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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 flies, 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 confirm a clinical suspicion in a single patient as well as to detect infection in asymptomatic dogs, should have high

sensitivity, specificity and reproducibility; it must be simple, easy to perform, non-expensive, feasible in regional laboratories or

adaptable for field 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) 274–287

* Corresponding author. Tel.: +351 213652600; fax: +351 213632105.

E-mail address: [email protected] (L. Campino).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.07.028

mailto:[email protected]

http://dx.doi.org/10.1016/j.vetpar.2008.07.028

C. Maia, L. Campino / Veterinary Parasitology 158 (2008) 274–287 275

2.2. Indirect diagnostics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

2.2.1. Humoral immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

2.2.2. Indirect immunofluorescent 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 flies.

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 confirmation of Leish-

mania infection or therapeutic control, different levels

of sensitivity, specificity, 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 2–4 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) 274–287276

According to Ferrer (1999) and Alvar et al. (2004),

the microscopy of BM and LN smears has a sensitivity

of 60–75% and 30–50%, 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 field) 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 fields

evaluated, may improve the sensitivity of the method.

Liarte et al. (2001) also described a direct and fast

fluorescent method, the Quantitative Buffy Coat

(QBC1) 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 fluorescent

microscope in 30 of the 31 animals (97%) considered

positive by serology and in 28 of the 28 dogs with

parasites identified in other tissues. However, authors

concluded that experiments on uninfected dogs from

endemic and non-endemic areas should be performed to

evaluate the specificity of QBC1.

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 fluorescein diacetate and

ethidium bromide under blue light, living parasites

fluoresced as green with the dead ones staining as

orange. This technique, as QBC1, requires an

expensive fluorescence 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 paraffin-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 immunoflorescence of

tissues can be used as a supplementary tool to confirm

the diagnosis on HE, particularly in organs which do not

have high parasite load. The Leishmania IHC method

for amastigote detection in formalin-fixed paraffin-

embedded tissues forms can be applied using strepta-

vidin–peroxidase/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 immunofluorescence in popliteal

LN with a specificity 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 confirm diagnosis of

CanL when the parasites are not clearly identifiable

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 identification 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


culture media used may be monophasic such as

Schneider’s insect medium, M199, RPMI, Grace’s

medium or diphasic such as Novy–McNeal–Nicolle

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

2nd–3rd 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% specific, 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

difficulty to perform due to poor adaptation of isolate to

the medium. On the other hand, is still required to obtain

sufficient number of parasites for isoenzymatic identi-

fication, to use as an antigen for immunological

diagnosis, for experimental infection models as well

as for in vitro drugs screening or even molecular

identification.

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 field 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 flies 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

flies 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

identification 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 first 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

DNA was 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


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 filter paper for PCR

can also be used to complement serological results

(Maia et al., 2007).

A single negative PCR result 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 conflicting 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 organ–parasite load

associated with Leishmania strain’s 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 efficacy of PCR technique 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 amplification of specific DNA

sequences as the reaction occurs. This allows for the

identification of the cycle of near logarithmic PCR

product generation (threshold cycle) and, by inference,

the precise quantification of the template DNA present

at the start of the reaction. From the quantification 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

amplification products, generally based on the use of

fluorescent probes. For instance, SYBR Green technol-

ogy is a non-specific detection system based on a

fluorescent DNA intercalator and it is applicable to all

potential targets. TaqMan technology is more specific

since it performs the direct assessment of the amount of

amplified DNA using a fluorescent probe specific for the

target sequence flanked by the primer pair. In 2004,

Rolao et al., developed a quantification of Leishmania

spp. parasites, and they were the first to use TaqMan

probes for absolute quantification in tissue biopsies. The

advantages of real-time PCR compared 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 specific humoral response in canine

leishmaniasis is, in general, very intense with high

levels of specific 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-specific 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-specific 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;

Rodrıguez-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 IgG subclasses were noted over

time. According to Day (2007), the conflicting results of


several studies which have questioned whether infected

dogs develop skewed IgG subclasses, could be related to

the specificity 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 specificity

for all four canine IgG subclasses.

Rodrıguez-Cortes et al. (2007b) observed that

despite the fact that dogs experimentally infected,

produced Leishmania-specific IgM, this immunoglo-

bulin appeared later than specific 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;

Rodrıguez-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 difficult. 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

specific 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 specificity

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 immunofluorescent 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 fluorescence 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 specificity 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 specific

anti-Leishmania antibodies in dogs with low titres,

without losing specificity.

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; specificity

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

specificity 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 specific

immunoglobulin. Its advantages are the rapidity with

which the test can be carried out, obtaining results in

few hours, 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).


2.2.4. Immunodiffusion assay (IDA)

An imunodiffusion assay with polyethylene glycol

(PEG) was developed by Bernadina 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

easy to perform, does not require sophisticated equipment

and has a high throughput which greatly facilitates the

processing of various serial samples at the same time. The

specificityobtainedbyBernadina et 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 field and

laboratory use (Meredith et al., 1995). In various studies

ondogs living inendemicareas forvisceral 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) specific. The test can be carried out

on plasma and serum. Although several researchers

defend its use in field conditions (Neogy et al., 1992;

Schallig et 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 field 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 serum dilutionand results are read after

3 h. In serum samples from dogs with CanL, sensitivity

and specificity from 93.6% to 97.7% and from 89.0% to

93.0%, respectively were obtained (Schallig et al., 2002b,

2004). Gomez-Ochoa et al. (2003) developed a modified

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 field

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, purified antigens, defined synthetic

peptides and recombinant proteins.

Mettler et al. (2005) had a high sensitivity in

asymptomatic (94.1–100%) and symptomatic (100%)

dogs using ELISA based on soluble promastigote or

amastigote antigens. Fisa et al. (1997), obtained a

sensitivity and specificity 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 significantly influence 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 specific 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 (Snap1

CLATK) showed high sensitivity, specificity and

feasibility (Vercammen et al., 1998; Ferroglio et al.,

2007). The Snap1 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 specificity. Recombinant DNA

technology has led to the molecular cloning of several

genes encoding antigenic Leishmania proteins that can

be used to develop more specific 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 specificity of ELISA with recombi-

nant antigens in CanL are shown in Table 1.


Table 1

Sensitivity and specificity of ELISA with recombinant antigens in diagnosis of canine visceral leishmaniasis

Antigen Reference Sensitivity (%) Specificity (%)

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 –

LiP2a–rLip2b–rLiP0–rH2A (FAST) Soto et al. (1998) 79.00–93.00 96.00–100.00

Hsp70, KMP-11 Nieto et al. (1999) 100.00 –

rLiH2A, rLiH2B, rLiH3, rLiH4 (FAST) Soto et al. (1999) 44.00–72.00 –

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) 99.10–100 96.00–100.00

rK-39 Rosario et al. (2005) 95.0–98.10 100.00

rK-40 Mettler et al. (2005) 52.90–64.70 96.00–100

rK9–rK26–rK39 Boarino et al. (2005) 96.00 99.00

Despite the results obtained and, as far as we are

aware, only rK39 and multiple chimeric rLiP2a–

rLiP2b–rLiP0–rH2A 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 field 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 field, 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 specificity (61–75%), 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 confirming clinically

suspected cases because of its high specificity in

symptomatic animals.

Costa et al. (2003) optimised and compared the

efficacy 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 specificity.

Mancianti et al. (2002) evaluated 5 commercial

immunochromatographic kits to compare 50 sera from

healthy and parasitological positive dogs and sensitivity

and specificity 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

34–35.4 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


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 (40–60; 80–90 and 110–150 kDa) were

found specifically 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

fluid. It allows simultaneous multiparametric analysis

of the physical and/or chemical characteristics of single

cells flowing 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

specific properties.

Andrade et al. (2007) evaluated the performance of

FC-based methodology to detect anti-fixed 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-

IgG and IgG2 is a helpful tool in discriminating infected

from non-infected vaccinated dogs with 100% of

specificity for both IgG and IgG2 and 97% and 93%

of sensitivity, respectively.

Silvestre et al. (2007) assessed the potential of FC

for CanL diagnosis 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 specificity 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 specific cellular immunity can be

detected in dogs with or without specific 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 specific 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 � 106–8/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.

Rodrıguez-Cortes et al. (2007b) detected a significant

correlation between LST reaction and the clinical status

of experimental infected dogs. LST is simple and

inexpensive, which makes it appropriate for fieldwork

involving large number of animals (Cardoso et al.,

1998, 2007; Fernandez-Bellon et al., 2005). However,

both the need for follow-up after 48–72 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


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 specificity 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 specific 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 Madin–Darby

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 maximum dilution 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 quantifies 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

LST were the most sensitive assays for the evaluation of

specific cellular immunity to Leishmania infection in

dogs. Nevertheless, Rodrıguez-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

CanL diagnosis 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 efficacy of the laboratory test

adopted and the selection of biological material. A

standard technique should have high sensitivity and

specificity, 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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