Review

Methods for diagnosis of canine leishmaniasis and

immune response to infection
C. Maia, L. Campino
*
Unidade de Leishmanioses, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Rua da Junqueira 96, 1349-008 Lisboa, Portugal
Received 21 April 2008; received in revised form 22 July 2008; accepted 25 July 2008
Abstract
Canine leishmaniasis (CanL) caused by Leishmania infantum (syn. L. chagasi, in Latin America), which is transmitted by the
bite of phlebotomine sand ies, is endemic and affects millions of dogs in Europe, Asia, North Africa and South America. It is an
emergent disease in North America. Early detection and treatment of infected animals may be critical in controlling the spread of the
disease and is an essential part of human zoonotic visceral leishmaniasis control. The laboratory diagnosis of CanL still poses a
challenge, despite progress made in the development of several direct and indirect methods. An effective diagnosis test, apart of
being able to conrm a clinical suspicion in a single patient as well as to detect infection in asymptomatic dogs, should have high
sensitivity, specicity and reproducibility; it must be simple, easy to perform, non-expensive, feasible in regional laboratories or
adaptable for eld conditions. Ideally, it should detect all Leishmania-infected dogs, preferentially using non-invasive collection of
biological samples. In this paper we review the advantages and shortcomings of the available procedures for CanL diagnosis in the
different phases, e.g. pre-patent and patent period of the infection and methods to determine the related immune response.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Dog; Leishmaniasis; Diagnostics; Immune response
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2. Laboratory diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2.1. Direct diagnostics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2.1.1. Microscopic examination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2.1.2. Histophatology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
2.1.3. Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
2.1.4. Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
2.1.5. Parasite isolation in laboratory animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1.6. Xenodiagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1.7. Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1.8. Real-time PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
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Veterinary Parasitology 158 (2008) 274287
* Corresponding author. Tel.: +351 213652600; fax: +351 213632105.
E-mail address: campino@ihmt.unl.pt (L. Campino).
0304-4017/$ see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2008.07.028
2.2. Indirect diagnostics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
2.2.1. Humoral immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
2.2.2. Indirect immunouorescent antibody test (IFAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
2.2.3. Counterimmunoelectrophoresis (CIE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
2.2.4. Immunodiffusion assay (IDA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
2.2.5. Direct agglutination test (DAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
2.2.6. Enzyme-linked immunosorbent assay (ELISA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
2.2.7. ELISA-recombinant antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
2.2.8. Immunochromatographic rapid tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
2.2.9. Western blotting (WB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
2.2.10. Flow cytometry (FC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
2.3. Cellular immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
2.3.1. Montenegro test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
2.3.2. Lymphocyte proliferation assay (LPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
2.3.3. Interferon-g (IFN-g) cytophatic effect inhibition bioassay (IFNB) . . . . . . . . . . . . . . . . . . . . . . . 283
3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
1. Introduction
Canine leishmaniasis (CanL) caused by Leishmania
infantum (syn. L. chagasi, in Latin America) is
transmitted by the bite of phlebotomine sand ies.
This severe zoonosis affects millions of dogs in Europe,
Asia, North Africa and South America and is an
emergent disease in North America (Rosypal et al.,
2003; Duprey et al., 2006).
Despite the lack of pathognomonic manifestations,
the commonest clinical signs are cutaneous alterations,
local or generalized lymphoadenomegaly, loss of body
weight, liver and spleen enlargement, glomerulopathy,
ocular lesions, epistaxis, onycogryphosis and lameness.
Atypical forms include monoclonal gammopathy,
chronic colitis, haemostatic alterations and disorders
of the cardiovascular, respiratory and musculo-skeletal
systems (Blavier et al., 2001). In this way, Leishmania
infection shares many of the clinical and pathological
features with other canine diseases (Barrouin-Melo
et al., 2005; Gomes et al., 2008).
However, the precise diagnosis of CanL may also
prove complex because not all the animals infected with
metacyclic promastigotes through a vector bite develop
clinical manifestations (Alvar et al., 2004). This fact
cannot be neglected since asymptomatic dogs are
infectious to phlebotomine vectors, although they seem
to constitute a lower risk than the symptomatic ones
(Campino, 2002). Hence, early detection of infected
animals, particularly before appearance of symptoms
and, in some cases, even before seroconversion, may be
critical in controlling the spread of the disease and has
become an essential part of human leishmaniasis control.
Laboratory diagnosis of CanL can be performed
using different methods: parasitological, with detection
of the parasite or its DNA direct diagnostics and
immunological tests indirect diagnostics. Depending
on the reasons for attempting laboratory diagnosis, such
as epidemiological studies, the conrmation of Leish-
mania infection or therapeutic control, different levels
of sensitivity, specicity, cost and ease of testing may be
required since each diagnostic method has advantages
and shortcomings and each should be selected in light of
these parameters (Schallig et al., 2004; Baneth and
Aroch, 2008; Gomes et al., 2008).
2. Laboratory diagnosis
2.1. Direct diagnostics
2.1.1. Microscopic examination
Conclusive diagnosis can be made using microscopic
observation of Leishmania amastigotes in stained
smears of infected organs/tissues, namely bone marrow
(BM), lymph node (LN), skin (SK), or peripheral blood
(PB). The majority of samplings are obtained using
overly invasive procedures, generally not useful for the
detection of the parasite in asymptomatic dogs (Alvar
et al., 2004). In smears stained with Giemsa or
Leishman stain, amastigotes found free or intracellu-
larly in monocytes, macrophages and neutrophils,
present as oval or round bodies with 24 mm in
diameter. Their cytoplasm appears pale blue, with a
relatively large nucleus which stains red. In the same
plane as the nucleus, but at right angles to it, is a deep
red or violet, rod-like body, the kinetoplast.
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 275
According to Ferrer (1999) and Alvar et al. (2004),
the microscopy of BM and LN smears has a sensitivity
of 6075% and 3050%, respectively. However, in a
study conducted by Rosypal et al. (2005), amastigotes
were observed in 93% of BM and LN samples from L.
infantum naturally infected dogs; on the other hand, in
experimentally infected dogs BM samples were more
likely to show Leishmania organisms than LN aspirates.
The density of the parasites in the smear can be
estimated by counting the number of amastigotes in
relation to the white blood cell counts. A logarithmic
scale from 0 (without parasites) to +6 (greater than 100
parasites per microscopic eld) can be applied
(Ciaramella et al., 1997; Saridomichelakis et al., 2005).
Taking into account that the number of organisms in
each clinical sample is often variable, increasing the
time devoted to observation and microscopic elds
evaluated, may improve the sensitivity of the method.
Liarte et al. (2001) also described a direct and fast
uorescent method, the Quantitative Buffy Coat
(QBC
1
) which presented a high sensitivity for
detection of amastigotes in PB of dogs infected with
CanL. After separation of the white blood cells by
centrifugation and using a microhematocrit tube coated
with a DNA stain, acridine orange plus potassium
oxalate, amastigotes were visualised using a uorescent
microscope in 30 of the 31 animals (97%) considered
positive by serology and in 28 of the 28 dogs with
parasites identied in other tissues. However, authors
concluded that experiments on uninfected dogs from
endemic and non-endemic areas should be performed to
evaluate the specicity of QBC
1
.
Cenini et al. (1989) developed a technique where the
amastigotes viability could be checked, using a
differential count of living and dead forms. After
staining amastigotes with uorescein diacetate and
ethidium bromide under blue light, living parasites
uoresced as green with the dead ones staining as
orange. This technique, as QBC
1
, requires an
expensive uorescence microscope but it would be
useful for follow-up treatment.
2.1.2. Histophatology
Histophatological analysis of infected organs stained
with hematoxylin and eosin (HE) has also been used to
detect the presence of parasites. Long searches may be
required to see the amastigotes since such parasitic
organisms are frequently not easily recognised (Xavier
et al., 2006). Although the low sensitivity of this
technique is recognised, Moreira et al. (2007) found that
popliteal LN material was the most effective for
detecting the parasite (43.9%, 40.0% and 39.13% in
symptomatic, oligosymptomatic and symptomatic
dogs, respectively) followed by the spleen (SP) and
BM parafn-embedded sections. Although those results
were obtained from samples collected post-mortem,
LN, BM and SK biopsies had been used in clinical
practice. Bourdoiseau et al. (1997b) evaluated HE for
the demonstration of Leishmania amastigotes in 34 skin
biopsies from seropositive dogs and obtained a
sensitivity of 32.35%.
2.1.3. Immunohistochemistry
Immunohistochemical (IHC) approaches such as
immunoperoxidase or direct immunoorescence of
tissues can be used as a supplementary tool to conrm
the diagnosis on HE, particularly in organs which do not
have high parasite load. The Leishmania IHC method
for amastigote detection in formalin-xed parafn-
embedded tissues forms can be applied using strepta-
vidinperoxidase/biotin system with canine hyperim-
mune serum as the primary antibody or by the use of
polyclonal or monoclonal anti-Leishmania antibodies
(Bourdoiseau et al., 1997b; Tafuri et al., 2004). Xavier
et al. (2006), using SK biopsy samples from different
anatomical regions, obtained a higher sensitivity
(62.1%) using IHC than using HE (44.8%). Similar
results were obtained by Moreira et al. (2007); these
authors used direct immunouorescence in popliteal
LN with a specicity of 100% and a sensitivity of
92.68%, 60% and 73.91% in symptomatic, oligosymp-
tomatic and asymptomatic dogs, respectively. This
technique is a useful supplement to conrmdiagnosis of
CanL when the parasites are not clearly identiable
under the microscope and when the histological pattern
clearly indicates the disease.
It is important to take into consideration that in
microscopic observation, histopathological and immu-
nohistochemical identication of amastigotes requires
considerable expertise and training and is subject to the
ability of the observer. These methods can also yield
either false negative results because their sensitivity
depends on the parasite load, or false positive results
because other artefacts viewed by light microscopy can
be erroneously considered as amastigotes (Alvar et al.,
2004; Baneth and Aroch, 2008; Gomes et al., 2008).
2.1.4. Culture
In vitro culture of different tissues can improve the
sensitivity of parasite detection. Not all Leishmania
strains grow at the same rate and not all tissues and
organs from the same dog have a similar parasite load.
Replicate inoculation of several tubes will increase/
improve the diagnostic sensitivity (Evans, 1989). The
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 276
culture media used may be monophasic such as
Schneiders insect medium, M199, RPMI, Graces
medium or diphasic such as NovyMcNealNicolle
medium or Brain Heart Infusion. Media are inoculated
with one or two drops of aspirate or a homogenised/
ground organ fragment and incubated at a temperature
between 228 and 26 8C. Cultures are examined weekly
for the presence of promastigotes. Parasites may be seen
by the 1st week although weekly subcultures in fresh
medium may be required to visualise them. In a study
performed by Maia et al. (2007), 77.1% of the cultures
from SP, LN, BM and LV samples collected post-
mortem became positive in the 1st week and 23% in the
2nd3rd weeks, reinforcing the need to subculture at
least up to the 3rd week. In general, a culture is
considered to be negative, following four successive
negative subcultures.
According to Madeira et al. (2006) and Maia et al.
(2007) SP, LN and BM are the biological materials with
a higher rate of positive cultures. Due to the satisfactory
results of SP culture, some authors (Barrouin-Melo
et al., 2005; Rosypal et al., 2005) supported the use of
SP as the organ of choice for the parasitological
diagnosis of Leishmania infection. However, the
invasive procedure to collect the biological material
is a valid reason for avoiding it, using popliteal LN
aspirate instead; in dogs without detectable LN, BM is a
suitable alternative for diagnosis.
Although 100% specic, cultures are nowadays less
used for diagnosis due to their drawbacks such as delay
in the result, susceptibility to microbiological contam-
ination, dependence on the parasite load and sometimes
difculty to perform due to poor adaptation of isolate to
the medium. On the other hand, is still required to obtain
sufcient number of parasites for isoenzymatic identi-
cation, to use as an antigen for immunological
diagnosis, for experimental infection models as well
as for in vitro drugs screening or even molecular
identication.
2.1.5. Parasite isolation in laboratory animals
The presence of the parasite can be demonstrated
after inoculation of golden hamster (Mesocricetus
auratus). Although animal inoculation is not usually
employed as a diagnosis test, since several months may
be required to obtain a result, it can be used with the
clinical material collected when there is a substantial
risk of contamination, especially under eld collection
(Alvar et al., 2004). After inoculation, the animal is
examined weekly for signs of infection, such as
hepatosplenomegaly. Amastigotes can be harvested
from the SP or LV.
2.1.6. Xenodiagnosis
Xenodiagnosis is a technique for the detection and
isolation of a pathogen using its natural arthropod
vector. Infectivity of dogs to sand ies has been
investigated in the Mediterranean basin using speci-
mens of the highly competent vectors Phlebotomus
perniciosus and P. ariasi (Rioux et al., 1972; Gradoni
et al., 1987; Molina et al., 1994) while in South America
most studies have been carried out with the natural
vector Lutzomyia longipalpis (Sherlock, 1996; Travi
et al., 2001). The infection rate of Phlebotomus reported
by different authors (Gradoni et al., 1987; Molina et al.,
1994) ranges between 21.9% and 92% while infection
of L. longipalpis ranges from 13% to 29% (Sherlock,
1996). Travi et al. (2001) postulated that this
discrepancy might be due to the fact that the threshold
of parasites for infecting P. perniciosus is lower than
that necessary to infect L. longipalpis or because dogs in
Europe, due to better nutrition, remain asymptomatic
despite having higher parasite burdens than the under-
nourished Latin America dogs, and consequently are
more infective to P. perniciosus. Xenodiagnosis,
although it has been used to solve important epide-
miological questions about the role of the clinical status
and drug treatment, is rarely used in CanL diagnosis
since it can only be carried out in specialised
laboratories, where a well-established colony of sand
ies is available.
2.1.7. Polymerase chain reaction (PCR)
PCR-based methods applied for Leishmania detec-
tion are more reliable for determining the presence and
identication of the parasite not only in active cases, but
also for monitoring parasitological cure after che-
motherapy. PCR for the detection of Leishmania DNA
can be carried out with a broad range of clinical
specimens: whole blood, buffy coat, BM, LN, SK or
conjunctiva swabs (Fisa et al., 2001; Andrade et al.,
2002; Strauss-Ayali et al., 2004; Maia et al., 2006;
Ferreira et al., 2008). According to Maia et al. (2007)
LN-PCR is useful as a rst line primary diagnosis or
therapeutic follow-up and BM-PCR for dogs without
detectable popliteal LN. More recently some authors
defended the use of conjunctiva swabs for diagnosis and
treatment follow-up (Ferreira et al., 2008). Leishmania
DNAwas also found in several other biological samples
which are regularly not used for the routine diagnosis,
such as SP, LV, lung, heart, penis, vagina, testis, semen,
uterus, placenta, kidney, intestine, milk and urine
(Andrade et al., 2002; Reithinger et al., 2002a;
Barrouin-Melo et al., 2005; Diniz et al., 2005; Rosypal
et al., 2005; Franceschi et al., 2006). A PCR-based
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 277
diagnostic test using peripheral biological samples are
more simple to perform and by far more acceptable to
dog owners than more invasive collection of material
biopsies, such as BM or LN. In epidemiological
surveys, whole blood spotted on lter paper for PCR
can also be used to complement serological results
(Maia et al., 2007).
Asingle negative PCRresult in a clinically suspected
dog is not enough to rule out infection. Studies
evaluating PCR from different tissues of infected dogs
have shown variable and sometimes conicting results
(Baneth and Aroch, 2008). Part of the wide range of
sensitivity observed between the different studies can be
explained by the heterogeneous distribution of the
parasites in each tissue or by the organparasite load
associated with Leishmania strains tropism and local
immune response (Maia et al., 2007). Concerning whole
blood, the duration, constancy and intensity of
parasitemia in canine host are still largely unknown
(Lachaud et al., 2002) leading to false negatives
especially in asymptomatic dogs. On the other hand,
during transmission season false positives can also
occur due to a natural contamination or transient
infection.
Moreover, the efcacy of PCRtechnique will depend
on different factors such as primers, number of copies of
the target, method of DNA extraction, biological
material and PCR protocol (Alvar et al., 2004; Cortes
et al., 2004; Baneth and Aroch, 2008).
2.1.8. Real-time PCR
Quantitative real-time PCR allows the continuous
monitoring of the amplication of specic DNA
sequences as the reaction occurs. This allows for the
identication of the cycle of near logarithmic PCR
product generation (threshold cycle) and, by inference,
the precise quantication of the template DNA present
at the start of the reaction. From the quantication of the
template DNA, an estimation of the relative load of
parasites in different samples can be obtained. Different
technologies have been set-up for the monitoring of
amplication products, generally based on the use of
uorescent probes. For instance, SYBR Green technol-
ogy is a non-specic detection system based on a
uorescent DNA intercalator and it is applicable to all
potential targets. TaqMan technology is more specic
since it performs the direct assessment of the amount of
amplied DNAusing a uorescent probe specic for the
target sequence anked by the primer pair. In 2004,
Rolao et al., developed a quantication of Leishmania
spp. parasites, and they were the rst to use TaqMan
probes for absolute quantication in tissue biopsies. The
advantages of real-time PCRcompared to standard PCR
techniques are a reduction in assay time, reduced risk of
contamination and improved sensitivity (Mortarino
et al., 2004; Rolao et al., 2004; Gomes et al., 2008). The
quantitative PCR is very useful for the diagnosis of
CanL and facilitates the monitoring of parasite load
during and after treatment in different samples allowing
the prediction of recurrences associated with tissue
loads of residual parasites after treatment (Pennisi et al.,
2005; Francino et al., 2006; Manna et al., 2007; Solano-
Gallego et al., 2007).
2.2. Indirect diagnostics
2.2.1. Humoral immunity
Serological diagnosis is widely and frequently used
but, although specic humoral response in canine
leishmaniasis is, in general, very intense with high
levels of specic immunoglobulins, it underestimates
the infection rate of Leishmania in dog populations
from endemic areas (Alvar et al., 2004). Although
antibody production is low on initial and late phase or in
asymptomatic infections, infected dogs usually develop
gradually increasing antibody titres over time (Oliva
et al., 2006). Symptomatic dogs, apart from haemato-
logical and protein alterations, develop a strong
humoral response (Campino et al., 2000). However,
the presence of anti-Leishmania antibodies alone is not
a conclusive sign of disease. Therefore, it is advisable to
perform more than one serological test, to improve the
diagnosis of CanL (Campino, 2002) and to continue
monitoring with repeated testing after 3 months (Baneth
and Aroch, 2008).
In fact, the IgG subclass determination is not
generally used in diagnosis. Levels of anti-Leishma-
nia-specic IgG subclasses in dogs have been tested as
markers for the susceptibility and consequent clinical
status of infected animals (Pinelli et al., 1994; Leandro
et al., 2001; Iniesta et al., 2002, 2005; Cardoso et al.,
2007). The majority of studies dealing with immuno-
globulin production in dogs have focused on the IgG1
and IgG2 response and have attempted to link levels of
Leishmania-specic subclasses with T-helper cell type
2 (Th2)-like susceptibility and Th1-like protective
responses, respectively (Desplazes et al., 1995; Nieto
et al., 1999; Iniesta et al., 2005; Cardoso et al., 2007;
Rodr guez-Cortes et al., 2007a). In two studies
performed by Quinnell et al. (2003) and Strauss-Ayali
et al. (2007), the serological response to natural and
experimental Leishmania infection was not polarized,
as rising titres of all four IgGsubclasses were noted over
time. According to Day (2007), the conicting results of
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 278
several studies which have questioned whether infected
dogs develop skewed IgGsubclasses, could be related to
the specicity of the commercially available polyclonal
antisera used to detect these subclasses. More mean-
ingful results might be obtained, using the panel of
monoclonal antibodies with well-validated specicity
for all four canine IgG subclasses.
Rodr guez-Cortes et al. (2007b) observed that
despite the fact that dogs experimentally infected,
produced Leishmania-specic IgM, this immunoglo-
bulin appeared later than specic IgG indicating that it
cannot be used for diagnosis during the initial phase of
CanL. IgA and IgE were also studied in non-infected
and infected dogs with or without symptoms. Iniesta
et al. (2005) found that IgE was only expressed by
animals that developed the pathology, which pointed to
its potential role as a marker of the active disease. Reis
et al. (2006) also found a strong correlation between IgE
and symptomatology. IgA was also detected in
symptomatic dogs and due to the role that IgA plays
in mucosal immunity, the production of this isotype in
CanL may arise when Leishmania spreads to different
tissues, including mucosal surfaces (Reis et al., 2006;
Rodr guez-Cortes et al., 2007a, 2007b).
Several serological tests have been used for
individual diagnosis as well as for epidemiology
surveys and required, as antigens, whole body parasites
or soluble extracts of them. The use of different
antigens, the variety of procedures and the selection of
different cut-off dilution has lead to inconsistent results
which makes standardization difcult. Generally,
methods that use whole body parasites provide
reproducible and more reliable results (Alvar et al.,
2004; Gomes et al., 2008). Serological assays have
several intrinsic problems including the persistence of
specic antibodies after recovery or cross-reactions
with antibodies against other pathogens such as
Trypanosoma cruzi and Ehrlichia canis (Ferreira
et al., 2007). High levels of sensitivity and specicity
are necessary to avoid false negative results which
underestimate the infection rate of Leishmania in dog
populations in endemic areas as well as false positive
reactions which can lead to unnecessary euthanasia of
non-infected dogs.
2.2.2. Indirect immunouorescent antibody test
(IFAT)
IFAT is considered the gold standard of serologic
diagnosis. This test, which uses whole body parasite as
antigen, is useful in epidemiological studies, in clinic
practice and also in treatment follow-up (Gradoni,
2002; Mancianti et al., 2002; Alvar et al., 2004).
However, its application requires a high level of skill
and experience and expensive laboratory facilities.
Another one of its limitations is the fact that serial
dilutions of serum must be made, which makes the test
laborious and not practical for screening of large
number of samples. A few commercial kits for canine
IFAT are available, but laboratory-made antigen
preparations are usually more effective (Gradoni,
2002).
Samples in which the parasites show homogeneous
green uorescence are considered positive while those
in which a matt red coloration is observed are
considered negative. The sensitivity of IFAT in
detecting infected dogs is reported to range from
21.6% (Silva et al., 2001) to 100% (Ciaramella et al.,
1997). In a study performed by Maia et al. (2007),
testing asymptomatic and symptomatic dogs from an
endemic area, the sensitivity and specicity were 85.5%
and 94.7%, respectively.
Fernandez-Perez et al. (1999) compared IFAT using
promastigotes or axenic amastigotes; although both
methods showed a good agreement, IFAT-amastigote
assay was more sensitive in the detection of specic
anti-Leishmania antibodies in dogs with low titres,
without losing specicity.
2.2.3. Counterimmunoelectrophoresis (CIE)
CIE is a qualitative technique for the detection of
anti-Leishmania antibodies developed by Barbosa et al.
(1973); it is based on the visualisation of a blue
(Coomassie Blue stain) precipitation arc on a cellogel1
strip due to the interaction between the Leishmania
antigens and the antibodies present in the serum sample
submitted to electrophoresis.
Mancianti and Meciani (1988) obtained a CIE
sensitivity of 96.1% in dogs with severe clinical
leishmaniasis, 80% in animals with mild signs of
disease and 72.7% in asymptomatic dogs; specicity
was 100% for healthy dogs and 90.5% for dogs with
other diseases. More recently, in a study performed on
143 dogs from an endemic area, the sensitivity and
specicity were found to be 85.5% and 94.7%,
respectively (Maia et al., 2007).
CIE can be used to analyse sera from different hosts
simultaneously since it does not require a specic
immunoglobulin. Its advantages are the rapidity with
which the test can be carried out, obtaining results in
fewhours, the lower monetary overheads and the simple
equipment necessary to perform it, making CIE one of
the tests of choice for epidemiological studies
(Mansueto et al., 1982; Abranches et al., 1991;
Campino et al., 2000).
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 279
2.2.4. Immunodiffusion assay (IDA)
An imunodiffusion assay with polyethylene glycol
(PEG) was developedbyBernadina et al. (1997) for CanL
diagnosis. This technique consists in a double immuno-
diffusion in a 1% agarose gel containing 3% PEG, and is
performed using serum samples and Leishmania soluble
antigen (LSA). The formation of bands is recorded after
24 h post-staining with Coomassie Blue. This assay is
easytoperform, does not requiresophisticatedequipment
and has a high throughput which greatly facilitates the
processingof various serial samples at the same time. The
specicityobtainedbyBernadinaet al. (1997) was 98%in
the endemic control dogs and 100% in dogs from a non-
endemic region and its sensitivity ranged from 69% in
symptomatic dogs with negative culture and 100% in
symptomatic dogs.
2.2.5. Direct agglutination test (DAT)
The method uses whole, stained promastigotes either
as a suspension or in a freeze-dried form. It is cheap and
simple to perform making it ideal for both eld and
laboratory use (Meredith et al., 1995). In various studies
ondogs livinginendemicareas for visceral leishmaniasis,
DAT has been found to be 70.6% (Mohebali et al., 2004)
and 100% (Silva et al., 2006) sensitive and 84.9%
(Mohebali et al., 2004) and 100% (Neogy et al., 1992;
Schallig et al., 2002a) specic. The test can be carried out
on plasma and serum. Although several researchers
defend its use in eld conditions (Neogy et al., 1992;
Schalliget al., 2002a), one of the limitations of DATis the
relatively long incubation time (18 h) and the fact that
serial dilutions of blood or serum must be made, which
makes the test laborious and not suitable for screening of
large number of samples (Harith et al., 1989). Never-
theless the development of a freeze-dried antigen makes
DAT very suitable for use under harsh eld conditions
since it remains stable at high temperatures (Meredith
et al., 1995; Schallig et al., 2002b). The fast agglutination
screening test (FAST) combines a higher parasite
concentration with a smaller test volume. Furthermore,
it requires a single serumdilutionandresults are readafter
3 h. In serum samples from dogs with CanL, sensitivity
and specicity from 93.6% to 97.7% and from 89.0% to
93.0%, respectivelywere obtained(Schalliget al., 2002b,
2004). Gomez-Ochoa et al. (2003) developed a modied
DAT, the Easy DAT, which offers the advantages of cost
reduction and decrease of the antigen elaboration time.
2.2.6. Enzyme-linked immunosorbent assay
(ELISA)
ELISA is useful for laboratory analysis or eld
applications and to screen a large number of samples in
a short time since can be performed simply and is easily
adapted for use with various antigens such as whole
cytoplasmatic, puried antigens, dened synthetic
peptides and recombinant proteins.
Mettler et al. (2005) had a high sensitivity in
asymptomatic (94.1100%) and symptomatic (100%)
dogs using ELISA based on soluble promastigote or
amastigote antigens. Fisa et al. (1997), obtained a
sensitivity and specicity of 100% for CanL diagnosis
using dot-ELISA with protein A-peroxidase. The
advantage of using the protein A labelled with
peroxidase instead of an anti-dog second antibody
allows the test to be used to analyse sera from humans
and other possible hosts. In the study performed by
Baleeiro et al. (2006) it was demonstrated that
variation in the Leishmania species used for antigen
preparation may signicantly inuence the result of the
test for the diagnosis of CanL. The use of antigen
preparations made from L. amazonensis or L.
braziliensis instead of L. chagasi (the species
associated with the infection under study) resulted in
a decrease in antibody activity measured by ELISA in
the serum of the animals.
Solano-Gallego et al. (2003) performed an ELISA
with protein A-peroxidase on the urine of 115 dogs; 26
samples were found positive. As urine anti-Leishmania
antibodies were found only in proteinuric dogs their
detection is a more specic and more reliable non-
invasive method for CanL diagnosis and prognosis for
dogs with glomerulonephropathies.
Several ELISA performed in a short period of time
were developed for the detection of CanL. Thirty
minutes dot-ELISA performed on a nitrocellulose
membrane and a commercial ELISA rapid test (Snap
1
CLATK) showed high sensitivity, specicity and
feasibility (Vercammen et al., 1998; Ferroglio et al.,
2007). The Snap
1
CLATK has also the advantage of
being carried out on whole blood.
2.2.7. ELISA-recombinant antigens
The use of crude antigens, either complete
promastigotes or amastigotes or soluble extracts of
them, limits their specicity. Recombinant DNA
technology has led to the molecular cloning of several
genes encoding antigenic Leishmania proteins that can
be used to develop more specic serodiagnosis
methods.
The recombinant antigen rK39 has been frequently
evaluated as a diagnostic marker for CanL (Rhalem
et al., 1999; Scalone et al., 2002; Mettler et al., 2005).
Sensitivity and specicity of ELISA with recombi-
nant antigens in CanL are shown in Table 1.
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 280
Despite the results obtained and, as far as we are
aware, only rK39 and multiple chimeric rLiP2a
rLiP2brLiP0rH2A antigens are commercialised.
2.2.8. Immunochromatographic rapid tests
Rapid immunochromatographic test kits are very
attractive because of their single-test format, ease of use
and very quick response times allowing immediate
intervention by the veterinarian, but the question of how
reliable they are regarding serological diagnosis of
CanL, remains (Gradoni, 2002).
An immunochromatographic test using rK39 antigen
is commercially available in the form of antigen-
impregnated nitrocellulose paper strips adapted for the
use under eld conditions. The appearance of two lines
(one control and one test, irrespective of the intensity of
the staining) indicates a positive result. The result of a
dipstick is considered not valid if the control is not
stained. For some authors, an rK39 dipstick is an ideal
format for use in the eld, as it is rapid, simple and does
not requires extensive training of the operator (Reithin-
ger et al., 2002b; Mohebali et al., 2004; Otranto et al.,
2005; Rosypal et al., 2005). However, others have
disagreed because of the required cold storage of the
running buffer, the impossibility of storing the test strips
at high ambient temperatures and the impossibility of
the test being performed with whole blood samples
limits its use for the serodiagnosis of visceral
leishmaniasis (Schallig et al., 2002a). For Reithinger
et al. (2002b) the major disadvantage of rK39 dipstick is
its very low specicity (6175%), which leads to a high
proportion of dogs being misdiagnosed as false
positives. In opposite, Mettler et al. (2005) concluded
that rK39 dipstick is helpful for conrming clinically
suspected cases because of its high specicity in
symptomatic animals.
Costa et al. (2003) optimised and compared the
efcacy of rK39 and rK26 formatted in rapid
immunochromatographic tests for the diagnosis of
CanL and concluded that although rK26 was less
sensitive than rK39, the two antigens complemented
each other and increased the overall sensitivity of the
test without lost of specicity.
Mancianti et al. (2002) evaluated 5 commercial
immunochromatographic kits to compare 50 sera from
healthy and parasitological positive dogs and sensitivity
and specicity ranged from 34.9% to 76.2% and from
61.1% to 100%, respectively.
2.2.9. Western blotting (WB)
This test is not applicable to routine diagnosis as it
requires technical expertise, is time consuming,
cumbersome and expensive; basically it is limited to
research (Ferroglio et al., 2007). There is not yet a
global agreement about the band pattern correlated with
infection/disease. Abranches et al. (1991), Carrera et al.
(1996) and Fernandez-Perez et al. (2003) related 26 and
3435.4 kDa bands with acute phase in experimentally
and naturally infected dogs, respectively. Dogs after
successful treatment displayed a low immunoreactivity
against 67 kDa indicating the potential prognosis
marker of this protein (Fernandez-Perez et al., 2003).
Iniesta et al. (2007) used WB to analyse the idiotype
expression of IgG1 and IgG2 in dogs naturally infected
and concluded that L. infantum infection is charac-
terised by the recognition of the polypeptide fractions
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 281
Table 1
Sensitivity and specicity of ELISA with recombinant antigens in diagnosis of canine visceral leishmaniasis
Antigen Reference Sensitivity (%) Specicity (%)
rLdA2 Carvalho et al. (2002) 87.00 100
Hsp70 Andrade et al. (1992) 75.00
rLiP0 Soto et al. (1995b) 78.00
rliP2a, rLiP2b Soto et al. (1995a) 80.00 100.00
rHSp83 Angel et al. (1996) 88.00
rHSp70 (FAST) Quijada et al. (1996) 100.00
rLiH3 (FAST) Soto et al. (1996) 81.00 100.00
LI gp63 Morales et al. (1997) 100.00
LiP2arLip2brLiP0rH2A (FAST) Soto et al. (1998) 79.0093.00 96.00100.00
Hsp70, KMP-11 Nieto et al. (1999) 100.00
rLiH2A, rLiH2B, rLiH3, rLiH4 (FAST) Soto et al. (1999) 44.0072.00
rPSA Boceta et al. (2000) 100.00
rk-9 Rosati et al. (2003) 95.00 100.00
rK-26 Rosario et al. (2005) and Rosati et al. (2003) 99.10100 96.00100.00
rK-39 Rosario et al. (2005) 95.098.10 100.00
rK-40 Mettler et al. (2005) 52.9064.70 96.00100
rK9rK26rK39 Boarino et al. (2005) 96.00 99.00
14, 16, 18 kDa by IgG1 and 14, 16 kDa by IgG2.
Zaragoza et al. (2003) carried out WB in urine of 20
healthy dogs and in 22 dogs with leishmaniasis and
several bands (4060; 8090 and 110150 kDa) were
found specically in the sick dogs. Talmi-Frank et al.
(2006) developed a quantitative computerised WB
analysis of antibody responses during experimental
canine L. infantum infection. Reactivity with the 14, 48,
68 kDa bands were associated with early infection
while 14, 24 and 29 kDa were associated with parasite
persistence post-allopurinol treatment and potential
unfavourable prognosis.
2.2.10. Flow cytometry (FC)
FC is a technique for counting, examining, and
sorting microscopic particles suspended in a stream of
uid. It allows simultaneous multiparametric analysis
of the physical and/or chemical characteristics of single
cells owing through an optical and/or electronic
detection apparatus. FC is becoming an increasingly
useful tool in both health care and research laboratories
since is a rapid, accurate and reproducible method of
analysis. It can analyse several thousand particles every
second and actively separate and isolate particles having
specic properties.
Andrade et al. (2007) evaluated the performance of
FC-based methodology to detect anti-xed L. chagasi
promastigotes antibodies (FC-AFPA-IgG, FC-AFPA-
IgG1 and FC-AFPA-IgG2) in sera samples from L
chagasi-infected dogs and from dogs vaccinated against
CanL. The authors concluded that the use of FC-AFPA-
IgGand IgG2 is a helpful tool in discriminating infected
from non-infected vaccinated dogs with 100% of
specicity for both IgG and IgG2 and 97% and 93%
of sensitivity, respectively.
Silvestre et al. (2007) assessed the potential of FC
for CanLdiagnosis using soluble antigens derived from
L. infantum promastigotes against serum from a cohort
of natural and experimentally infected dogs. FC
detected seropositivity for leishmaniasis in experi-
mental infected dogs as little as 1 week after
inoculation. However, further work must be done in
order to increase their specicity since it showed a
cross-reactivity in sera from dogs with other pathol-
ogies even in animals from non-endemic areas of
leishmaniasis.
Despite some assays here mentioned are not
currently used for CanL diagnosis their application
had improved the knowledge of host immune response
to L. infantum infection, namely WB and IgG subclass
determination as well as the techniques related with
cellular immunity.
2.3. Cellular immunity
Assays to assess the cellular immune response to
Leishmania in dogs are fewer and less standardised than
serologic techniques and are not usually used as
diagnostic tools.
Although a specic cellular immunity can be
detected in dogs with or without specic antibodies
(Cabral et al., 1993, 1998; Leandro et al., 2001), a
positive immune cellular response should be the basis
for protection against the development of the disease
(Cabral et al., 1992; Pinelli et al., 1994; Cardoso et al.,
2007). According to Fernandez-Bellon et al. (2005), the
accurate determination of specic cellular immune
response to Leishmania in dogs is a crucial indicator of a
Th1-like phenotype associated with an effective control
of host infection and survival. A practical and
standardised assay to evaluate the cellular immune
response would certainly be applicable in clinical
settings both to monitor the evolution of leishmaniasis
and response to treatment and to help to establish a
prognosis.
2.3.1. Montenegro test
The Montenegro or leishmanin skin test (LST),
which indicates the delayed-type hypersensitivity to
Leishmania antigen, consists of an intradermal inocu-
lation of a suspension of inactivated promastigotes
(3 10
68
/ml) diluted in phenol or merthiolate saline
solution. As a control leishmanin diluent is injected at a
different site on the skin. A positive reading at 48 or
72 h is an induration of over 5 mm in diameter
(Montenegro, 1926; Pinelli et al., 1994; Cardoso
et al., 1998; Solano-Gallego et al., 2001; Fernandez-
Bellon et al., 2005). In general, during active disease,
the LST is negative, while it is positive during
subclinical infection (self-healing), early stage of
visceral leishmaniasis or after successful treatment.
Rodr guez-Cortes et al. (2007b) detected a signicant
correlation between LST reaction and the clinical status
of experimental infected dogs. LST is simple and
inexpensive, which makes it appropriate for eldwork
involving large number of animals (Cardoso et al.,
1998, 2007; Fernandez-Bellon et al., 2005). However,
both the need for follow-up after 4872 h and the
putative iatrogenic induction of false positive results
after repeated LST testing, undermine its utility
(Fernandez-Bellon et al., 2005).
2.3.2. Lymphocyte proliferation assay (LPA)
Following separation of peripheral blood mono-
nuclear cells (PBMCs), these are stimulated with LSA
C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274287 282
and a mitogen. Non-stimulated cells are also incubated
as negative controls. Cell proliferation is generally
expressed as a stimulation index (SI, stimulated cells/
non-stimulated cells). SI values higher than 2 (Pinelli
et al., 1994; Fernandez-Bellon et al., 2005), 2.5 (Cabral
et al., 1992, 1998; Fernandez-Perez et al., 2003) or 3
(Leandro et al., 2001; Santos-Gomes et al., 2003) are
considered positive. Quinnell et al. (2001), only
considered positive dogs with a SI 5 due to the
low specicity of cellular responsiveness to Leishmania
antigens. Resistant and asymptomatic dogs present a
strong proliferative response to leishmanial antigens
while susceptible dogs fail to respond in vitro to LSA
(Abranches et al., 1991; Pinelli et al., 1994; Quinnell
et al., 2001). Generally, mitogenic proliferation of
PBMC from dogs with obvious disease is abolished
(Abranches et al., 1991; Pinelli et al., 1994; Rhalem
et al., 1999; Strauss-Ayali et al., 2005). A restoration of
lymphoproliferation was observed in dogs clinically
cured after chemotherapy with antimonials (Bourdoi-
seau et al., 1997a), antimonials and allopurinol
(Fernandez-Perez et al., 2003), pentamidine (Rhalem
et al., 1999) or amphotericin B (Moreno et al., 1999).
Although a specic lymphoproliferative response has
been observed in experimental and naturally infected
asymptomatic or symptomatic dogs with or without
treatment, it seems to be closely dependent, not only on
the severity of the disease but also on the genetic and
immunological background of the host.
2.3.3. Interferon-g (IFN-g) cytophatic effect
inhibition bioassay (IFNB)
This bioassay detects IFN-g produced by circulating
lymphocytes in cultured supernatants. PBMC are
incubated with or without LSA during 72 h. Super-
natants are removed and incubated with MadinDarby
canine kidney cells for 16 h. After this period,
supernatant is discharged and cells are infected with
vesicular stomatitis virus and incubated for 24 h.
Production of IFN-g is expressed as the ratio of the
reciprocal of the maximumdilution that protects 50%of
the cell monolayer of stimulated versus non-stimulated
cells; values 2 are considered positive. According to
Fernandez-Bellon et al. (2005) this assay quanties the
cytokine that contributes most to cellular immune
response; it could be the method of choice for the
evaluation of this type of response in dogs. The
cumbersome nature of the test and the use of a virus
included in list A of the World Organization of Animal
Health, are its biggest disadvantages.
In the study performed by those authors IFNB and
LSTwere the most sensitive assays for the evaluation of
specic cellular immunity to Leishmania infection in
dogs. Nevertheless, Rodr guez-Cortes et al. (2007a)
defended the use of these two techniques together with
LPA in order to achieve a proper perspective of the
immunological events that shape the cellular-mediated
immunity response against L. infantum.
3. Conclusion
CanLdiagnosis is still a challenge in spite of advances
made in the development of several parasitological,
serological and molecular techniques. Equal importance
should be given to both the efcacy of the laboratory test
adopted and the selection of biological material. A
standard technique should have high sensitivity and
specicity, must be reproducible, easy to perform and
adaptable for use in local laboratories without sophis-
ticated equipment, and should detect all Leishmania-
infected dogs in an initial stage preferentially using non-
invasive procedures to obtain the samples. Furthermore,
costs should be taken into account in order to choose
which diagnosis test could be applied.
More research is required to achieve all these
objectives.
Acknowledgments
We thank C.C. Moore for the English revision. C.
Maia (SFRH/BD/12523/2003) holds a fellowship from
Fundacao para a Ciencia e Tecnologia, Ministerio da
Ciencia, Tecnologia e Ensino Superior, Portugal.
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