First clinical case of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis in a domestic cat from French Guiana

We report the first case of natural infection of a domestic female cat (Felis catus) by Leishmania (Viannia) braziliensis in French Guiana. The infected animal had a cutaneous ulcer on the nose and nodules of different sizes in the ears. The diagnosis was confirmed by molecular analysis of cutaneous samples that detected the presence of Leishmania parasites and allowed identifying the Leishmania (Viannia) braziliensis species. The discovery of a cat infected by L. (V.) braziliensis suggests the possibility that cats could be potential secondary reservoirs of Leishmania parasites in French Guiana. Thus, it would be important to investigate the possible epidemiological role of domestic cats in domestic foci of Leishmania in this region.     

Post mortem parasitological evaluation of dogs seroreactive for Leishmania from Rio de Janeiro, Brazil

A parasitological study was conducted on 66 dogs seroreactive for Leishmania captured as a control measure of visceral leishmaniasis in the State of Rio de Janeiro, Brazil. Biological samples from different anatomical sites were collected during autopsy of the animals and cultured on biphasic medium (NNN/Schneider). The Leishmania isolates were characterized by isoenzyme electrophoresis. Leishmania was isolated from 80.3% of the animals: 12 animals with Leishmania (Viannia) braziliensis isolated exclusively from cutaneous lesions, 39 with L. (L.) chagasi isolated from different sites in the same animal, and 2 with simultaneous isolation of L. (V.) braziliensis from cutaneous lesions and L. (L.) chagasi from different sites. Isolation in culture revealed the absence of Leishmania parasites in 13 animals. The results obtained confirm the existence of mixed infections in dogs in Rio de Janeiro and indicate the need to complement the investigation of seroreactive dogs using methods for the parasitological diagnosis and identification of Leishmania species.     

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.     

Use of ELISA employing Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens for the detection of IgG and IgG1 and IgG2 subclasses in the diagnosis of American tegumentary leishmaniasis in dogs

American tegumentary leishmaniasis (ATL) shows a reduced humoral response in dogs and levels of specific antibodies may therefore not be detected by indirect immunofluorescence. Although the sensitivity of enzyme-linked immunosorbent assay (ELISA) is higher than that of indirect immunofluorescence, the best antigen for the diagnosis of ATL in dogs has not been defined. The detection of IgG subclasses represents an alternative to increase the efficiency of the serological diagnosis. In Rio de Janeiro, sporotrichosis is the main differential diagnosis of ATL in dogs, and a sensitive, specific and little invasive method that permits the discrimination of the two diseases is desired. In the present study, 69 serum samples, 34 obtained from dogs with ATL and 35 from dogs with sporotrichosis, all of them with a confirmed etiological diagnosis, were tested. The samples were analyzed by ELISA using Leishmania (Viannia) braziliensis and L. (L.) chagasi antigens for the detection of anti-Leishmania IgG, IgG1 and IgG2. The use of L. (V.) braziliensis antigens for the detection of IgG and IgG2 yielded the best results. Using L. (L.) chagasi antigen, the sensitivity and specificity for the detection of IgG were 82.4% and 100%, respectively, whereas both sensitivity and specificity were 97.1% with the L. (V.) braziliensis antigen. No improvement in the performance of the test was observed when IgG2 was analyzed separately. The IgG1 assays presented low accuracy, irrespective of the antigen used: sensitivity and specificity of 58.8% and 60% for L. (V.) braziliensis and of 64.7% and 77.1% for L. (L.) chagasi, respectively. The present results suggest that IgG ELISA using the L. (V.) braziliensis shows the best performance for the diagnosis of ATL, permitting the discrimination between cases of ATL and sporotrichosis in dogs.     

In vitro activity of dicationic compounds against a North American foxhound isolate of Leishmania infantum

Canine leishmaniasis caused by Leishmania infantum is enzootic in the North American foxhound population. Currently available chemotherapy for canine leishmaniasis is not completely effective and relapses are common in treated dogs. Pentamidine and related aromatic diamidines possess broad spectrum antiprotozoal activity. The in vitro antileishmanial activities of 35 aromatic cationic molecules were determined, using pentamidine as the reference drug. The compounds were examined for activity against promastigotes of L. infantum isolated from a foxhound from Virginia. The compounds most active against Leishmania parasites were reversed amidines. Compound 9, a reversed amidine, exhibited the highest activity against L. infantum, with a 50% inhibitory concentration (IC(50)) of 0.0042 microM compared with 14.2 microM for pentamidine. Antileishmanial activities of nine compounds were at least 1000-fold higher relative to the reference drug. Results from this study indicate that several pentamidine-related compounds warrant further investigation as possible new agents for the treatment of canine leishmaniasis.     

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.     

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.     

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.     

Detection of different Leishmania spp. and Trypanosoma cruzi antibodies in cats from the Yucatan Peninsula (Mexico) using an iron superoxide dismutase excreted as antigen

Although human leishmaniasis has been reported in 20 states in Mexico, no case of leishmaniasis has been reported in cats to date. In the Yucatan Peninsula, it has been found that dogs may act as reservoirs for at least three Leishmania species (Leishmania mexicana, Leishmania braziliensis, and Leishmania panamensis). In this study we identified specific antibodies against these three Leishmania spp. and Trypanosoma cruzi in the sera from 95 cats from two States on the Yucatan Peninsula, namely Quintana Roo and Yucatan, by ELISA and Western blot techniques using whole extract and an iron superoxide dismutase excreted by the parasites as antigens. As well as demonstrating the presence of trypanosomatid antibodies in the feline population on the Yucatan Peninsula, we were also able to confirm the high sensitivity and specificity of the iron superoxide dismutase antigen secreted by them, which may prove to be very useful in epidemiological studies.     

Feline leishmaniasis due to Leishmania (Leishmania) amazonensis in Mato Grosso do Sul State, Brazil

A case of leishmaniasis in a domestic cat (Felis domesticus) is described. The animal showed a single, nodular lesion on the nose and many nodules of different size on the ears and digital regions of all the paws. Diagnosis was made by microscopic detection of amastigotes in Giemsa-stained smears from the lesions. By monoclonal antibodies the aetiological agent was identified as Leishmania (Leishmania) amazonensis, one of the seven species implicated in human leishmaniasis in Brazil. The clinical signs in feline leishmaniasis are unspecific and similar to those observed in other diseases such as cryptococcosis and in sporotrichosis, commonly found in cats. Leishmaniasis should therefore, be added to the differential diagnosis by feline veterinary practitioners and adequate investigations should carried out for dermal leishmaniasis in the area where the feline infection is detected.     

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.     

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.     

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.     

Leishmania infantum and Neospora caninum simultaneous skin infection in a young dog in Italy.

Leishmania infantum, the agent of canine leishmaniasis in Mediterranean countries, and Neospora caninum, a recently recognized protozoal pathogen in dogs, were diagnosed in a 9-month-old Argentine Dogo dog. Both skin lesions and neurological signs were present. Histopathology of cutaneous lesions revealed a suppurative, diffuse dermatitis with numerous intracellular protozoa. Serology was positive for both L. infantum (1:640) and N. caninum (1:800). Double-label immunohistochemical staining of skin samples with hyperimmune serum from L. infantum-infected dogs was positive for protozoa within macrophages, while the polyclonal antibody specific for N. caninum showed positive reactions for protozoa in endothelial cells and fibroblasts. Transmission electron microscopy (TEM) confirmed the infection with both protozoa. This is, to the authors' knowledge, the first case of simultaneous infection with L. infantum and N. caninum in a dog. It is possible that the immunosuppressive effects of Leishmania infection or long-term steroid therapy may have been a contributing factor to the development of N. caninum in this dog.     

Longitudinal analysis of cytokine gene expression and parasite load in PBMC in Leishmania infantum experimentally infected dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, an intracellular protozoan parasite that causes a severe infectious disease. To evaluate the gene expression profile associated to CVL in vivo, we have measured monthly by real-time PCR over one year the IL-4, IL-10, IL-12, IL-13, IFN-gamma, TGF-beta and TNF-alpha mRNA levels in peripheral blood mononuclear cells in 6 experimentally infected dogs that exhibited different progressions of the illness. While in two dogs no parasite, or a very low number of parasites, was detected and the two dogs did not show any clinico-pathological abnormalities at the end of the study (L dogs), for the remaining dogs high parasite loads were detected and they developed clinical leishmaniasis (H dogs). The L dogs have null expression of both IL-4 and IL-13 for the first 4 months after the infection, whereas an early IL-4 and IL-13 expression occurs in this period of infection in most of the dogs that developed clinical leishmaniasis (H dogs). Furthermore, a higher IFN-gamma expression was associated with the increase of parasite load and clinical status in these dogs. Moreover, the high variability of expression at the pre-infection stage causes us to reject the possibility that the basal levels of these cytokines indicate the prognosis of the subsequent response against infection.     

Infection of sandflies by a cat naturally infected with Leishmania infantum

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.     

Prevalence of Leishmania spp. infection in domestic dogs in Chapare, Bolivia

Data on Leishmania spp. infection in dogs in Bolivia is scarce. Dogs from an area where 90% of human cutaneous leishmaniasis (CL) cases are due to Leishmania (Viannia) braziliensis were screened for Leishmania infection using established enzyme-linked immunosorbent antibody test (ELISA) protocols. Although none of the 51 dogs surveyed had clinical lesions indicative of CL, 6 out of 51 (11.8%) sampled dogs tested positive by ELISA.     

Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program

In 2008, in the west zone of Rio de Janeiro municipality-Brazil, the leishmaniasis control program identified 155 dogs with titers ≥ 40 by Indirect ImmunoFluorescence (IIF) on blood collected onto filter paper. The objective of this study was to describe the laboratory test findings performed in dogs euthanized by the leishmaniasis program control of Rio de Janeiro municipality. Dogs were examined, subjected to euthanasia and collection of clinical specimens. Parasite isolation was obtained in 29 animals: Leishmania chagasi was isolated in 14 dogs; Leishmania braziliensis was isolated in five dogs; Trypanosoma caninum was obtained in seven animals and one dog had mixed infection (L. braziliensis and L. chagasi). By Polymerase Chain Reaction, seventeen animals were positive in intact skin fragments. In the serological reassessment of serum samples, 28% and 22% were positive for IIF and enzyme immunoassay, respectively. Ninety-one (59%) dogs were negative for all tests performed in this study. The findings indicate that the visceral leishmaniasis control program needs to be adjusted in order to avoid non-infected dogs from being removed or permit that dogs infected with L. chagasi to remain undetected in endemic areas.     

Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symptomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicaspase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P<0.05). Apoptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P<0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F=20.45; P=0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis.     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.