Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amastigote-flow cytometry

Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections.     

The first record in the Americas of an autochthonous case of Leishmania (Leishmania) infantum chagasi in a domestic cat (Felix catus) from Cotia County, São Paulo State, Brazil

A case of feline cutaneous leishmaniasis is reported in a domestic cat (Felis catus) as an apparently natural infection in a non-endemic area. Amastigotes were seem in smears of a nodular lesion on the cat's nose. No parasite could be seen in cytological preparations of liver or spleen but DNA obtained from a sample of the spleen produced the expected fragment in a Leishmania specific rDNA based PCR assay. The PCR product, a 520 bp fragment, was sequenced and the nucleotide sequence was identical to that of Leishmania (Leishmania) infantum chagasi. These results are surprising since no autochthonous human or canine cases of visceral leishmaniasis have ever been reported from this region. This case suggests that natural transmission of this disease is occurring in this area, and that cats could act as a reservoir of L. (L.) infantum chagasi.     

Visceral leishmaniasis in a captive crab-eating fox Cerdocyon thous

Visceral leishmaniasis (VL) is a zoonotic disease with worldwide distribution. The crab-eating fox (Cerdocyon thous) is considered a wild reservoir of many zoonotical diseases, particularly VL. This study reported the presence of Leishmania infantum amastigotes in different organs of one captive C. thous found dead in a zoo. This animal was positive by the indirect fluorescence antibody test and had many clinical signs of VL. Intracellular amastigote forms of L. infantum were seen in neutrophils and macrophages in sample tissues from skin, lymph nodes (popliteal, submandibular, prescapular, and mesenteric), spleen, and liver. The numbers of positive cells and intracellular parasites were higher in macrophages than in neutrophils. In addition, polymerase chain reaction demonstrated extensive distribution of Leishmania DNA in C. thous tissues from multiple organs. The presence of intracellular amastigotes in neutrophils and macrophages as well as DNA of the parasite in tissues, specifically skin demonstrate that this crab-eating fox is an adequate host for L. infantum and reinforce the importance of VL for symptomatic wild canids kept in captivity in endemic areas.     

The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis

Leishmania parasites cause a group of diseases collectively known as leishmaniases. The primary hosts of Leishmania are sylvatic mammals of several orders (Rodentia, Marsupialia, Carnivora, etc.). Under certain circumstances, particularly in peridomestic and domestic transmission foci, synanthropic and domestic animals can act as source of infection for phlebotomine sand fly vectors. Dogs have long been implicated as the main domestic reservoirs of Leishmania (Leishmania) infantum, the aetiological agent of zoonotic visceral leishmaniasis, and there exists an increasing trend to regard dogs as the main domestic reservoirs of Leishmania (Viannia) braziliensis, the most widespread aetiological agent of American tegumentary leishmaniasis. However, insights derived from recent research indicate that not dogs but humans are probably the most important domestic reservoirs of L. (V.) braziliensis. In the present article, the role of dogs as reservoirs of Leishmania parasites, with emphasis on L. (L.) infantum and L. (V.) braziliensis, is reviewed.     

Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model

For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is not yet defined. In the present work, we have reappraised the use of golden hamster (Mesocricetus auratus) as an experimental model for infection with Leishmania infantum. Groups of hamsters were challenged by the intracardial route with doses ranging from 10(3) to 10(5) infectious promastigotes and the animals were monitored for 1-year follow-up period. The outcome of the infection was assessed by clinical symptoms of leishmaniasis, parasite loads in both liver and spleen, humoral response to Leishmania antigens and antibody levels in kidneys. The humoral response was analysed using either crude antigens (by ELISA and Western blotting) or several recombinant Leishmania antigens (Hsp70, Hsp83, LiP2a, LiP2b, H2A, H3 and KMP-11). From the analysis of all these parameters, we established the existence of three groups of animals: symptomatic or susceptible, oligosymptomatic, and resistant. Given the parallelism existing between the outcomes of Leishmania-infection in hamsters, dogs and humans, we believe that our data illustrate that the hamster is an excellent experimental model to study visceral leishmaniasis and for the design of vaccine development.     

Infections in immunocompetent and immune-deficient mice with promastigotes of a North American isolate of Leishmania infantum

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is widespread among foxhounds in the United States. Experimental infections with a North American isolate of L. infantum were evaluated using two inoculation routes in immunocompetent and immunosuppressed mouse strains. Groups of 2-5 interferon gamma gene knockout (IFN-gamma-KO) (BALB/c-Ifng), inducible nitric oxide synthase (NOS) gene knockout (iNOS-KO) (C57BL/6), B-cell-deficient (microMT) (C57BL/6), and BALB/c mice were intravenously (i.v.) or subcutaneously (s.c.) inoculated with various doses of promastigotes of the LIVT-1 strain of L. infantum. None of the mice developed clinical signs of leishmaniasis during the 8-9 weeks of the study. Promastigotes were cultured from spleens of all i.v.-infected mice by 3 days post culture. Spleens from s.c.-infected mice inoculated with greater than 1 x 10(6) parasites became culture positive 3-24 days post culture, but promastigotes were not cultured from mice infected with 1 x 10(5) or 5 x 10(5) LIVT-1 promastigotes. Histological lesions were prominent in the livers of i.v.-infected mice but were mild to nonexistent in s.c. infection. Serological responses were low and transient determined by indirect fluorescent antibody testing in all groups. These results indicate that the i.v. route of infection is superior to the s.c. route in a mouse model of North American leishmaniasis and that mice lacking INF-gamma, iNOS or mice that are B-cell-deficient are not more susceptible to acute infection.     

Histopathological studies of visceralized Leishmania (Leishmania) amazonensis in mice experimentally infected

BALB/c, C57BL/6, and DBA/2 mice were subcutaneously infected in the left footpad by injecting 10(4) Leishmania (Leishmania) amazonensis amastigotes. Mice were sacrificed 20, 30, 40, 60 and 90 days post-infection. Fragments of liver, kidney, spleen, skin, and draining lymph node were collected for histological examination. Light microscopy showed that at 20 days after infection BALB/c mice presented discrete inflammatory infiltrates in the skin made up of eosinophils, lymphocytes, and rare parasitized macrophages. Ninety days post-infection, the dermis showed necrotic tissue, large numbers of mononuclear cells and vacuolated macrophages filled with amastigotes. Forty days post-infection, the draining lymph nodes showed hyperplastic germinal centers, increase of high endothelial venules and apoptosis in germinal center cells. After the first 3 months post-infection, the involvement of spleen, kidney and liver was discreet, being characterized by multifocal inflammatory infiltrates. Eight months after infection, the animals presented metastatic lesions in the contralateral footpad and nose. In deep dermis, there was remarkable proliferation of fibroblasts associated with collagen fibers. The liver showed multifocal granulomas and mononuclear infiltrate around the blood vessels, but no parasites were observed, except in one animal. In some mice there were immature cells of the hematopoietic lineage. Both BALB/c and C57BL/6 mice presented osteonecrosis, which is characterized by pycnotic osteocytes and empty lacunae at the point of inoculation and subsequently, replacement of this tissue by fibrous connective tissue and colonization of the bone marrow. A diffuse inflammatory process composed of mononuclear cells and rare parasites were seen in the kidneys. In one mouse, bone marrow cells were observed in the renal medulla along with where free amastigotes. DBA/2 mice developed a mild infection and they did not visceralize. In conclusion, our data demonstrates that in susceptible mice L. (L.) amazonensis, a causative agent of tegumentary leishmaniasis, causes pathological changes similar to those produced by Leishmania (L.) infantum in both humans and canids.     

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.     

Canine visceral leishmaniasis: dog infectivity to sand flies from non-endemic areas

Canine visceral leishmaniasis (VL), caused by Leishmania infantum (Leishmania chagasi in the New World), is a zoonotic, endemic disease in Western Europe and Latin America. The potential spreading to new regions was suggested by the appearance of canine VL among foxhounds in the US. Although the sand fly vectors in the major foci of transmission have been described, no information exists on other sand flies that could propagate the infection outside endemic areas. We evaluated the capacity of Lutzomyia shannoni (Dyar) and Lutomyia youngi (Feliciangeli & Murillo), which are widely distributed in the New World, to acquire L chagasi (Cunha and Chagas) infections. A high proportion of L youngi were infected after feeding on an oligosymptomatic dog (51 per cent) or a polysymptomatic individual (95 per cent), but the intensity of infection was low (< 200 promastigotes/fly). L shannoni became infected only by feeding on the polysymptomatic dog, and the infection rate was lower (9 per cent) than in Lutzomyia longipalpis (36 per cent), and Lutzomyia evansi (Nunez-Tovar) (Lutz and Neiva) (38 per cent), but the intensity of infection (200 to > 500 promastigotes/fly) was comparable (L longipalpis) or higher (L evansi) than in the New World vectors. It is hypothesised that the presence of infected dogs in areas where L shannoni or L youngi occur could initiate new endemic cycles of VL in both South and North America.     

Alternative strategy for visceral leishmaniosis control: HisAK70-Salmonella Choleraesuis-pulsed dendritic cells

Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.     

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.     

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.     

PCR identification of Leishmania in diagnosis and control of canine Leishmaniasis

Leishmaniases are endemic in many countries, mainly in rural areas. In Brazil, Leishmania infection is responsible for many cases of Leishmaniases, including recent reports in urban regions. Despite their sensitivity, traditional serological and parasitological methods for detecting Leishmaniases have proven inadequate for species discrimination. This study aimed to identify Leishmania species in biological samples by a fast methodology, avoiding "in vitro" cultivation. Knowledge of the Leishmania species is an important tool in regions where both New World visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are prevalent. As these new foci appear in areas not traditionally endemic for VL, the main problem is to distinguish between true autochthonous infections and infections acquired in other well-known endemic areas. Since, domestic dogs are known to be the main VL and CL reservoir, they are regularly investigated in endemic areas to prevent, principally, severe and often fatal VL in humans. However, several infected dogs present no clinical signs or clinical signs similar to other canine diseases. Here, we evaluated the ability of PCR to diagnose VL and distinguish L. (L.) chagasi from other Leishmania species in domestic dogs. Samples from 114 dogs from 30 cities (Sao Paulo, Brazil) were divided into two groups: 44 symptomatic and 70 asymptomatic. They were assayed by parasitological methods (culture and microscopic examination) and PCR to determine L. (L.) chagasi, L. (V.) braziliensis; and in some cases, Leishmania spp. Parasitological tests and PCR-L. chagasi were concordant in 105 samples (92%). VL was confirmed in 49 dogs, while 56 had negative results. Of the 114 samples, 9 had discordant results, but were further tested by PCR-Leishmania spp. with positive results. VL was also confirmed in 4 dogs having negative parasitological tests and positive PCR-L. chagasi. Consequently, this PCR was positive for 100% (53/49) of dogs with parasites detected in parasitological tests. Also, PCR demonstrated high specificity detecting 61 dogs negative for VL. Leishmania infection was negative in 56 dogs, and 5 with positive culture and PCR-Leishmania spp. had CL since they were positive in PCR-L. braziliensis. This study shows the importance of including PCR in diagnosis of Leishmaniases by differential diagnosis contributing to the surveillance and control of VL programs.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

An update on antileishmanial vaccine candidates and prospects for a canine Leishmania vaccine

Dogs are the domestic reservoir for Leishmania infantum, the parasite causing zoonotic visceral leishmaniasis (VL) in both the Old and New Worlds. Since the available methods for canine leishmaniasis treatment and control have limited efficacy, the development of a canine Leishmania vaccine is highly desirable. Mechanisms of antileishmanial immune responses in murine, human, and canine infections are briefly presented. Vaccine candidates, including live or killed parasites, Leishmania purified fractions, defined recombinant parasite antigens, live recombinant bacteria expressing Leishmania antigens and antigen-encoding DNA plasmids, are reviewed. Finally, some practical requirements for the evaluation of vaccine candidates in dogs are indicated.     

Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies

Visceral leishmaniasis (VL) is a growing zoonosis with an increasing number of new cases and a rapid geographical spreading of the disease. In the present study, a canine survey was carried out in the city of Montes Claros (320,000 inhabitants), an endemic area of American visceral leishmaniasis in the state of Minas Gerais, Brazil. A total number of 4795 dogs were examined by serology, which showed a rate of seropositivity of 5%. Isoenzymatic analysis confirmed Leishmania infantum chagasi as the local aetiological agent of CVL. Canine tissues were assayed for the presence of Leishmania parasite DNA using different techniques. The infectivity of asymptomatic, oligosymptomatic and symptomatic seropositive dogs was tested by xenodiagnosis using laboratory reared Lutzomyia longipalpis. Rates of infection of 5.4%, 5.1% and 28.4% were found for the phlebotomine sand flies that fed in asymptomatic, oligosymptomatic and symptomatic dogs, respectively. Our results indicate that, under experimental conditions, symptomatic dogs are about four times more infective to VL vectors than oligosymptomatic or asymptomatic animals. The lower infectivity rates of dogs displaying any of the last two clinical forms of leishmaniasis, however, must be taken into account in the epidemiology of CVL.     

Oxidative stress and non-enzymatic antioxidative status in dogs with visceral Leishmaniasis

Leishmaniasis is a potentially fatal chronic protozoan disease in human, canine and rodent species. The infection by Leishmania is endemic in the Mediterranean Sea region, Africa, Asia and South America. Canine visceral leishmaniasis (CanVL) is a systemic disease caused by Leishmania infantum and Leishmania chagasi from the Leishmania donovani complex group. The blood glutathione (GSH), plasma malondialdehyde (MDA), ascorbic acid (AA), beta-carotene, retinol and ceruloplasmin levels of dogs with CanVL were investigated to establish the status of the antioxidant defense mechanism in the infected animals. Dogs diagnosed as CanVL with amastigotes in lymph node smear examination and/or antibody titers > or = 128 were used as subjects, while those with no serological response against leishmaniasis were used as healthy controls. The glutathione and retinol amounts were decreased although not significantly (p > 0.05), but the MDA levels were significantly higher in dogs with VL, suggesting increased lipid peroxidation.     

Immunological characteristics of experimental murine infection with Leishmania (Leishmania) amazonensis

The murine models of Leishmania infection are well-studied and suitable models for studying this disease, which, despite its incidence of nearly 2 million new cases worldwide per year and its prevalence of 12 million cases, has been a somewhat neglected disease. Data obtained using such models are important for a better understanding of the disease in humans due to similarities in physiology and the advantage provided by the uniform infection profile within each mouse strain. In this review, we focus on studies of experimental murine infection with Leishmania (Leishmania) amazonensis, a species that has been associated with infections exhibiting various clinical features in humans. Mainly, we point out and discuss reports on: the effects of variations of the inoculum (such as strain, site, and size) in the establishment and development of the infection; characteristics of the infection in distinct mouse strains; and, the effects and subversions of the infection on components of the host innate and adaptive immune responses. The results obtained in these studies show that L. (L.) amazonensis infection in mice presents some unique features and immunoregulatory mechanisms, making it an interesting model for obtaining further knowledge of potential drugs targets and immunotherapy in Leishmania infection.     

Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.