985).
7. Dermatophytes
Dermatophytosis (ringworm) is an infection by
fungal species belonging to the genera Microsporum,
Trichophyton and Epidermophyton of the keratinized
superficial tissues of man and animals; the corneum
stratum, hair and skin (Sparkes et al., 1993).
Dermatophytosis is important in the dog, not so much
for the severity of the process, which is never lifethreatening, but fundamentally for their zoonosic
character. It is much more frequent in domestic cats,
which are the main source of infection for man. It is
important in cattle, since there are a great number of these
animals affected; in fact, in North and Central Europe it is
considered one of the most important zoonoses, and
vaccines that in principle are fully effective are used in
many countries (Garcia and Blanco, 2000).
Microsporum canis followed by Trichophyton
mentagrophytes, are the dermatophytes most frequently
isolated from dog and cat (Cabañes et al., 1997),
whereas Trichophyton verrucosum and T. mentagrophytes are the most important species isolated from
cattle and horses (Garcia and Blanco, 2000).
Dermatophytes are not part of the normal flora of the
skin. Due to their keratinophilic and keratinolytic
nature, they are able to use cutaneous keratin as a
nutrient producing the infection in this way (Garcia and
Blanco, 2000). Although the infection is confined to the
superficial keratinised tissues, it induces a humoral and
a cellular immune response (DeBoer et al., 1991;
DeBoer and Moriello, 1993; Mignon et al., 1999a;
Sparkes et al., 1993, 1995).
Skin in itself represents a very effective physical
barrier against fungal invasion, in which the action of
neutrophils, epidermal cellular proliferation and keratinisation have a very important role in the initial
response of the host, restricting the microorganism to
the superficial stratum of the skin, and promoting rapid
elimination of the fungus (Osawa et al., 1998).
After the organism is exposed to a dermatophyte, the
following sequence of events occurs in the skin
(Gudding and Lund, 1995; Hunsaker and Perino, 2001):
(1) Presentation of the antigen: Antigens are caught by
the antigen-presenting cells of the immune system
59
of the skin (Langerhans cells), which move to the
regional lymphatic nodes, where the antigen is
presented to the T lymphocytes through the MHC II
(Teunissen, 1992). This movement is probably
favoured by cytokines; GM-CSF has a stimulatory
effect on the Langerhans cells, promoting its
capacity to present the antigen (Kaplan et al.,
1992). Since Langerhans cells have little phagocytic
capacity, macrophages have an important role in the
capture and degradation of the antigen into smaller
fragments. In the same way, keratinocyte have the
capacity to degrade antigens by the phagocytosis,
and to transfer them to Langerhans cells to be
presented to T lymphocytes (Suter, 1993).
(2) Recruitment of cells: In cattle experimentally
infected with T. verrucosum it has been demonstrated that there is an increment of macrophages,
CD4+ and CD8+ lymphocytes, and T g/a cells in the
dermis, and the most abundant are CD4+ T helper
cells (Pier et al., 1993). A dense neutrophil
population has been observed in the skin.
(3) Resolution of the process: Resolution of the disease
is generally accompanied by the development of a
delayed hypersensitive response, while the persistence of the infection seems to be associated with
the absence of this response and with a poor in vitro
lymphoproliferation (DeBoer and Moriello, 1995;
Mignon et al., 1999b). This implies a protective
status against re-infection (Sparkes et al., 1993;
DeBoer and Moriello, 1995).
In response to contact with the fungus, epidermopoiesis in the skin is increased, which produces an increase
in the rate of regeneration of epidermal cells and a
consequent removal of the fungus from the skin surface.
The CMI response is essential to the establishment of an
immunoprotective status against dermatophyte infection (Sohnle, 1993; Mignon et al., 1999b). The sequence
of events in the HI response to dermatophytosis is
unclear, although the action of specific antibodies could
have a direct fungistatic effect by means of opsonization
and complement activation (Pier et al., 1993; Sparkes
et al., 1994).
7.1. Vaccine against dermatophytosis
The cellular wall of dermatophytes is composed
mainly of chitin, glucans and glycopeptides, which are
the main antigens of these fungi (Wagner and Sohnle,
1995). The most important antigens are the proteic
portion of glycopeptides that stimulate the HI response,
and keratinases, which produce a delayed hypersensi-
60
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
tivity response when they are inoculated intradermally
(Dahl, 1993).
The development of effective immunoprophylactics
offers an interesting alternative in the control of this
disease, once the protective status of antigenic extracts
is proven. A great variety of veterinary vaccines
effective against fungal disease have been marketed in
different countries, in some cases for many years.
The inactivated vaccines stimulate the CMI, as
demonstrated by skin tests and leukocyte migration
inhibition tests. Vaccines containing T. verrucosum
conidia inactivated with formalin have been described
for use in cattle (Wawrzkievicz and Wawrzkievicz,
1992).
An inactivated vaccine plus adjuvant containing
conidia and mycelium of two T. equinum strains has
been used in the immunization of horses (Pier and
Zancanella, 1993). The vaccine does not prevent the
disease, but the lesions are less severe in vaccinated
animals in compared to non-vaccinated animals.
The most widely used inactivated vaccine is Insol
Dermatophyton1, developed in Switzerland by Boehringer Ingelheim. The manufacturers indicate that it is
effective in horse, dog and cat, and that it can be used as
treatment of the disease, improving the clinical outcome.
It contains strains of T. verrucosum, T. mentagrophytes, T.
sarkisovii, T. equinum, M. canis, M. canis var. distortum,
M. canis var. obesum, and M. gypseum.
The commercial vaccine Feo-O-Vax MC-K1 was
developed by Fort Dodge in USA. It is an inactivated
vaccine containing the mycelium of M. canis and an
adjuvant. This vaccine produces anti-dermatophyte
antibody titres similar to those developed in the course
of the natural infection, with a low CMI. All vaccinated
cats developed the disease after a topical application of
M. canis conidia; however, the lesions were smaller
than those in the control animals. The fact that all of the
animals vaccinated had lesions suggests that high titres
of antibody against M. canis may not be enough for
protection against the infection (DeBoer and Moriello,
1995).
The inactivated vaccine Dermatovac-IV has been
developed in guinea pigs. It contains an adjuvant and an
optically standardized inactivated suspension of conidia
and mycelium of the fungi M. canis, T. equimun, M.
gypseum and T. mentagrophytes (Pier et al., 1995).
Without doubt, the most effective and widely used
have been the live vaccines. The Ringvac bovis LTF1301 vaccine, marketed by Alpharma, and elaborated
with the LTF-130 strain of T. verrucosum, which has a
characteristic high level of immunogenicity, low
virulence and great stability, fundamental requirements
of a strain used in a vaccine. It has been used effectively
in Russia and Norway, where its effectiveness was
demonstrated with experimental animals as well as in
field studies. It is administered intramuscularly, and it
contains a residual virulence able to stimulate the
appropriate immune response, producing a delayed
hypersensitivity reaction, which is considered essential
for the removal of ringworm lesions (Rybnikar et al.,
1998).
The live vaccine Permavax-Tricho1, marketed in the
Czech Republic by Bioveta Ivanovice, contains an
attenuated strain of T. verrucosum. This vaccine triggers
a protective immunity status 28 days after the second
inoculation, preventing the appearance of the clinical
disease for 1 year after vaccination (Rybnikar et al.,
1998, Mignon et al., 1999b).
8. Malassezia pachydermatis
The lipophilic yeast M. pachydermatis is part of the
normal cutaneous microflora of most warm-blooded
vertebrates. The opportunistic nature of this yeast has
been demonstrated, confirmed by the excellent response
to specific antifungal therapy. The normally commensal
yeast may become a pathogen whenever alternation of
the skin surface microclimate or host defence occurs
(Akerstedt and Vollset, 1996; Guillot and Bond, 1999;
Ashbee, 2006, 2007).
M. pachydermatis is the only species in the genus
that does not require an exogenous source of lipid for
growth (Blanco et al., 2000; Ashbee, 2006). In
veterinary medicine, it is the most important species
in the genus, although another species has been
implicated in skin disorders. M. pachydermatis is the
species most frequently isolated from the skin, mucosa
or ear canal of healthy dogs and cats.
In dogs, this yeast acts as an opportunistic secondary
pathogen within the ear canal. Otitis externa associated
with M. pachydermatis is often characterized by a waxy,
moist, brown or yellow exudate with variable erythema
and pruritus (Griffin, 1993; Guillot and Bond, 1999).
The factors that favour proliferation of M. pachydermatis and its transition from a commensal organism to
an apparent pathogen on canine skin are poorly
understood, but presumably reflect disturbances of
the normal physical, chemical or immunological
mechanisms that restrict microbial colonization of
skin. Breed predilections have been identified, and
geographical variations are apparent (Guillot and Bond,
1999). Concurrent diseases have been recognized in
many, but not all, cases of Malassezia dermatitis in
dogs, and correction of the concurrent disease may
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
prevent, or reduce the frequency of relapse in some dogs
(Garcia and Blanco, 2000). Some atopic dogs carry
large numbers of M. pachydermatis yeasts in both
lesional skin and in unaffected areas (Rossychuck,
1994; Ashbee, 2007). Immediate intradermal test
reactivity has been observed in atopic dogs following
the injection of M. pachydermatis extracts at concentrations that cause no reaction in healthy dogs,
suggesting that hypersensitivity responses to yeast
allergens may be involved in the pathogenesis in some
cases of atopic disease (Morris and Rosser, 1995).
These observations are analogous to the suggestion that
IgE-mediated hypersensitivity to Malassezia-derived
allergens may be important in the pathogenesis of the
‘‘head-neck’’ form of atopic dermatitis in humans
(Kieffer et al., 1990; Guillot and Bond, 1999).
In cats, dermatitis associated with Malassezia spp. is
frequently reported in those with endocrine and
metabolic diseases, neoplasia, and infection with feline
leukaemia virus (FLV) and feline immunodeficiency
virus (FIV). Some reports suggest that Malassezia spp.
overgrowth may be found with feline allergic skin
diseases (Ahman et al., 2007; Ordeix et al., 2007).
M. pachydermatis has the ability to stimulate the
immune system via the classical and alternative
complement pathways, acting as an adjuvant and elicits
both humoral and cellular immune responses in healthy
individuals and individuals with conditions associated
with Malassezia spp. (Sohnle and Collins-Lech, 1983;
Takahashi et al., 1986; Ashbee et al., 1994a,b; Suzuki
et al., 1998). The activation of the complement
pathways is responsible for the inflammation associated
with seborrheic dermatitis. In contrast, it is able to resist
phagocytic killing by neutrophils and down-regulate
cytokine responses when co-cultured with peripheral
blood mononuclear cells (Richardson and Shankland,
1991; Kesavan et al., 1998). These apparently contradictory behaviours may be related to the lipid-rich
capsular-like layer that surrounds the yeast cells, and is
responsible for the lack of inflammation associated with
Malassezia in its commensal state (Mittag, 1995;
Ashbee and Evans, 2002; Ashbee, 2007).
Understanding the apparently contradictory ability
of Malassezia to upregulate or suppress the immune
response directed against it may well be the key to
understanding how Malassezia spp. occur both as
commensals and as pathogens (Ashbee, 2006).
With the interaction of Malassezia with keratinocytes,
the yeast induces the production of different cytokines,
more in M. pachydermatis than in other species of
Malassezia, especially IL-6. Yeast-cell contact is
required for this stimulation, and cytokine production
61
is not caused by a soluble factor. This higher level of
cytokine induction by M. pachydermatis may explain the
greater severity of disease associated with this specie. In
normal skin or mucous membranes, Malassezia reduces
the inflammatory response, enabling it to live as a
commensal (Watanabe et al., 2001; Ashbee, 2006).
The ability of Malassezia spp. to stimulate the
immune system is well documented (Ashbee and Evans,
2002), but the antigenicity is lower in comparison to
other organisms. Then, 20–100-fold more protein from
Malassezia than from C. albicans was required to
stimulate the cellular immune response (Sohnle and
Collins-Lech, 1980).
The interaction of Malassezia with phagocytic cells
may serve to amplify the inflammatory response and
encourage further recruitment of phagocytic cells. The
ability of neutrophils to kill Malassezia seems limited,
in contrast to its function against other yeasts, such as C.
albicans (Richardson and Shankland, 1991; Ashbee and
Evans, 2002). One possible reason for this limited
killing ability of phagocytes may be the production of
azelaic acid by Malassezia. Also, the lipids associated
with the cell wall of Malassezia may be antiphagocytic
and involved in protection against killing by neutrophils
(Akamatsu et al., 1991; Ashbee and Evans, 2002).
The majority of individuals have some antibodies to
Malassezia, even from a relatively young age, with IgG
and IgM. Levels of IgA are generally low, suggesting
that mucosal sensitisation by Malassezia is not
important (Ashbee and Evans, 2002).
9. Cryptococcus
Recently, a differentiation has been made between
two species, Cryptococcus gattii and Cryptococcus
neoformans. This differentiation was made on the basis
of genetic variation and a lack of evidence for genetic
recombination between both genetypes (Duncan et al.,
2006a,b). These genetic differences are consistent with
differences in habitat, geographical distribution and,
most importantly, in pathogenicity and the effectiveness
of the response of the host immune system. C.
neoformans infects predominantly immunocompromised hosts, while C. gattii has not been associated
with a suppressed immune system (Duncan et al.,
2006a,b). This can explain many historical contradictions about the pathogenicity of this fungus, and
especially the need for a state of immunosuppression in
the host for the success of the disease.
C. neoformans is a widespread fungus found in
environmental niches such as soil and avian excreta,
whereas C. gattii is found on eucalyptus trees and in the
62
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
soil (Traynor and Huffnagle, 2001; Duncan et al.,
2006a).
The primary route of entry for Cryptococcus is via
the lungs, where the fungus may establish a primary
infection. If the initial pulmonary infection is not
controlled, the fungus can disseminate to other organs
and the central nervous system, resulting in fatal
cryptococcal meningoencephalitis (Traynor and Huffnagle, 2001; Aguirre et al., 2004; Chen et al., 2007).
Human disseminated cryptococcosis has two unusual
features that distinguish it from other disseminated
fungal infections. First, patients have cryptococcal
polysaccharide antigen in their body fluids, and detection
of this is useful in diagnosis of the disease. Second, there
is a limited inflammatory response in tissues harbouring
C. neoformans. One could interpret these findings to
mean that individuals with the highest levels of
cryptococcal polysaccharide in their body fluids simply
had higher number of organisms in their tissues and
therefore were the most likely not to respond to antifungal
therapy. Another interpretation could be that the
cryptococcal polysaccharide had an adverse effect on
host defence mechanisms, thus allowing a progressive
disease, concluding that cryptococcal polysaccharide in
the bloodstream exacerbates disease (Diamond and
Bennett, 1974; Murphy, 1989; Clemons et al., 2000).
Clinical cryptococcosis has been reported worldwide
in many animal species. It is the most common systemic
fungal infection in cats and is often described in dogs
(Duncan et al., 2006b). In cats, the disease takes the
form of rhinitis when the process is primary, and is
systemic with alterations mainly of the central nervous
system and with important affectation of lymph nodes
when it is secondary to the infection by FIV.
The role of FIV in feline cryptococcosis is under
debate: some studies show an equivalent prevalence of
FIV in cats with and without cryptococcosis in
hospitalized animals, whereas other studies show that
concurrent infection with FIV or FELV in cats with
cryptococcosis was much higher than the prevalence of
those viral diseases in a population of hospitalized cats.
In the majority of these studies, the causative agent was
assumed to be C. neoformans and not C. gattii. This is
important, because C. neoformans is most commonly
isolated from immunosuppressed individuals, whereas
C. gattii should be considered a primary pathogen
because it infects immunocompetent hosts, even in
areas where the organism is endemic (Duncan et al.,
2006a). Therefore, it may be necessary to reconsider the
exact etiologic entity in those cases.
In other animal species, cryptococcosis can appear to
be related to immunodepression, although not always.
Then, have been described outbreaks of ovine and
caprine cryptococcosis with respiratory signs, and
without immunodepression situation detected in those
animals (Garcia and Blanco, 2000). In these cases,
respiratory symptoms associated with cachexia were the
predominant clinical picture; liver and brain involvement was also documented (Baro et al., 1998).
Cryptococcosis may occur in horses as a disseminated cryptococcal infection with osteomylitis of both
the axial and appendicular skeleton produced by C.
gattii. The administration of corticoids led to clinical
deterioration due to immunomodulating effects.
Administration of systemic corticoids occurred after
lesions had appeared; however, the horse deteriorated
significantly after corticosteroid treatment (Lenard
et al., 2007).
9.1. Immune response
Clearance of Cryptococcus infection requires the
development of a Th1-type CMI and the subsequent
pulmonary recruitment and activation of leukocytes.
The leukocytic infiltrate in response to cryptococcal
infection includes a mix of myeloid and lymphoid cells,
all of which are capable of inhibiting the growth of, or
killing, the organism in vitro (Traynor and Huffnagle,
2001; Romani, 2004). Neutrophils and macrophages are
the two phagocytic cells in the natural host defence that
are most likely to be responsible for clearing the
cryptococcal cells from the tissues. There was very little
inflammatory response, i.e. influx of neutrophils,
lymphocytes and macrophages, into infected host
tissues (Clemons et al., 2000).
In this Th1-CMI, IFN-g, TNF-a, IL-2, IL-12, IL-15,
IL-18, monocyte chemotactic protein-1, macrophage
inflammatory protein-1a and nitric oxide have been
shown to have important roles in murine models. GMCSF plays a complex role in the development of
anticryptococcal immunity in the lungs: it is required
for early recruitment of leukocytes into the lung,
movement of recruited leukocytes into the alveolar
space and the formation of inflammatory foci in the
lungs (Traynor and Huffnagle, 2001; Chen et al., 2007).
The limited leukocyte infiltration into infected
tissues in cryptococcosis patients is due to the
circulating glucuronoxylomannan (GXM, predominant
polysaccharide of high molecular weight) stimulating
L-selectin to shed from the surface of neutrophils, thus
preventing the first step in extravasation. This is the
mechanism responsible for the reduced number of
leukocytes that are seen in Cryptococcus-infected
tissues (Clemons et al., 2000).
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
The Th2 cytokines IL-4 and IL-5 were not secreted at
significantly higher levels in Cryptococcus-infected
brains of immune mice compared to control mice
(Uicker et al., 2005).
CD4+ T-cells are critical for the control of
Cryptococcus. An antigen-specific CD4+ T cell
response occurs when the T cell receptor recognizes
processed antigen fragments presented by MHC II.
Then CD4+ T cells secrete cytokines and proliferate
(Kwon-Chung et al., 2000).
Nevertheless, in the lung, both CD4+ and CD8+ T cells
are required to clear the cryptococcal infection. If yeasts
escape and colonize the brain, rapid proliferation leads to
serious central nervous system disease. Work with severe
T cell and B cell-deficient combined immunodeficient
mice and wild-type controls has demonstrated a CD4+ T
cell-mediated resistance to cryptococcal organisms that
colonize the brain. Depletion of CD8+ T cells had no
detectable effect on T cell-mediated resistance in brain in
vivo. In contrast, CD8+ T cells play a well-defined and
important role in containment of pulmonary infection
(Aguirre et al., 2004).
10. Pneumocystis
Pneumocystis is a genus that has had an interesting
tenure on the scientific stage. The first documentation of
the existence of the organism known as Pneumocystis
was as a part of the trypanosome life-cycle. Several years
later, Pneumocystis was identified as an organism
separate from trypanosomes. This includes a life-cycle
similar to that of protozoans, based on the identification
of a small trophic form, the larger cyst form may include
five to eight progeny within the cyst. However, the
disease caused by Pneumocystis was not thoroughly
reported until World War II, where it was observed to be
associated with pneumonia in malnourished children.
Thereafter, Pneumocystis infections became increasingly
evident in the immunocompromised patient population
but it was the AIDS epidemic that brought Pneumocystis
to the forefront of lethal, opportunistic fungal infections.
During this time, another interesting observation roused
the Pneumocystis field with the report that Pneumocystis
was more closely related to fungi than to protozoan.
Actually, Pneumocystis is clearly within the fungal
kingdom, falling between ascomycetes and basidiomycetes (Edman et al., 1988; Steele et al., 2005). Although P.
carinii had been isolated from many animal species,
including man, traditionally a great specificity of the
isolates has been demonstrated for the species that could
infect. Then, P. carinii was considered as a group of
heterogeneous populations, genetically isolated from
63
each other, that have undergone a prolonged process of
genetic and functional adaptation to each mammalian
species (Dei-Cas, 2000). Further genetic analysis has
shown that Pneumocystis isolated from different species
has significant differences in gene sequence and
chromosomes. This has prompted nomenclature changes
and P. carinii is now reserved for the rat pathogen, P.
carinii f.sp. muris for the murine pathogen, and P. jiroveci
for the human pathogen (Stringer et al., 2002; Steele
et al., 2005). It may be that this situation will be repeated
in different animal species, like the description of the
genetically different P. canis in dog (English et al., 2001).
Pneumocystis pneumonia is a well-recognized major
opportunistic infection in HIV-positive individuals, and
is growing in importance in HIV-negative patients
undergoing immunosuppressive treatment for malignancy, connective tissue disease or organ transplantation.
In the veterinary field, Pneumocystis has been described
as producing pneumonia in dogs, causing serious
pulmonary alterations. Pneumocystis is important in
horses, where treatment with immunosuppressive drugs,
like corticoids, is a relatively frequent practice in animals
dedicated to sport, originating a pneumonic process with
bronchoneumonic diffuse image. In pigs, this fungus
gives rise to pneumonic processes, affecting animals 7–
11 weeks old, with lung damage that includes a decrease
of the size of the pulmonary septa with infiltration of
mononuclear cells and appearance of exudates in the
alveoli. This focal pneumonia evolves toward a diffuse
pneumonia, very similar to what happens with this
disease in children (Garcia and Blanco, 2000; Kondo
et al., 2000; Cavallini et al., 2007).
Pneumocystis infects hosts by a respiratory route,
and animal-to-animal airborne transmission has been
clearly established (Dumoulin et al., 2000).
In humans, the persistence of Pneumocystis in the
lung is a limited-time phenomenon inversely related to
immunological improvement. A normal immune
response completely eradicates Pneumocystis from
the host. However, immunocompetent hosts can be
parasitized transiently by Pneumocystis: increased titres
of Pneumocystis antibodies were detected in hospital
staff in close contact with Pneumocystis pneumonia
patients, and immunocompetent experimental hosts
were parasitized transiently by Pneumocystis after close
contact with hosts developing pneumonia by this fungus
(Dei-Cas, 2000).
10.1. Immune response
Host defence against Pneumocystis is indistinguishable from that observed for other medically important
64
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
fungal pathogens of the lung (Steele et al., 2005). As
with fungal pathogens such as A. fumigatus, C.
neoformans and H. capsulatum, alveolar macrophages
are an essential component of the immune response
against Pneumocystis and are ultimately responsible for
clearing this fungus from the lungs (Brummer and
Stevens, 1996; Ibrahim-Granet et al., 2003; He et al.,
2003; Steele et al., 2005).
Natural killer cells are a subset of innate immune
cells that may have a role in host defence against
Pneumocystis, as has been demonstrated for A.
fumigatus and C. neoformans.
Similar to other opportunistic fungal infections,
Pneumocystis pneumonia is most often observed when
the CD4+ T helper cell count falls below 200 cells/mm3.
CD4+ T cells are absolutely critical for resolution of
Pneumocystis, having an essential role in the recruitment
and activation of effector cells against the organism.
CD8+ T cells by themselves do not aid clearance of
Pneumocystis from the lungs (Beck and Harmsen, 1998;
Dei-Cas, 2000; Traynor and Huffnagle, 2001).
IFN-g is not absolutely required for clearing
Pneumocystis from the lungs but is required for
modulating the inflammation (Dei-Cas, 2000).
B cells and/or Pneumocystis-specific antibody are
important for clearance of Pneumocystis from the lungs.
Antibodies against Pneumocystis are reported to be
readily detectable in the early years of life, at ages
similar to that reported for other pathogenic fungi such
as C. albicans and C. neoformans (Goldman et al.,
2001; Steele et al., 2005), and most data suggest that the
organism is widely encountered in nature and that
antibody production is part of the natural host response,
predominantly of the IgG class, but also IgM (Furuta
et al., 1985; Steele et al., 2005). In this sense, many
ELISA-based assays have been developed and employ a
wide variety of Pneumocystis preparations and antigens
to detect the presence of Pneumocystis-specific antibodies (Steele et al., 2005).
In pigs, the infection is due to the naı̈ve system of
young pigs; immunohistochemistry revealed Pneumocystis infection in suckling pigs and a serological study
observed an increase in the titre of antibodies to the
organism in pigs after weaning, concluding that HI
response contributes to the defence against Pneumocystis. A relation has been described with porcine
reproductive and respiratory syndrome virus infection,
which produces a decrease in the number of CD4+ T
cells (Kondo et al., 2000).
In dogs, the disease is usually recognized in young
animals, and is thought to be associated with an
underlying immunodeficiency. In a study with Cavalier
King Charles Spaniels dogs it was concluded that there
is a defect in immunity, with a lower concentration of
IgG and a higher concentration of IgM compared to the
levels in control individuals, and an impaired cellmediated immunity is possible. The concentration of
serum IgA was not abnormal (Watson et al., 2006).
In horses, cases have been described with laboratory
evidence of immunosuppression based on low levels of
IgG, and IgM deficiency, which could have been
secondary to the observed decreased expression of
MHC II molecules, leading to reduced B cell
stimulation. Congenital diseases such as common
variable immune deficiency should also be considered
(MacNeill et al., 2003).
It may be that infection by Pneumocystis in domestic
animals always needs an immunocompromised situation in the affected organism, as happens in man.
11. Conclusion
We have discussed the response of animal immune
systems against fungal infection, citing some examples.
Although a considerable body of knowledge has
accumulated, there is still much to learn. It is important
to avoid generalizations, which can lead to mistakes and
confusion; we know that differences exist among the
immune systems of different animal species, which can
lead to success or failure in the resistance to fungal
infection. Generalizations should be avoided also for
the fungi-like pathogens; there are many pathogenicity
mechanisms that can develop at any given moment, and
the mechanisms can be very different between species.
The example of canine aspergillosis discussed above
illustrates both aspects. Although traditionally they are
not considered alterations of immunodepression in
affected animals, the deficiencies that are observed in
certain breeds of dog for the synthesis of IgA can be
important in the susceptibility against disease. Immunodepression differs, depending on species, and even
among breeds of the same species. There are very few
reports of A. fumigatus producing disseminated
aspergillosis in dogs, as seen in other species within
the genus Aspergillus and in other genera. It is important
to understand why A. fumigatus does not usually
produce an invasive process in dogs, while it develops
disseminated mycoses in many other animal species,
and it is the main invasive pathogenic fungus in humans.
We need to know if this is related to mechanisms of
pathogenicity of the fungus, or to the defensive
mechanisms of the dog, or to a mixture of both.
There is an obvious temptation to assume that all of
the discoveries made in murine models and confirmed
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
in humans apply to all animal species. However, as
discussed here, differences in the immune system of
different animal species could be essential in the fight
against specific fungal diseases, and the recently
described TLRs serve as a clear example.
In conclusion, further research is needed, and that
will require collaboration between clinicians, veterinarians and immunology investigative laboratories.
Clinical cases of animal mycosis should be studied in
depth taking all immunologic variables into consideration in order to reach an understanding of the pathogen–
host interaction for individual animal species with the
different fungal species.
References
Aerts, A.M., François, I.E.J.A., Cammue, B.P.A., Thevissen, K., 2008.
The mode of antigungal action of plant, insect and human defensins. Cell. Mol. Life Sci. 65, 2069–2079.
Aguirre, K., Crowe, J., Haas, A., Smith, J., 2004. Resistance to
Cryptococcus neoformans infection in the absence of CD4+ T
cells. Med. Mycol. 42, 15–25.
Ahman, S., Perrins, N., Bond, R., 2007. Carriage of Malassezia spp.
yeasts in healthy and seborrhoeic Devon Rex cats. Med. Mycol.
45, 449–455.
Akamatsu, H., Kamura, J., Asada, Y., Miyachi, Y., Niwa, Y., 1991.
Inhibitory effect of azelaic acid on neutrophil fractions: a posible
cause for its efficacy in treating pathogenetically unrelated diseases. Arch. Dermatol. Res. 283, 162–166.
Akerstedt, J., Vollset, I., 1996. Malassezia pachydermatis with special
reference to canine skin diseases. Br. Vet. J. 152, 269–281.
Amitani, R., Taylor, G., Elezis, E.N., Llewellyn-Jones, C., Mitchell, J.,
Kuze, F., Cole, P.J., Wilson, R., 1995. Purification and characterization of factors produced by Aspergillus fumigatus which affect
human ciliated respiratory epithelium. Infect. Immun. 63, 3266–
3271.
Ashbee, H.R., 2006. Recent developments in the immunology and
biology of Malassezia species. FEMS Immunol. Med. Microbiol.
47, 14–23.
Ashbee, H.R., 2007. Update on the genus Malassezia. Med. Mycol.
45, 287–303.
Ashbee, H.R., Evans, E.G.V., 2002. Immunology of diseases
associated with Malassezia species. Clin. Microbiol. Rev. 15,
21–57.
Ashbee, H.R., Fruin, A., Holland, K.T., Cunliffe, W.J., Ingham, E.,
1994a. Humoral immunity to Malassezia furfur serovars A, B and
C in patients with pityriasis versicolor, seborrheic dermatitis and
controls. Exp. Dermatol. 3, 227–233.
Ashbee, H.R., Ingham, E., Holland, K.T., Cunliffe, W.J., 1994b. Cellmediated immune response to Malassezia furfur serovars A, B and
C in patients with pityriasis versicolor, seborrheic dermatitis and
controls. Exp. Dermatol. 3, 106–112.
Baro, T., Torres-Rodriguez, J.M., Hermoso de Mendoza, M., Morera,
Y., Alia, C., 1998. First identification of autochtonous Cryptococcus neoformonans var. gattii isolated from goats with predominantly severe pulmonary disease in Spain. J. Clin. Microbiol.
36, 458–461.
Beck, J.M., Harmsen, A.G., 1998. Lymphocites in host defense
against Pneumocystis carinii. Semin. Respir. Infect. 13, 330–338.
65
Bellocchio, S., Bozza, S., Montagnoli, C., Perruccio, K., Gaziano, R.,
Pitzurra, L., Romani, L., 2005. Immunity to Aspergillus fumigatus:
the basis for immunotherapy and vaccination. Med. Mycol. 43
(Suppl. I), S181–S188.
Berry, W.L., Leisewitz, L., 1996. Multifocal Aspergillus terreus
discospondylitis in two German shepherd dogs. J. S. Afr. Vet.
Assoc. 67, 222–228.
Blanco, J.L., Garcia, M.E., 2000. Presente y futuro del diagnóstico
inmunológico de las micosis animales. Rev. Iberoam. Micol. 17,
S23–S28.
Blanco, J.L., Guedeja-Marron, J., Blanco, I., Garcia, M.E., 2000.
Optimum incubation conditions for the isolation of yeasts from
canine otitis externa. J. Vet. Med. B 47, 599–605.
Blanco, J.L., Hontecillas, R., Bouza, E., Blanco, I., Pelaez, T., PerezMolina, J., Garcia, M.E., 2002. Correlation between the elastase
activity index and invasiveness of clinical isolates of Aspergillus
fumigatus. J. Clin. Microbiol. 40, 1811–1813.
Bozza, S., Gaziano, R., Spreca, A., Bacci, A., Montagnoli, C., di
Francesco, P., Romani, L., 2002. Dendritic cells transport conidia
and hyphae of Aspergillus fumigatus from the airways to the
draining lymph nodes and initiate disparate Th responses to the
fungus. J. Immunol. 168, 1362–1371.
Bozza, S., Perruccio, K., Montagnoli, C., Gaziano, R., Bellochio, S.,
Burchielli, E., Nkwanyuo, G., Pitzurra, L., Velardi, A., Romani,
L., 2003. A dendritic cell vaccine against invasive aspergillosis in
allogeneic hematopoietic transplantation. Blood 102, 3807–3814.
Bromuro, C., Torosantucci, A., Gomez, M.J., Urbani, F., Cassone, A.,
1994. Differential release of an immunodominant 65 kDa mannoprotein antigen from yeast and mycelial forms of Candida
albicans. J. Med. Vet. Mycol. 32, 447–459.
Brown, M.R., Thompson, C.A., Mohamed, F.M., 2005. Systemic
candidiasis in an apparently immunocompetent dog. J. Vet. Diagn.
Invest. 17, 272–276.
Brummer, E., Stevens, D.A., 1996. Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for
Histoplasma capsulatum. Clin. Exp. Immunol. 102, 65–70.
Butterworth, S.J., Barr, F.J., Pearson, G.R., Day, M.J., 1995. Multiple
discospondylitis associated with Aspergillus species infection in a
dog. Vet. Rec. 136, 38–41.
Cabañes, F.J., Abarca, M.L., Bragulat, M.R., 1997. Dermatophytes
isolated from domestic animals in Barcelona, Spain. Mycopathologia 137, 107–113.
Casadevall, A., 1995. Antibody immunity and invasive fungal infections. Infect. Immun. 63, 4211–4218.
Casadevall, A., Feldmesser, M., Pirofski, L.A., 2002. Induced humoral
immunity and vaccination against major human fungal pathogens.
Curr. Opin. Microbiol. 5, 386–391.
Cassone, A., Conti, S., De Bernardis, F., Polonelli, L., 1997. Antibodies, killer toxins and antifungal immunoprotection: a lesson
from nature? Immunol. Today 18, 164–169.
Cavallini, E.M., Pescador, C., Rozza, D., Spanamberg, A., Borba,
M.R., Ravazzolo, A.P., Driemeier, D., Guillot, J., Ferreiro, L.,
2007. Detection of Penumocystis spp. in lung samples from pigs in
Brazil. Med. Mycol. 45, 395–399.
Cenci, E., Mencacci, A., Del Sero, G., Bacci, A., Montagnoli, C.,
d’Ostiani, C.F., Mosci, P., Bachmann, M., Bistoni, F., Kopf, M.,
Romani, L., 1999. Interleukin-4 causes susceptibility to invasive
pulmonary aspergillosis through suppression of protective type I
responses. J. Infect. Dis. 180, 1957–1968.
Cenci, E., Mencacci, A., Bacci, A., Bistoni, F., Kurup, V.P., Romani,
L., 2000. T cell vaccination in mice with invasive pulmonary
aspergillosis. J. Immunol. 165, 381–388.
66
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
Cenci, E., Mencacci, A., Spreca, A., Montagnoli, C., Bacci, A.,
Perruccio, K., Velarde, A., Magliani, W., Conti, S., Polonelli,
L., Romani, L., 2002. Protection of killer antiidiotypic, antibodies
against early invasive aspergillosis in a murine model of allogeneic
T-cell-depleted bone marrow transplantation. Infect. Immun. 70,
2375–2382.
Chen, G.H., Olszewski, M.A., McDonald, R.A., Wells, J.C., Paine III,
R., Huffnagle, G.B., Toews, G.B., 2007. Role of granulocyte
macrophage colony-stimulating factor in host defense against
pulmonary Cryptococcus neoformans infection during murine
allergic bronchopulmonary mycosis. Am. J. Pathol. 170, 1028–
1040.
Christin, L., Wysong, D.R., Meshulam, T., Hastey, R., Simons, E.R.,
Diamond, R.D., 1998. Human platelets damage Aspergillus fumigatus hyphae and may supplement killing by neutrophils. Infect.
Immun. 66, 1181–1189.
Clemons, K.V., Calich, V.L.G., Burger, E., Filler, S.G., Graziutti, M.,
Murphy, J., Roilides, E., Campa, A., Dias, M.R., Edwards, J.E.,
Fu, Y., Fernandes-Bordignon, G., Ibrahim, A., Katsifa, H.,
Lamaignere, C.G., Meloni-Bruneri, L.H., Rex, J., Savary, C.A.,
Xidieh, C., 2000. Pathogenesis I: interactions of host cells and
fungi. Med. Mycol. 38 (Suppl. 1), 99–111.
Clercx, C., McEntee, K., Snaps, F., Jacquinet, E., Coignoul, F., 1996.
Bronchopulmonary and disseminated granulomatous disease associated with Aspergillus fumigatus and Candida species infection in
a Golden Retriever. J. Am. Anim. Hosp. Assoc. 32, 139–145.
Cox, A.R., 1989. Immunology of the Fungal Diseases. CRC Press,
Florida, USA.
Crameri, R., Blaser, K., 2002. Allergy and immunity to fungal
infections and colonization. Eur. Respir. J. 19, 151–157.
Dahl, M.V., 1993. Dermatophytosis and the immune response. Acad.
Dermatol. 28, 519–523.
Dallman, M.J., Dew, T.L., Tobias, L., Doss, R., 1992. Disseminated
aspergillosis in a dog with diskospondylitis and neurologic deficits. J. Am.Vet. Med. Assoc. 200, 511–513.
Day, M.J., Penhale, W.J., 1991. An immunohistochemical study of
canine disseminated aspergillosis. Aust. Vet. J. 68, 383–386.
Day, M.J., Penhale, W.J., 1998. Humoral immunity in disseminated
Aspergillus terreus infection in the dog. Vet. Microbiol. 16, 283–
294.
Day, M.J., Eger, C.E., Shaw, S.E., Penhale, W.J., 1985. Immunologic
study of systemic aspergillosis in German shepherd dogs. Vet.
Immunol. Immunopathol. 9, 335–347.
DeBoer, D.J., Moriello, K.A., 1993. Humoral and cellular responses to
Microsporum canis in naturally occurring feline dermatophytosis.
J. Vet. Med. Mycol. 31, 121–132.
DeBoer, D.J., Moriello, K.A., 1995. Investigations of a killed dermatophyte cell-wall vaccine against infection with Microsporum
canis in cats. Res. Vet. Sci. 59, 110–113.
DeBoer, D.J., Moriello, K.A., Cooley, A.I., 1991. Immunological
reactivity to intradermal dermatophyte antigens in cats with
dermatophytosis. Vet. Dermatol. 2, 59–67.
Deep Jr., G.S., 1997. Prospects for the development of fungal vaccines. Clin. Microbiol. Rev. 10, 585–596.
Dei-Cas, E., 2000. Pneumocystis infections: the iceberg? Med.
Mycol. 38 (Suppl. 1), 23–32.
Diamond, R.D., Bennett, J.E., 1974. Prognostic factors in cryptococcal meningitis. A study in 111 cases. Ann. Intern. Med. 80, 176–
181.
Dumoulin, A., Mazars, E., Seguy, N., Gargallo-Viola, D., Vargas, S.,
Cailliez, J.C., Aliouat, E.M., Wakefield, A.E., Dei-Cas, E., 2000.
Transmission of Pneumocystis carinii disease from immunocom-
petent contacts of infected hosts to susceptible hosts. Eur. J. Clin.
Microbiol. Infect. Dis. 19, 671–678.
Duncan, C.G., Stephen, C., Campbell, J., 2006a. Evaluation of risk
factors for Cryptococcus gattii infection in dogs and cats. J. Am.
Vet. Med. Assoc. 228, 377–382.
Duncan, C.G., Stephen, C., Campbell, J., 2006b. Clinical characteristics and predictors of mortality for Cryptococcus gattii infection
in dogs and cats of southwestern British Columbia. Can. Vet. J. 47,
993–998.
Duong, M., Ouellet, N., Simard, M., et al., 1998. Kinetic study of host
defense and inflammatory response to Aspergillus fumigatus in
steroid-induced immunosuppressed mice. J. Infect. Dis. 178,
1472–1482.
Edman, J.K., Kovacs, J.A., Masur, H., Santi, D.V., Elwood, H.J.,
Sogin, M.L., 1988. Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature 334, 519–522.
Elad, D., Lahav, D., Blum, S., 2008. Transuterine transmisión of
Aspergillus terreus in a case of disseminated canine aspergillosis.
Med. Mycol. 46, 175–178.
English, K., Peters, S.E., Meskell, D.J., Collins, M.E., 2001. DNA
analysis of Pneumocystis infecting a Cavalier King Charles Spaniel. J. Eukaryot. Microbiol. Suppl. 106S.
Feldmesser, M., 2005. Prospects of vaccines medically important
fungi. Med. Mycol. 43, 571–587.
Filler, S.G., Swerdloff, J.N., Hobbs, C., Luckett, P.M., 1995. Penetration and damage of endothelial cells by Candida albicans. Infect.
Immun. 63, 976–983.
Fratti, R.A., Belanger, P.H., Ghannoum, M.A., Edwards Jr., J.E.,
Filler, S.G., 1998. Endothelial cell injury caused by Candida
albicans is dependent on iron. Infect. Immun. 66, 191–196.
Furuta, T., Fujiwara, K., Yamanouchi, K., Veda, K., 1985. Detection of
antibodies to Pneumocystis carinii by enzyme-linked immunosorbent assay in experimentally infected mice. J. Parasitol. 71,
522–523.
Gantner, B.N., Simmons, R.M., Underhill, D.M., 2005. Dectin-1
mediates macrophage recognition of Candida albicans yeast
but not filaments. EMBO J. 24, 1277–1286.
Ganz, T., 2002. Immunology. Versatile defensins. Science 298, 977–
979.
Ganz, T., 2003. Defensins: antimicrobial peptides of innate immunity.
Nat. Rev. Immunol. 3, 710–720.
Garcia, M.E., Blanco, J.L., 2000. Principales enfermedades fúngicas
que afectan a los animales domésticos. Rev. Iberoam. Micol. 17,
S2–S7.
Garcia, M.E., Caballero, J., Toni, P., Garcia, I., Martinez de Merlo, E.,
Rollan, E., Gonzalez, M., Blanco, J.L., 2000. Disseminated
mycoses in a dog by Paecilomyces sp. J. Vet. Med. A 47, 243–249.
Garcia, M.E., Caro, A., Fragio, C., Blanco, I., Blanco, J.L., 2001a. A
clinical case of canine mycotic pneumonia. J. Vet. Med. A 48,
501–506.
Garcia, M.E., Caballero, J., Cruzado, M., Andrino, M., GonzalezCabo, J.F., Blanco, J.L., 2001b. The value of the determination of
anti-Aspergillus IgG in the serodiagnosis of canine aspergillosis:
comparison with galactomannan detection. J. Vet. Med. B 48,
743–750.
Garcia, M.E., Duran, C., Cruzado, M., Andrino, M., Blanco, J.L.,
2004a. Evaluation of molecular and immunological techniques for
the diagnosis of mammary aspergillosis in ewes. Vet. Microbiol.
98, 17–21.
Garcia, M.E., Guedeja-Marron, J., Lazaro, M., Blanco, J.L., 2004b.
Monitoring of canine dermatophytosis by an immunodiagnostic
method. Vet. Rec. 154, 116–117.
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
Garcia, M.E., Lanzarot, P., Lopez-Rodas, V., Costas, E., Blanco, J.L.,
2007. Fungal flora in trachea of birds from a wildlife rehabilitation
centre in Spain. Vet. Med. 52, 464–470.
Gelatt, K.N., Chrisman, C.L., Samuelson, D.A., Shell, L.G., Buergelt,
C.D., 1991. Ocular and systemic aspergillosis in a dog. J. Am.
Anim. Hosp. Assoc. 27, 427–431.
Gene, J., Blanco, J.L., Cano, J., Garcia, M.E., Guarro, J., 2003. The
new filamentous fungus Sagenomella chlamydospora responsible
for a disseminated infection in a dog. J. Clin. Microbiol. 41, 1722–
1725.
Goldman, D.L., Khine, H., Abadi, J., Lindenberg, D.J., Pirofski, L.,
Niang, R., Casadevall, A., 2001. Serologic evidence for Cryptococcus neoformans infection in early childhood. Pediatrics 107,
E66.
Gonzalez, C.E., Lyman, C.A., Lee, S., Del Guercio, C., Roilides, E.,
Bacher, J., Gehrt, A., Fenerstein, E., Toscos, M., Walsh, T.J., 2001.
Recombinant human macrophage colony-stimulating factor augments pulmonary host defences against Aspergillus fumigatus.
Cytokine 15, 87–95.
Griffin, C.E., 1993. Otitis externa and otitis media. In: Griffin,
C.E., Kwochka, K.W., MacDonald, J.M. (Eds.), Current Veterinary Dermatology. Mosby Year Book, St Louis, pp. 244–262.
Gudding, R., Lund, A., 1995. Immunoprofilaxis of bovine dermatophytosis. Can. Vet. J. 36, 302–306.
Guerin, S.R., Walker, M.C., Kelly, D.F., 1993. Cavitating mycotic
pulmonary infection in a german shepherd dog. J. Small Anim.
Pract. 34, 36–39.
Guillot, J., Bond, R., 1999. Malassezia pachydermatis: a review. Med.
Mycol. 37, 295–306.
Han, Y., Cutler, J.E., 1995. Antibody response that protect against
disseminated candidiasis. Infect. Immun. 63, 2714–2719.
He, W., Casadevall, A., Lee, S.C., Goldman, D.L., 2003. Phagocytic
activity and monocyte chemotactic protein expression by pulmonary macrophages in persistent pulmonary cryptococosis. Infect.
Immun. 71, 930–936.
Hernando, F.L., Estevez, J.J., Cebrian, M., Poulain, D., Ponton, J.,
1993. Identification of Candida albicans cell wall antigens lost
during subculture in synthetic media. J. Med. Vet. Mycol. 31, 227–
237.
Hostetter, M.K., 1994. Adhesins and ligands involved in the interaction of Candida spp. with epithelial and endothelial surfaces. Clin.
Microbiol. Rev. 7, 29–42.
Hunsaker, B.D., Perino, L.J., 2001. Efficacy of intradermal vaccination. Vet. Immunol. Immunopathol. 79, 1–13.
Ibrahim-Granet, O., Phillipe, B., Boleti, H., Boisvieux-Ulrich, E.,
Grenet, D., Stern, M., Latge, J.P., 2003. Phagocytic and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. Infect. Immun. 71, 891–903.
Jadhav, V.J., Pal, M., 2006. Canine mycotic stomatitis due to Candida
albicans. Rev. Iberoam. Micol. 23, 233–234.
Janeway Jr., C.A., Medzhitov, R., 2002. Innate immune recognition.
Ann. Rev. Immunol. 20, 197–216.
Jang, S.S., Dorr, T.E., Biberstein, E.L., Wong, A., 1986. Aspergillus
deflectus infection in four dogs. J. Med. Vet. Mycol. 24, 95–104.
Jensen, H.E., Basse, A., Aalbaek, B., 1989. Mycosis in the stomach
compartiments of cattle. Acta Vet. Scand. 30, 409–423.
Kaplan, G., Walsh, G., Guido, L.S., Meyn, P., Burkhardt, R.A.,
Abalos, R.M., Barker, J., Frindt, P.A., Fajardo, T.T., Celona, R.,
1992. Novel responses on human skin to intradermal recombinant
granulocyte/macrophage colony stimulating factor: Langerhans
cells recruitment, keratinocyte growth, and enhance wound healing. J. Exp. Med. 175, 1717–1728.
67
Kauffman, A.C., Green, C.E., Selcer, B.A., Styles, M.E., Mahaffey,
E.A., 1994. Systemic aspergillosis in a dog and treatment with
hamycin. J. Am. Anim. Hosp. Assoc. 30, 132–136.
Kesavan, S.A., Walters, C.E., Holland, K.T., Ingham, E., 1998. The
effects of Malassezia on pro-inflammatory cytokine production by
human peripheral blood mononuclear cells in vitro. Med. Mycol.
36, 97–106.
Kieffer, M., Bergbrant, I.M., Faergemann, J., Jemec, G.B., Ottevanger,
V., Stahlskov, P., Svejgaard, E., 1990. Immune reactions to
Pityrosporum ovale in adult patients with atopic and seborrheic
dermatitis. J. Am. Acad. Dermatol. 22, 739–742.
Klotz, S.A., 1992. Fungal adherence to the vascular compartment: a
critical step in the pathogenesis of disseminated candidiasis. Clin.
Infect. Dis. 14, 340–347.
Kondo, H., Hikita, M., Ito, M., Kadota, K., 2000. Immunohistochemical study of Pneumocystis carinii infection in pigs: evaluation of
Pneumocystis pneumonia and a retrospecive investigation. Vet.
Rec. 147, 544–549.
Kozel, T.R., 1996. Activation of the complement system by pathogenic fungi. Clin. Microbiol. Rev. 9, 34–46.
Kullberg, B.J., Netea, M.G., Vonk, A.G., van der Meer, J.W., 1999.
Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice. FEMS Immunol.
Med. Microbiol. 26, 299–307.
Kuwamura, M., Ide, M., Yamate, J., Shiraishi, Y., Kotani, T., 2006.
Systemic candidiasis in a dog developing spondylitis. J. Vet. Med.
Sci. 68, 1117–1119.
Kwon-Chung, K.J., Sorrell, T.C., Dromer, F., Fung, E., Levitz, S.M.,
2000. Cryptococcosis: clinical and biological aspects. Med.
Mycol. 38 (Suppl. 1), 205–213.
Las Heras, A., Domı́nguez, L., Lopez, I., Paya, M.J., Peña, L.,
Mazzucchelli, F., Garcia, L.A., Fernández-Garayzabal, J.F.,
2000. Intramammary Aspergillus fumigatus infection in dairy
ewes associated with antibiotic dry therapy. Vet. Rec. 147,
578–580.
Latge, J.P., 1999. Aspergillus fumigatus and Aspergillosis. Clin.
Microbiol. Rev. 12, 310–350.
Lehmann, P.F., 1985. Immunology of fungal infections in animals.
Vet. Immunol. Immunopathol. 10, 33–69.
Lenard, Z.M., Lester, N.V., O’Hara, A.J., Hopper, B.J., Lester, G.D.,
2007. Disseminated cryptococcosis including osteomyelitis in a
horse. Aust. Vet. J. 85, 51–55.
Littman, M.P., Goldschmidt, M.H., 1987. Systemic paecilomycosis in
a dog. J. Am. Vet. Med. Assoc. 191, 445–447.
MacNeill, A.L., Alleman, A.R., Fanklin, R.P., Long, M., Giguere, S.,
Uhl, E., Lopez-Martinez, A., Wilkerson, M., 2003. Pneumonia in a
Paso-Fino mare. Vet. Clin. Pathol. 32, 73–76.
Madan, T., Kaur, S., Saxena, S., Singh, M., Kishore, U., Thiel, S., Reid,
K.B.M., Sarma, P.U., 2005. Role of collectins in innate immunity
against aspergillosis. Med. Mycol. 43 (Suppl. I), S155–S163.
Markaryan, A., Morozova, I., Yu, H., Kolattukudy, P.E., 1994. Purification and characterization of an elastinolytic metalloprotease
from Aspergillus fumigatus and immuno-electron microscopic
evidence of secretion of this enzyme by the fungus. Infect. Immun.
62, 2149–2157.
McCormack, F.X., Whitsett, J.A., 2002. The pulmonary collectins,
SP-A and SP-D, orchestrate innate immunity in the lung. J. Clin.
Invest. 109, 707–712.
Medzhitov, R., Janeway Jr., C.A., 1997. Innate immunity: the virtues
of a nonclonal system of recognition. Cell 91, 295–298.
Mehrad, B., Standiford, T.J., 1999. Role of cytokines in pulmonary
antimicrobial host defense. Immunol. Res. 20, 15–27.
68
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
Meier, A., Kirschning, C.J., Nikolaus, T., Wagner, H., Heesemann, J.,
Ebel, F., 2003. Toll-like receptor (TLR) and TLR4 are essential for
Aspergillus induced activation of murine macrophages. Cell.
Microbiol. 5, 569–570.
Mignon, B.R., Coignoul, F., Leclipteux, T., Focant, C., Losson, B.J.,
1999a. Histopathological pattern and humoral immune response to
a crude exo-antigen and purified keratinase of Microsporum canis
in symptomatic and asymptomatic infected cats. Med. Mycol. 37,
1–9.
Mignon, B.R., Leclipteux, T., Focant, C.H., Nikkels, A.J., Pierard,
G.E., Losson, B.J., 1999b. Humoral and cellular immune response
to a crude exoantigen and purified keratinase of Microsporum
canis in experimentally infected guinea pigs. Med. Mycol. 37,
123–129.
Mittag, H., 1995. Fine structural investigation of Malassezia furfur.
Part II. The envelope of the yeast cells. Mycoses 38, 13–21.
Morris, D.O., Rosser, E.J., 1995. Immunologic aspects of Malassezia
dermatitis in patients with canine atopic dermatitis. In: Proceedings of the 11th Annual Meeting American Academy of Veterinary
Dermatology, American College of Veterinary Dermatology,
Santa Fe, USA.
Morrison, B.E., Park, S.J., Mooney, J.M., Mehrad, B., 2003. Chemokine-mediated recruitment of NK cells is a critical host defense
mechanism in invasive aspergillosis. J. Clin. Invest. 112, 1862–
1870.
Mullanery, T.P., Levin, S., Indrieri, R.J., 1983. Disseminated aspergillosis in a dog. J. Am. Vet. Med. Assoc. 182, 516–518.
Murphy, J.W., 1989. Clearance of Cryptococcus neoformans from
immunologically suppressed mice. Infect. Immun. 57, 1946–1952.
Murphy, P.M., Baggiolini, M., Charo, I.F., Hebert, C.A., Horuk, R.,
Matsushima, K., Miller, L.H., Oppenheim, J.J., Power, C.A., 2000.
International Union of Pharmacology. XXII. Nomenclature for
chemokine receptors. Pharmacol. Rev. 52, 145–176.
Netea, M.G., Van der Graaf, C.A.A., Vonk, A.G., Verschveren, I., Van
der Meer, J.W., Kullberg, B.J., 2002. The role of toll-like receptor
(TLR) 2 and TLR 4 in the host defence against disseminated
candidiasis. J. Infect. Dis. 185, 1483–1489.
Netea, M.G., Warris, A., Van der Meer, J.W., Fenton, M.J., VerverJanssen, T.J., Jacobs, L.E., Andresen, T., Verweij, P.E., Kullberg,
B.J., 2003. Aspergillus fumigatus evades immune recognition
during germination through loss of toll-like receptor-4-mediated
signal transduction. J. Infect. Dis. 188, 320–326.
Netea, M.G., Sutmuller, R., Hermann, C., Van der Graaf, C.A., Van der
Meer, J.W., van Krieken, J.H., Hortung, T., Adema, G., Kullberg,
B.J., 2004. Toll-like receptor 2 suppresses immunity against
Candida albicans through induction of SL-10 and regulatory T
cells. J. Immunol. 172, 3712–3718.
Netea, M.G., Ferwerda, G., Van der Graaf, C.A.A., Van der Meer,
J.W., Kullberg, B.J., 2006. Recognition of fungal pathogens by
Toll-like receptors. Curr. Pharm. Des. 12, 4195–4201.
O’Garra, A., McEvoy, L.M., Zlotnik, A., 1998. T-cell subsets: chemokine receptors guide the way. Curr. Biol. 8, 646–649.
de Oliveira, M.A.M., Carvalho, L.P., Gomes, M.S., Baceller, O.,
Barros, T.F., Carvalho, E.M., 2007. Microbiological and immunological features of oral candidiasis. Microbiol. Immunol. 51,
713–719.
Ordeix, L., Galeotti, F., Scarampella, F., Dedola, C., Bardası́, M.,
Romano, E., Fondati, A., 2007. Malassezia spp. Overgrowth in
allergic cats. Vet. Dermatol. 18, 316–323.
Orozco, A.S., Zhon, X., Filler, S.G., 2000. Mechanisms of the proinflammatory response of endothelial cells to Candida albicans
infection. Infect. Immun. 68, 1134–1141.
Osawa, H.R.C., Summerbell, R.C., Clemons, K.V., Koga, T., Ron,
Y.P., Rashid, A., Sohnle, P.G., Stevens, D.A., Tsuboi, R., 1998.
Dermatophytes and the host defence. Med. Mycol. 36, S166–
S173.
Paris, S., Boisvieux-Ulrich, E., Crestani, B., Houcine, O., Taramelli,
D., Lombardi, L., Latge, J.P., 1997. Internalization of Aspergillus
fumigatus conidia by epithelial and endothelial cells. Infect.
Immun. 65, 1510–1514.
Pastor, J., Pumarola, M., Cuenca, R., Lavin, S., 1993. Systemic
aspergillosis in a dog. Vet. Rec. 132, 412–413.
Patterson, J.M., Rosendal, S., Humphrey, J., Teeter, W.G., 1983. A
case of disseminated paecilomycoses in the dog. J. Am. Anim.
Hosp. Assoc. 19, 569–574.
Peeters, D., Day, M.J., Clercx, C., 2005. An immunohistochemical
study of canine nasal aspergillosis. J. Comp. Pathol. 132, 283–
288.
Peeters, D., Peters, I.R., Helps, C.R., Gabriel, A., Day, M.J., Clercx,
C., 2007. Distinct tissue cytokine and chemokine mRNA expression in canine sino-nasal aspergillosis and idiopathic lymphoplasmacyte rhinitis. Vet. Immunol. Imunopathol. 117, 95–105.
Perez, J., Mozos, E., Chacon, F., Paniagua, J., Day, M.J., 1996.
Disseminated aspergillosis in a dog: an immunohistochemical
study. J. Comp. Pathol. 115, 191–196.
Phadke, A.P., Mehrad, B., 2005. Cytokines in host defense against
Aspergillus: recent advances. Med. Mycol. 43 (Suppl. I), S173–
S176.
Pier, A.C., Zancanella, P.J., 1993. Immunization of horses against
dermatophytosis caused by Trichophyton equinum. Equine Pract.
15, 23–27.
Pier, A.C., Ellis, J.A., Mills, K.W., 1993. Development of immune
response in experimental bovine Trichophyton verrucosum infection. Vet. Dermatol. 3, 131–138.
Pier, A.C., Hodges, A.B., Lauze, J.M., Raisbeck, M., 1995. Experimental immunity to Microsporum canis and cross reactions with
other dermatophytes of veterinary importance. J. Med. Vet. Mycol.
33, 93–97.
Polonelli, L., Casadevall, A., Han, Y., Bernardis, F., Kirkland, T.N.,
Matthews, R.C., Adriani, D., Boccanera, M., Burnie, J.P., Cassone,
A., Conti, S., Cutler, J.E., Frazzi, R., Gregory, C., Hodgetts, S.,
Illidge, C., Magliani, W., Rigg, G., Santoni, G., 2000. The efficacy
of acquired humoral and cellular immunity in the prevention and
therapy of experimental fungal infections. Med. Mycol. 38 (Suppl.
1), 281–292.
Richardson, M.D., Shankland, G.S., 1991. Enhanced phagocytosis and
intracellular killing of Pityrosporum ovale by human neutrophils
after exposure to ketoconazole is correlated to changes of the yeast
cell surface. Mycoses 34, 29–33.
Rodriguez-Adrian, L.J., Grazzintti, M.L., Rex, J.H., Anaissie, E.J.,
1998. The potential role of cytokine therapy for fungal infections
in patients with cancer: is recovery from neutropenia all that is
needed? Clin. Infect. Dis. 26, 1270–1278.
Roeder, A., Kirschning, C.J., Rupec, R.A., Schaller, M., Weindl, G.,
Korting, H.C., 2004. Toll-like receptors as key mediators in innate
antifungal immunity. Med. Mycol. 42, 485–498.
Roilides, E., Walsh, T., 2004. Recombinant cytokines in augmentation
and immunomodulation of host defenses against Candida spp.
Med. Mycol. 42, 1–13.
Roilides, E., Uhlig, K., Venzon, D., Pizzo, P.A., Walsh, T.J., 1993.
Enhancement of oxidative response and damage caused by human
neutrophils to Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon. Infect. Immun. 61,
1185–1193.
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
Roilides, E., Sein, T., Holmes, A., Chanok, S., Blake, C., Pizzo, P.A.,
Walsh, T.J., 1995. Effects of macrophage colony-stimulating
factor on antifungal activity of mononuclear phagocytes against
Aspergillus fumigatus. J. Infect. Dis. 172, 1028–1034.
Roilides, E., Dimitriadou-Georgiadou, A., Sein, T., Kadiltsoglou, I.,
Walsh, T.J., 1998. Tumor necrosis factor alpha enhances antifungal activities of polymorphonuclear and mononuclear phagocytes against Aspergillus fumigatus. Infect. Immun. 66, 5999–
6003.
Roilides, E., Tsaparidou, S., Kadiltsoglou, I., Sein, T., Walsh, T.J.,
1999. Interleukin-12 enhances antifungal activity of human mononuclear phagocytes against Aspergillus fumigatus: implications for
a gamma interferon-independent pathway. Infect. Immun. 67,
3047–3050.
Roilides, E., Lyman, C.A., Sein, T., Gonzalez, C., Walsh, T.J., 2000.
Antifungal activity of splenic, liver and pulmonary macrophages
against Candida albicans and effects of macrophage colonystimulating factor. Med. Mycol. 38, 161–168.
Romani, L., 1999. Immunity to Candida albicans: Th1, Th2 cells and
beyond. Curr. Opin. Microbiol. 2, 363–367.
Romani, L., 2004. Immunity to fungal infections. Nat. Rev. Immunol.
4, 1–23.
Rossi, D., Zlotnik, A., 2000. The biology of chemokines and their
receptors. Annu. Rev. Immunol. 18, 217–242.
Rossychuck, R.A.W., 1994. Management of otitis externa. Vet. Clin.
North Am. Small Anim. Pract. 24, 921–995.
Rybnikar, A., Vrzal, V., Chumela, J., 1998. Protective efficacy of
vaccines against bovine dermatophytosis after double and single
vaccination. Mycoses 41, 83–86.
Sandhu, D.K., Sandhu, R.S., Khan, Z.U., Damodaran, V.N., 1976.
Conditional virulence of a p-aminobenzoic acid-requiring mutant
of Aspergillus fumigatus. Infect. Immun. 13, 527–532.
Smith, P.D., Lamerson, C.L., Banks, S.M., Saini, S.S., Wahl, L.M.,
Calderone, R.A., Wahl, S.M., 1990. Granulocyte-macrophage
colony-stimulating factor augments human monocyte
fungicidal activity for Candida albicans. J. Infect. Dis. 161,
999–1005.
Sohnle, P.G., 1993. Dermatophytosis. In: Murphy, J.W., Friedman,
H., Bendinello, M. (Eds.), Fungal Infections and Immune
Response. Plenum, New York, pp. 27–47.
Sohnle, P.G., Collins-Lech, C., 1980. Relative antigenicity of P.
orbiculare and C. albicans. J. Invest. Dermatol. 75, 279–283.
Sohnle, P.G., Collins-Lech, C., 1983. Activation of complement by
Pityrosporum orbiculare. J. Invest. Dermatol. 80, 93–97.
Sparkes, A.H., Stokes, R., Gruffydd-Jones, T.J., 1993. Humoral
immune response in cats with dermatophytosis. Am. J. Vet.
Res. 54, 1869–1873.
Sparkes, A.H., Stokes, R., Gruffydd-Jones, T.J., 1994. SDS-PAGE
separation of dermatophyte antigens, and western immunoblotting
in feline dermatophytosis. Mycopathologia 128, 91–98.
Sparkes, A.H., Stokes, R., Gruffydd-Jones, T.J., 1995. Experimental
Microsporum canis infection in cats: correlation between immunological and clinical observations. J. Med. Vet. Mycol. 33, 177–
184.
Starkey, R.J., McLoughlin, M.A., 1996. Treatment of renal aspergillosis in a dog using nephrostomy tubes. J. Vet. Intern. Med. 10,
336–338.
Steele, C., Shellito, J.D., Kolls, J.K., 2005. Immunity against the
opportunistic fungal pathogen Pneumocystis. Med. Mycol. 43, 1–
19.
Steinstraesser, L., Koehler, T., Jacobsen, F., Daigeler, A., Goertz, O.,
Langer, S., Kesting, M., Steinau, H., Eriksson, E., Hirsch, T., 2008.
69
Host defense peptides in wound healing. Mol. Med., doi:10.2119/
2008-00002.
Stevens, D.A., Walsh, T.J., Bistoni, F., Cenci, E., Clemons, K.V., Del
Sero, G., Fe d’Ostiani, C., Kullberg, B.J., Mencacci, A., Roilides,
E., Romani, L., 1998. Citokynes and mycoses. Med. Mycol. 36
(Suppl. 1), S174–S182.
Stringer, J.R., Beard, C.B., Miller, R.F., Wakefield, A.E., 2002. A new
name (Pneumocystis jiroveci) for Pneumocystis from humans.
Emerg. Infect. Dis. 8, 891–896.
Suter, M.M., 1993. Intercellular communication in inflammatory and
immune-mediated skin diseases. In: Proceedings 10th European
Society of Veterinary Dermatology Congress, Denmark, pp. 165–
180.
Suzuki, T., Ohno, N., Oshima, Y., Yadomae, T., 1998. Activation of the
complement system, alternative and classical pathways, by Malassezia furfur. Pharm. Pharmacol. Lett. 8, 133–136.
Takahashi, M., Ushijima, T., Ozaki, Y., 1986. Biological activity
of Pityrosporum. II. Antitumor and immune stimulating
effect of Pityrosporum in mice. J. Natl. Cancer Inst. 77,
1093–1097.
Tell, L.A., 2005. Aspergillosis in mammals and birds: impact on
veterinary medicine. Med. Mycol. 43 (Suppl. 1), S71–S73.
Teunissen, M.B.M., 1992. Dynamic nature and function of epidermal
Langerhans cells in vivo and in vitro: a review with emphasis on
human Langerhans cells. Histochemistry 24, 697–796.
Traynor, T.R., Huffnagle, G.B., 2001. Role of chemokines in fungal
infections. Med. Mycol. 39, 41–50.
Uicker, W.C., Doyle, H.A., McCracken, J.P., Langlois, M., Buchanan,
K.L., 2005. Cytokine and chemokine expression in the central
nervous system associated with protective cell-mediated immunity
against Cryptococcus neoformans. Med. Mycol. 43, 27–38.
Van de Wetering, J.K., van Golde, L.M., Batenburg, J.J., 2004.
Collectins: players of the innate immune system. Eur. J. Biochem.
1, 1229–1249.
Wagner, D.K., Sohnle, P.G., 1995. Cutaneous defenses against dermatophytes and yeasts. Clin. Microbiol. Rev. 8, 317–335.
Walsh, T.J., Roilides, E., Cortez, K., Kottilil, S., Bailey, J., Lyman,
C.A., 2005. Control, immunoregulation, and expression of innate
pulmonary host defenses against Aspergillus fumigatus. Med.
Mycol. 43 (Suppl. I), S165–S167.
Wang, J.E., Warris, A., Ellingsen, E.A., Jorgensen, P.F., Flo, T.H.,
Espevik, T., Solberg, R., Verweij, P.E., Aasen, A.O., 2001. Involvement of CD14 and Toll-like receptors in activation of human
monocytes by Aspergillus fumigatus hyphae. Infect. Immun. 69,
2402–2406.
Watanabe, S., Kano, R., Sato, H., Nakamura, Y., Hasegawa, A., 2001.
The effects of Malassezia yeasts on cytokine production by human
keratinocytes. J. Invest. Dermatol. 116, 769–773.
Watson, P.J., Wotton, P., Eastwood, J., Swift, S.T., Jones, B., Day,
M.J., 2006. Immunoglobulin deficiency in Cavalier King Charles
spaniels with Pneumocystis pneumonia. J. Vet. Intern. Med. 20,
523–527.
Watt, P.R., Robins, G.M., Galloway, A.M., O’Boyle, D.A., 1995.
Disseminated opportunistic fungal disease in dogs: 10 cases
(1982–1990). J. Am. Vet. Med. Assoc. 207, 67–70.
Wawrzkievicz, K., Wawrzkievicz, J., 1992. An inactivated vaccine
against ringworm. Comp. Immun. Microbiol. Infect. Dis. 15, 31–
40.
Wilson, S.M., Odeon, A., 1992. Disseminated Aspergillus terreus
infection in a dog. J. Am. Anim. Hosp. Assoc. 28, 447–450.
Wright, J.R., 1997. Immunomodulatory functions of surfactant. Physiol. Rev. 77, 931–962.
70
J.L. Blanco, M.E. Garcia / Veterinary Immunology and Immunopathology 125 (2008) 47–70
Wuthrich, M., Filutowicz, H.I., Klein, B.S., 2000. Mutation of the
WI-1 gene yields an attenuated Blastomyces dermatitidis
strain that induces host resistance. J. Clin. Invest. 106,
1381–1389.
Zanatta, R., Miniscalco, B., Guarro, J., Gene, J., Capucchio, M.T.,
Gallo, M.G., Mikulicich, B., Peano, A., 2006. A case of disse-
minated mycosis in a german shepherd dog due to Penicillium
pururogenum. Med. Mycol. 44, 93–97.
Zasloff, M., 2002. Antimicrobial peptides of multicellular organisms.
Nature 415, 389–395.
Zlotnik, A., Yoshie, O., 2000. Chemokines: a new classification
system and their role in immunity. Immunity 12, 121–127.