Download Canine leishmaniosis – new concepts and insights on an

Review
Canine leishmaniosis – new concepts
and insights on an expanding
zoonosis: part one
Gad Baneth1*, Alexander F. Koutinas2*, Laia Solano-Gallego3*,
Patrick Bourdeau4* and Lluis Ferrer5*
1
School of Veterinary Medicine, Hebrew University, P.O. Box 12, Rehovot 76100, Israel
Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 St Voutyra Street,
54627 Thessaloniki, Greece
3
Royal Veterinary College, Department of Pathology and Infectious Diseases, Hawkshead Lane, North Mymms, Hatfield,
Herts AL9 7TA, UK
4
Ecole Nationale Veterinaire de Nantes, Route de Gachet, BP 40706, Nantes Cedex 03, France
5
Departament de Medicinia i Cirurgia Animals, Universitat Autonoma de Barcelona, 08193-Bellaterra, Spain
2
Recent research has provided new insights on the
epidemiology, pathology and immunology of canine
leishmaniosis (CanL) and its genetic basis. The prevalence of infection in endemic areas is considerably higher
than that of apparent clinical illness. In addition, infection spreads rapidly among dogs in the presence of
optimal conditions for transmission. Infection involves
a variety of granulomatous and harmful immunemediated responses, and susceptibility to the disease
is influenced by a complex genetic basis. These concepts
will be instrumental for devising control programs. This
review, the first in a series of two articles on CanL,
presents an updated view on progress in elucidating
the epidemiology and pathogenesis of this challenging
disease, and the second part focuses on advances in
diagnosis, treatment and prevention.
An expanding complex zoonosis
Canine leishmaniosis (CanL) is caused by Leishmania
infantum, or its New World synonym Leishmania chagasi,
and is a major potentially fatal zoonotic infection in regions
of Europe, Africa, Asia and America. The domestic dog is
the main reservoir of human infection, and phlebotomine
sand flies are the biological vectors of this protozoal disease. It has been estimated that at least 2.5 million dogs
are infected in southwestern Europe alone [1], and the
disease is spreading north into the foothills of the Alps and
the Pyrenees [2]. The number of infected dogs in South
America also is estimated in millions, and there are high
infection rates in some areas of Venezuela and Brazil,
where a high prevalence of canine infection is associated
with high risk of human disease [3]. The term ‘CanL’ used
in this review describes infection with L. infantum, which
affects both the viscera and skin of dogs, but does not
encompass canine tegumentary leishmaniosis caused by
Leishmania spp. in the New World or Leishmania tropica,
a rare cause of disease in dogs in the Old World [4].
Corresponding author: Baneth, G. ([email protected]).
The authors are members of the LeishVet European Clinical Standardization
Group.
*
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CanL is a complex disease that has a high prevalence of
infection, involving as much as 63%–80% of the population
[5–7], and is accompanied by a lower rate of apparent
clinical disease. Dogs that do show disease manifest different clinical signs and variable degrees of severity. Substantial research published recently on the pathogenesis of
CanL and immune responses during infection has contributed considerably to our understanding of this complex
zoonosis and its epidemiology [5,8,9]. Current studies have
revealed new facts about the prevalence and spread of
infection and established novel concepts on its evolution
and dynamics [2,3,5,6,8]. These new insights impact on
efforts to prevent and control the disease and its spread
into human populations.
Epidemiology of CanL in endemic regions – major
concepts
Two major concepts have arisen from epidemiological
research with molecular diagnostic techniques for CanL.
The first concept is that infection in endemic areas is
widespread, but not all infected dogs develop the disease.
An early study employing cellular immunity and serological assays demonstrated that a spectrum of immune
responses to Leishmania infection is present among
asymptomatic dogs in an endemic area [10]. An experimental model of infection showed that dogs mounted variable
immune responses, and although some developed the disease, other infected dogs remained asymptomatic over a 5year observation period [11]. Studies using PCR in endemic
areas have confirmed that the prevalence of infection in
dogs is much higher than the proportion that actually
develops symptomatic disease [5,7,12]. A study of 100 dogs
from a municipal pound in the island of Mallorca, Spain,
indicated that 13% had apparent clinical disease, 26% were
seropositive and 63% were found to be positive for Leishmania DNA when tested by PCR [5]. Of 73 clinically
healthy hunting dogs in Greece, 12.3% tested positive by
using serology, whereas 63% tested positive by using PCR
[6]. Longitudinal studies in endemic areas have shown that
the natural history of the infected dogs can evolve in
1471-4922/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2008.04.001 Available online 29 May 2008
Review
different ways [8,13]. Two major patterns of progression
have been documented. In some dogs severe clinical signs
of the disease appear shortly after infection. It is thought
that animals with severe disease are unable to develop an
effective cellular immune response, although they have a
strong but ineffective humoral response [14,15]. A second
group of dogs remains infected for a long period of time
(years or lifelong), but they are able to avoid the appearance of lesions and clinical disease [11,14]. However, a
change in the health status of these dogs or, for instance,
the administration of an immunosuppressive drug or a
severe immunosuppressive disease can lead to the activation of a latent infection and the development of clinical
signs.
The second concept is that when favorable conditions for
disease transmission (such as high vector sand-fly and
canine-host densities) are present, the infection spreads
quickly and extensively among the dog population
[8,16,17]. The evolution of a cohort of naı̈ve dogs exposed
to three consecutive transmission seasons in Naples, Italy,
indicated that at the end of the study, 97.3% of the dogs
were positive by bone-marrow-nested PCR and 75.7% were
seropositive. This extremely high prevalence of infection
was attributed to a high density of Phlebotomus perniciosus vectors in the disease focus and the lack of control
measures to protect the dogs [8].
These two concepts (Box 1) have clarified that clinical
CanL is only the tip of the iceberg in endemic areas, and the
majority of the population is exposed and becomes infected
without showing clinical evidence of disease or serum antiLeishmania antibodies (Figure 1).
Canine leishmaniosis in nonendemic regions
Canine visceral leishmaniosis is often diagnosed in nonendemic countries, and the need for detection and treatment
of this disease also is imperative in northern Europe and
other areas where no natural transmission occurs [18–20].
This mostly is due to the increased travel of dogs in and out
of endemic areas. A study from Holland found that about
58 000 dogs are taken yearly to southern Europe with their
owners on vacation, and the risk of acquiring leishmaniosis is 0.027%–0.23% [18]. Importation and relocation of
dogs from endemic countries is responsible for the growing
numbers of dogs with this disease in Germany [19]. The
Pet Travel Scheme has eased the movement of dogs from
EU countries, the USA and rabies-free islands to the UK.
The change in quarantine restrictions and the increased
numbers of traveling and imported companion animals
has raised serious concerns about the introduction of CanL
into the UK [20]. The risk for autochthonous transmission
from traveled dogs in the absence of sand-fly vectors is
probably small; however, infection has been described in
Box 1. New concepts on the epidemiology of canine
leishmaniosis
Infection in the canine population in endemic areas is widespread,
and the rate of infected dogs is much higher than the fraction that
shows clinical illness.
Infection spreads quickly and extensively among the dog population when environmental conditions for transmission are optimal.
Trends in Parasitology
Vol.24 No.7
Figure 1. A schematic presentation of the distribution of leishmaniosis among the
canine population in an endemic focus, as seen in a cross-sectional study. The
peak of the pyramid (orange) consists of a relatively small subset of dogs showing
clinical signs of the disease. Seropostive dogs with no signs of disease represent a
second subset (beige). Most of the dogs in the clinical disease and the
asymptomatic seropositive subsets would be PCR+. A third subset includes
asymptomatic PCR+ but seronegative infected dogs (purple), and a fourth subset
includes dogs that are seronegative and do not harbor parasites (blue). A minority
of dogs might be seropositive and PCR- (not shown in this model). A study from
the Island of Mallorca, Spain [5], showed that 13% of the dogs had clinical disease,
26% were seropositive, 63% were PCR positive and 67% had evidence of infection
either by serology or by PCR.
dogs with no travel history living together with imported
infected dogs, pups born to infected dams or animals
receiving blood transfusions from infected canine donors
[21–23].
Canine visceral leishmaniosis in the USA was reported
rarely before 2000, when infection was implicated as the
cause of mortality in a foxhound kennel in New York State
[24]. A serosurvey revealed that canine infection, predominantly involving foxhounds, is present in 18 states and
two Canadian provinces [25]. No autochthonous human
cases were reported, and sand-fly transmission in the USA
has not been demonstrated. This unique situation is currently being investigated with major concern regarding the
possibility of spread to humans.
Update on canine pathology during the disease
Canine visceral leishmaniosis is a multisystemic disease
with variable clinical signs [4,26–29]. The majority of dogs
are presented with poor body condition, generalized muscular atrophy, lymphadenomegaly and excessive skin scaling (Table 1 and Figure 2). The typical histopathological
finding in their tissues is a granulomatous inflammatory
reaction associated with the presence of Leishmania amastigotes within macrophages.
The dermal changes in CanL include exfoliative, ulcerative, nodular and pustular dermatitis [27,30,31]. Extracellular matrix assessment in the skin of symptomatic dogs
demonstrated a decrease in collagen type I and an increase
in collagen type III fibers proportional to the severity of the
skin lesion and tissue destruction [32]. Dermal lesions in
CanL are not necessarily provoked by an inflammatory
response to the physical presence of parasites in the skin
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Trends in Parasitology Vol.24 No.7
Table 1. Clinical findings in dogs with symptomatic canine
leishmaniosis
Clinical examination finding
Skin disease
Enlargement of lymph nodes
Eye disease
Pallor of mucous membranes
Enlarged spleen
Cachexia
Fever
Nose bleeding (epistaxis)
Abnormal nails (onychogryposis)
Percentage of
symptomatic dogs
81%–89%
62%–90%
16%–81%
58%
10%–53%
10%–48%
4%–36%
6%–10%
20%–31%
Refs
[4,27]
[4,26,27]
[4,27,41,42]
[27]
[4,26,27]
[4,26,27]
[26,27]
[26,27]
[4,26,27]
because normal-looking skin also harbors large numbers of
parasites in symptomatic dogs. Furthermore, a similar
parasite load was found in skin with macroscopic lesions
and normal-looking skin from symptomatic dogs [30,31].
The marked enlargement of lymph nodes in CanL is
caused by the increased number and size of lymphoid
follicles and the marked hypertrophy and hyperplasia of
medullary macrophages in the cords and sinuses [33]. The
parasitic load in enlarged lymph nodes frequently is not
correlated with the type and severity of lesions in other
organs [33,34]. The enlargement of the spleen in CanL is
associated with increased monocyte and macrophage cellularity and changes in the microvasculature structure
with abundant pulp venules and veins and increased
reticular fibers [35].
The kidneys are affected in virtually all dogs with CanL,
and renal disease might be the only apparent abnormality
in infected dogs. A study of seropositive dogs from Brazil
identified glomerulonephritis in all 55 dogs, of which 13
were asymptomatic. Interstitial nephritis was present in
78% of the dogs, and glomerular deposition of parasite
antigen was seen in 91% [36]. Renal disease can progress
from asymptomatic proteinuria to nephrotic syndrome or
chronic renal failure with glomerulonephritis, tubulointerstitial nephritis and amyloidosis [27]. Glomerulonephritis
frequently is associated with the renal deposition of
immune complexes [37]. Despite the high prevalence of
kidney pathology, azotemia with the elevation of serum
creatinine and urea typical of kidney failure are evident
only when the majority of nephrons are dysfunctional,
which happens rather late during disease progression.
Joint and bone lesions frequently are present in infected
dogs [38]. A study on skeletal lesions in CanL found that of
58 dogs with CanL, 45% had gait abnormalities. Both
erosive and nonerosive polyarthritis were observed with
Leishmania amastigotes detected by microscopy in the
synovial fluid [38]. Affected bones typically have periosteal
and intramedulary proliferative lesions with frequent cortical and medullary osteolysis [37–39]. Progressive muscle
atrophy is associated with chronic polymyositis characterized by the presence of mononuclear infiltrates with Leishmania amastigotes, neutrophilic vasculitis and IgG
immune complexes in muscle tissues in conjunction with
serum anti-myofiber antibodies [40].
Ocular lesions are present in 16% to 80.5% of dogs with
symptomatic CanL [26,27,41,42]. These consist of anterior
uveitis, conjunctivitis, dry keratoconjunctivitis, blepharitis
or a combination of these. Eye lesions are the sole presenting complaint in up to 16% of symptomatic cases. In
dry keratoconjunctivitis, inflammatory infiltrates located
around the lacrimal ducts cause secretory retention and a
decrease in tear production [42].
Nose bleeding (epistaxis), hematuria and hemorrhagic
diarrhoea in CanL are associated with tissue ulceration
and alterations in primary and secondary hemostasis.
Hemostatic disorders described in CanL include platelet
aggregation abnormalities leading to platelet dysfunction,
low platelet number, decreased coagulation factor activities and fibrinolysis [43]. Profuse epistaxis can appear as
Figure 2. Clinical manifestations of symptomatic canine leishmaniosis: (a) purulent keratoconjunctivitis with periocular dermatitis, (b) facial skin lesions with multifocal
exfoliative dermatitis, (c) epistaxis, (d) onychogryposis and (e) skin ulceration on the ear.
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Review
the only presenting sign of disease and can be a cause of
death because of uncontrollable blood loss. The underlying
mechanism involves pyogranulomatous or lymphoplasmacytic rhinitis, (with or without ulceration), thrombocytopathy and hyperglobulinemia [44]. Anemia is present in
the majority of symptomatic dogs because of chronic renal
disease or decreased erythropoiesis caused by chronic disease and can be aggravated by blood loss or immunemediated destruction of red blood cells [26,27].
Genetics of susceptibility and resistance to canine
visceral leishmaniosis
Evidence that the host’s genetics could play a major role in
susceptibility or resistance to CanL has come from different sources. Investigations on the genetics of human visceral leishmaniasis have indicated that several genes are
involved in disease susceptibility. Studies on kala-azar
(visceral leishmaniosis) caused by Leishmania donovani
in endemic areas in Sudan showed that although more
than 90% of the villagers showed immunological evidence
of infection, only 30% developed kala-azar. Candidate gene
testing revealed linkage to polymorphism in the natural
resistance-associated macrophage protein 1 (NRAMP1)
gene later named the Slc11a1 gene [45]. The Slc11a1 gene
encodes a proton divalent cation antiporter with a role in
regulating macrophage function – including upregulation
of chemokine and cytokine genes, such as tumor necrosis
factor (TNF) and interleukin-1b (IL-1b) – and induction of
nitric oxide synthase [46]. These investigations demonstrated that in humans there is a complex genetic basis
for susceptibility to developing visceral leishmaniasis,
which involves several mechanisms and additional genes
such as the interleukin-4 (IL-4) and interferon-g receptor
[47].
Epidemiological studies in canine populations suggested
a role for genetics in the resistance to developing the disease
(Box 2). The Ibizian hound, a local breed from the Balearic
islands, was reported to develop a primarily cellular
immune response, detected by the leishmanin skin test,
and very rarely develop clinical disease [48].
So far, two genes have been implicated in susceptibility
to CanL. In a cohort of Brazilian mongrel dogs, a study on
the genetic variation in major histocompatabillity complex
class II (MHC II), termed ‘the dog leukocyte antigen (DLA)
system’ in the dog, has detected a significant association
between the presence of the DLA–DRB1*01502 allele and
susceptibility to CanL [49]. In another study haplotypes
of the canine Slc11a1 gene were identified as associated
with susceptibility to CanL in Spain [50]. Moreover, singlenucleotide polymorphism mutations in the promoter
Box 2. Evidence that resistance or susceptibility to canine
leishmaniosis is genetic
The dog breed Ibizian hound typically develops a strong cellular
immune response to leishmaniosis and rarely shows clinical
disease in Mallorca [48].
Susceptibility to leishmaniosis is associated with a major
histocompatabillity complex class II (MHC II) genotype [49].
Certain Slc11a1 (formerly NRAMP1) genotypes are associated
with susceptibility to leishmaniosis [50].
Trends in Parasitology
Vol.24 No.7
region of the Slc11a1 gene were associated with susceptibility to CanL, as also described in humans [46], and one
haplotype (TAG-8-141) was associated with boxers’ predisposition to CanL [51]. These recent findings associating
genetics with susceptibility to CanL are probably only
pieces of a more complex multigenic puzzle that determines the individual dog’s natural resistance to infection.
Nevertheless, susceptibility to developing the disease also
is influenced by non-genetic factors such as nutritional
status, concomitant infections and parasitism, parasite
virulence and previous exposure to CanL.
Cellular and humoral immune responses
A spectrum of innate and acquired immune responses to L.
infantum infection is mounted by the canine host [10,52].
The two opposite extreme poles of this spectrum are protective immunity that is T-cell mediated and susceptibility
to overt disease associated with a marked nonprotective
humoral immune response and a depressed cell-mediated
immunity (CMI) [14,15]. Based on in vitro and in vivo
studies, it is widely accepted that macrophages play a
central role in the control of Leishmania infection. Cytokines such as interferon-g (IFN-g), IL-2 and tumor necrosis
factor-a (TNF-a) secreted by activated T cells induce
macrophage anti-leishmanial activity. Nitric oxide (NO)
produced by macrophages has been found to be the principal effector molecule mediating intracellular killing of
Leishmania amastigotes by apoptotic cell death controlled
by proteasome inhibitors [53].
Cellular immune responses
It is now widely accepted that protective immunity against
Leishmania parasites is mediated by CD4+ T helper1 (Th1)
cellular responses [54]. In contrast to the findings in experimental murine cutaneous leishmaniasis, visceral disease
in humans, dogs and experimental rodents does not show a
clear Th1/Th2 dichotomy pattern [54]. Leishmania infantum appears to induce mixed Th1 and Th2 responses in
which the control of parasite replication, disease progression or cure are determined by the balance between
these two dichotomous directions (Figure 3).
Understanding the profile of cytokines expressed in
CanL is complex because of the limited number of studies reported, the broad spectrum of clinical disease
stages investigated, the different tissues analyzed and
the differences between experimental and natural infection. In naturally infected dogs expression levels of
IFN-g in the bone marrow or spleen were similar in
symptomatic and asymptomatic dogs [9,55,56], and a
predominant induction of both IFN-g (Th1 cytokine)
and IL-4 was evident in stimulated Leishmania antigen-peripheral blood mononuclear cells from asymptomatic dogs [57]. Spleen cells from infected dogs showed a
predominant expression of IL-10 that was positively
correlated with parasitic load and clinical status severity
[56]. IL-10 is considered a regulator of the Th1 activity
that maintains balance between Th1 and Th2 responses
and inhibits the microbicidal activity of infected macrophages [15,58].
Longitudinal experimental CanL infection studies
mostly have indicated a mixed Th1 and Th2 pattern
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Trends in Parasitology Vol.24 No.7
Figure 3. A view of the complex interaction between the Th1 and Th2 responses in visceral leishmaniosis focused on current understanding of these responses in CanL. Mixed
Th1 and Th2 responses occur in infected dogs, and it is assumed that the balance between these responses tilts in the Th2 direction in dogs that succumb to clinical illness,
whereas a stronger protective Th1 activity directs to resistance. The cytokines IFN-g, IL-2 and TNF-a induce macrophage activation and NO killing of parasites in the Th1
response. By contrast, IL-10, TGF-b and IL-4 are involved in the parasite dissemination associated with increased B-cell (B) and plasma-cell (P) activity and hyperglobulinemia
related to the Th2 response. Th1 responses are downregulated by IL-10 produced by T-regulatory (Treg) cells. IL-10 also is produced by cells of the Th1 lineage and is proposed
to be beneficial in limiting secondary immunopathology triggered by infection, but it also prevents sterile immunity and allows persistence of low-level infection.
[54]. Cytokine expression in blood showed an initial asymptomatic phase with an absence of or low expression during
a period of ‘silent establishment of parasite’ followed by a
short-term production of Th1-type cytokines before the
appearance of clinical signs [59]. In another study cytokine
and chemokine expression in the spleen revealed an initial
elevation in IL4 one month postinfection followed by an
increase in the Th1-associated IFN-g and chemokines Tbet, IP-10 and RANTES [54].
The cellular basis and mechanisms for the development of T-cell unresponsiveness in CanL are not understood fully. The majority of infected dogs are likely to
develop positive specific CMI expressed as proliferation
of lymphocytes stimulated in vitro by Leishmania antigen or in vivo by a positive skin test early in infection.
However, as the disease progresses in susceptible dogs,
these responses diminish. Blood parasite load and Leishmania-specific CMI were shown to be inversely correlated during a longitudinal follow-up of experimentally
infected dogs [60]. It has been postulated that CMI
unresponsiveness in progressive disease is due to the
decrease in peripheral CD4+ T-cell numbers or the
decreasing expression of co-stimulatory molecules such
as B7 [12,15,61,62].
Humoral immune response
Canine leishmaniosis frequently is associated with a
marked humoral response, which is not protective and
signifies failure to control the infection. The levels of
Leishmania-specific immunoglobulins detected in symptomatic dogs are greater than those detected in infected but
asymptomatic dogs, and a marked association was found
between these levels, the clinical status and tissue parasite
density [63].
328
The levels of the canine IgG subclasses IgG1 and IgG2
have been studied extensively in an attempt to establish a
correlation between the subclass level, the type of Th
response and the clinical outcome of infection. Studies
evaluating IgG1 and IgG2 using polyclonal antibodies
frequently have reached conflicting outcomes, possibly
because of a lack of specificity of commercial reagents
[58]. Moreover, investigations employing monoclonal antibodies to canine IgG1 and IgG2 have shown a steady
increase in the production of both subclasses during
natural and experimental CanL without indicating a practical use for these levels [17,54].
Transmission of canine leishmaniosis
The natural cycle of Leishmania infection involves a phlebotomine sand-fly vector in which promastigotes replicate
after transformation from intracellular amastigotes taken
during the bloodmeal. There are numerous sand-fly
species, and only a minority of these are competent vectors
of CanL [64]. Dogs with symptomatic as well as asymptomatic infections are infectious to sand flies. However,
infectiousness appears to be higher in dogs with clinical
disease. In a study from Brazil, 28% of seropositive symptomatic dogs were infectious to Lutzomyia longipalpis sand
flies, whereas only 5.4% of the asymptomatic dogs were
infectious [65]. Infectiousness of dogs is related to immunosuppression as manifested by levels of circulating Th
cells. An inverse relationship was found between the CD4+
T-cell count in naturally infected dogs and their infectiousness to sand flies, as found for HIV+ people coinfected with
L. infantum [62,66].
Although sand flies are the only biologically adapted
vectors of leishmaniosis, a possible role in transmission for
other hematophagous ectoparasites, such as ticks and
Review
fleas, has been proposed. Transmission of leishmaniosis to
laboratory hamsters was achieved under experimental
conditions when Rhipicephalus sanguineus ticks and Ctenocephalides felis fleas from naturally infected dogs were
macerated and orally inoculated into hamsters [67,68].
However, the significance of transmission to dogs by these
arthropods has not been verified in natural conditions.
Congenital transmission of visceral leishmaniasis from
infected mother to offspring is reported in humans and has
been studied experimentally in mice. Canine vertical infection was demonstrated experimentally in puppies born
to infected male and female beagles, and transmission was
assumed to be transplacental [22]; however, investigations
in naturally infected dogs have reported conflicting outcomes [69,70]. In addition to the option of transmission by
blood transfusion [23], direct dog-to-dog transmission by
contact has been suggested to take place in the USA in an
effort to explain the spread of CanL among kennel foxhounds in the absence of proven sand-fly vectors and
human infection [25]. Modalities of infection other than
sand-fly transmission should be further studied; however,
at present it is not known whether they play an important
role in the epidemiology of CanL.
Summary
Recent research has revealed the extensive distribution
and expansion of CanL in large areas of the world. The slow
and progressive nature of this severe zoonosis and the high
infectiousness of dogs to sand flies are of major concern. It
is clear that control efforts and preventative measures
should encompass total dog populations in endemic regions
to be effective. The second part of this review will address
updates on diagnosis, treatment, prevention and public
health concerns. It will integrate concepts described in this
part with further insights leading to improved possibilities
for future control programs.
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