Canine Visceral Leishmaniasis In Brazil Research Articles

Therapeutic success and failure in using miltefosine to treat dogs naturally infected with Leishmania infantum.

In urban environments, domestic dogs (Canis familiaris) are a major reservoir for the parasite Leishmania infantum. Miltefosine has been used as the standard treatment for canine visceral leishmaniasis in Brazil. However, therapeutic failures have been reported. In the present study, two dogs (CG03 and CG06) with a diagnosis of infection by L. infantum underwent two cycles of treatment with miltefosine (Milteforan™ - Virbac®). Analyses showed increases in the parasite load of both CG03 and CG06, even after treatment. The clinical score of CG03 dropped from 1 to 0 (after one round of treatment), such that this dog became asymptomatic. CG06 showed clinical worsening, such that its score increased from 1 to 2. After the second therapeutic round, the parasite load in CG03 was found to have decreased, but it was still higher than before drug treatment even though this dog was physically asymptomatic. There was no decrease in the parasite load in CG06 and there was clinical worsening. The clinical response of these dogs to the treatment differed, but the parasite load remained high in both cases, which poses a risk to public health, making it essential take measures to prevent the sandfly vector from accessing the dog.

Epidemiological indicators as prophylaxis tools for Canine Visceral Leishmaniasis in Brazil

Epidemiological indicators as prophylaxis tools for Canine Visceral Leishmaniasis in Brazil

Factors associated with Leishmania infantum infection in dogs from urban areas endemic for visceral leishmaniasis in Brazil

There are gaps in the knowledge of the factors associated with canine visceral leishmaniasis (CVL). Moreover, there is a need for studies conducted with appropriate sample selection and with standardized diagnostic methods, to assess the current infection status of animals. This study analyzed factors associated with Leishmania infantum infection in dogs from urban areas endemic for CVL in Brazil. Data from four cities in different regions of the country (Fortaleza, Brasília, Palmas, and Bauru) were included, encompassing a large and heterogeneous sample group. Dogs were considered infected if they had positive results in parasitological tests (skin lesion culture, histological or immunohistochemical analysis of skin). Data analysis consisted of logistic regression with adjustment for the effect of cities as cluster variables, using generalized estimating equations. The canine positivity for L. infantum was 9.8%. Bauru had the highest percentage (18.7%), followed by Brasília (8.4%), Fortaleza (7.9%), and Palmas (4%). Male animals were twice as likely to be infected as females (Confidence Interval (CI): 1.5–2.8). Dogs living outdoors were 1.5 times more likely to be positive (CI: 1.4–1.6). Dogs living in households rearing chickens were 40% less likely to be infected (CI: 0.5–0.8). A significant multiplicative interaction indicated that a protective association between purebred and CVL was stronger among dogs older than seven years. These findings provide evidence for the role of individual and environmental factors on L. infantum infection in dogs from endemic urban areas. They may be used for identifying groups of animals at higher risk for delivering control interventions.

The diagnosis of canine visceral leishmaniasis in Brazil: Confronting old problems

In Brazil, the main strategy adopted to contain Visceral Leishmaniasis (VL) is the controversial culling of dogs with reagent serology for Canine VL (CVL). Despite there are studies showing that significant reduction of human cases has not been observed, as well as there are works demonstrating the occurrence of false-positive results in the confirmatory test, the protocol has been maintained. Researches that can reinforce the existence and persistence of this problem, as well as bring concrete alternatives are pivotal. In this context, the aim of this work was to evaluate and compare the serological, molecular and parasitological methods employed for CVL detection in Brazil, in dogs with diverse clinical profiles, from two endemic areas of Pernambuco state. Comparisons among TR-DPP®, EIE-LVC and qPCR (animals from Goiana-PE: 91) demonstrated that agreements varied from ‘poor’ to ‘moderate’ (kappa = 0.162–0.442), and a triple agreement occurred in 61.36% (54/88) of the samples. The highest percentage of agreement was obtained between TR-DPP® and EIE-LVC within the polysymptomatic group (93.33%; 14/15). Of the 34 dogs with reagent serology from Caruaru-PE, 17 (50%) and 29 dogs (85.29%) were positive for qPCR and parasitological exam, respectively. By comparing serology, qPCR and parasitological exam, the lowest percentage of agreements were obtained within the asymptomatic group (40%–72.72%). It was possible to observe that the percentage of agreement tended to decrease according to the absence of clinical manifestations in the dogs. Thus, from the fact that all diagnostic tools evaluated have their limitations, it is very important to be careful before to propose an alternative set of diagnostic criteria. Besides, the answer for better results in the control of CVL may not be in the choose of the best set of diagnostic tools, but it may be in the strategy of culling itself. In this context, it is very important to invest in alternative control measures, such as mass vaccination and treatment of dogs, thus reducing the transmission to the vector and helping to avoid new canine and human cases.

High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil - A Phase II study.

BackgroundCanine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health.Methodology/Principal findingsTo evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85–99%) vs. 83% (IC 95%; 69–92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49–97%) vs. 54% (IC 95%; 33–74%) for asymptomatic dogs and 94% (IC 95%; 83–99%) vs, 88% (IC 95%; 76–95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81–91%) of sensitivity, 94% (IC 95%; 90–97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59–74%), specificity of 87% (IC 95%; 81–92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52–66%), specificity of 98% (IC 95%; 95–99%) and accuracy of 80% (IC 95%; 76–84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63–77%), specificity of 99% (IC 95%; 97–100%) and accuracy of 86% (IC 95%; 83–89%).ConclusionThus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil.

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs.

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

Diagnostic accuracy of rKLO8 versus rK26 ELISAs for screening of canine visceral leishmaniasis

Canine visceral leishmaniasis (CVL) represents an important public health issue. Despite numerous diagnostic tests available, CVL diagnosis still needs to be improved to achieve a more accurate detection rate. Recently, rKLO8, a new antigenic protein of Sudanese Leishmania donovani, was studied for the first time in diagnosis of human visceral leishmaniasis (HVL) and showed good performance. The present study aimed to evaluate serum reactivity to rKL08 and the reference antigen rK26, and to compare both diagnostic proteins with the combined DPP® CVL rapid test and ELISA (EIE-Bio-Manguinhos) confirmatory test, which are both recommended for the diagnosis of CVL in Brazil. Serum samples of dogs were grouped into: (I) DPP®/EIE negative (n=100) and (II) DPP®/EIE positive sera (n=100). Enhanced levels of IgG, mainly IgG2, to both rKLO8 and rK26 were found in group II. Sensitivity was 68% and 77% and specificity was 92% and 91%, for rKLO8 and rK26 antigens, respectively. Moreover, the combination of rKLO8 and rK26 antigens (rKLO8+rK26) exhibited higher sensitivity (85%) and specificity (93%). Thus, our results show that apart from the improved diagnostic power of rKLO8 in HVL, this new antigen is also suitable for the diagnosis of CVL. Further, the combination of rKLO8 and rK26 antigens increases the diagnostic accuracy of CVL.

Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs.

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the DNA of Leishmania spp. has been reported in whole ticks. We investigated the presence of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus ticks collected from tick-infested dogs in two cities of Brazil. We used 66 dogs that tested positive and 33 that tested negative for Leishmania spp. according to direct cytological examination assays. Ten ticks were collected from each dog and dissected to collect the intestines, ovaries, and salivary glands for immunohistochemistry (IHC) and diagnostic real-time polymerase chain reaction (RT-PCR). IHC results showed Leishmania spp. in 98, 14, and 8% of the intestines, ovaries, and salivary glands, respectively. Real-time PCR showed that 89, 41, and 33% of the tick intestine, ovary, and salivary glands, respectively, were positive for Leishmania spp. The verification of promastigotes of Leishmania spp. by two independent techniques in ticks collected from these urban region dogs showed that there is need for clarification of the role of ticks in the transmission of canine visceral leishmaniasis in Brazil.

Accuracy and reproducibility of a rapid chromatographic immunoassay for the diagnosis of canine visceral leishmaniasis in Brazil.

Visceral leishmaniasis is a major public health concern in Brazil and the domestic dog is the main source of infection. In this study, we aimed to evaluate the accuracy and reliability of a rapid chromatographic immunoassay based on a dual-path platform for the diagnosis of canine visceral leishmaniasis (CVL). Sampling consisted of 428 domestic dogs selected from two neighborhoods in the municipality of Fortaleza, Ceara state, Brazil. The reference standard was composed of three parasitological tests and was applied samples from 333 dogs. The rapid test was used to analyse whole blood and serum samples. Accuracy of the rapid test in whole blood samples through visual reading (n=305), serum samples through electronic reading (n=333) and serum samples through visual reading (n=333), yielded sensitivities of 87.5% (21/24; 95% CI: 66.5 to 96.7), 88% (22/25; 95% CI: 67.5 to 96.8) and 88% (22/25; 95% CI: 67.5 to 96.8), and specificities of 73.3% (206/281; 95% CI: 67.7 to 78.4), 68.2% (210/308; 95% CI: 62.2 to 74.3) and 69.2% (213/308; 95% CI: 63.7 to 74.3), respectively. Agreement between the visual and electronic readings in 428 serum samples were classified as almost perfect (Kappa Index=0.88; 95% CI: 0.83 to 0.93). The positive predictive value of the test using whole blood samples was 21.9% for the 7.9% prevalence detected by the reference standard in the study sample. A sensitivity analysis of the positive predictive value revealed that it remained below 50% in scenarios with a prevalence of up to 20%. The similarity of the accuracy values of the rapid test using whole blood or serum samples, together with its reliable performance in sera through visual and electronic reading, suggests that it may contribute as a screening test for routine use under field-conditions. However, future studies need to improve the accuracy of the test so that it can be successfully implemented in public health programs.

Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis.

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil

BackgroundAmerican visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity.MethodsA validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories.ResultsThe sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used.InterpretationELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.

Diminished CD4+/CD25+ T cell and increased IFN-γ levels occur in dogs vaccinated with Leishmune ® in an endemic area for visceral leishmaniasis

The Leishmune ® vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania ( Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-γ, IL-4 and TNF-α were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-γ ( P < 0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune.

Immunogenicity assay of the Leishmune ® vaccine against canine visceral leishmaniasis in Brazil

Leishmune ® is the industrialized version of the FML-saponin vaccine which has been shown to develop 92–95% protection in vaccinated dogs and 76–80% vaccine efficacy against field canine visceral leishmaniasis (CVL) in Brazil. Leishmune ® has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. In the present investigation, 550 healthy seronegative dogs of endemic and epidemic areas of Brazil were monitored for Leishmune ®-induced immunogenicity during a 2-year trial. Another group of 588 untreated exposed dogs was also studied in parallel. Both groups were seronegative on day 0. The strong immunogenicity induced by Leishmune ® vaccine was demonstrated by the 98% of FML-seroconversion, increase in absorbencies, the 82.7% DTH positive reactions and increase in skin test size diameters, the average increase in CD8+ total lymphocytes population in blood (27.1%), expected for QS21 saponin-containing vaccine, the sustained proportions of CD4+ T cells, and the average increased proportions of CD21+ B lymphocytes (42.3%). The Leishmune ®-induced protection against CVL is demonstrated by the results: 98.8% asymptomatic dogs (at the end of first year) and 99% healthy survivors (at the end of the second year) among vaccinated dogs, compared to the 79.4% asymptomatic and 61% survivor dogs ( p < 0.001) monitored in the untreated exposed cohort. In spite of the low vaccine coverage, it was possible to detect a 66.1% ( p < 0.005) reduction in Belo Horizonte and an 80.2% ( p < 0.005) reduction in Araçatuba of the incidence of CVL among vaccinated dogs, when compared to the global incidence of CVL of each town, respectively. Our preliminary results support the potential use of Leishmune ® to prevent CVL epidemics.

Safety trial using the Leishmune ® vaccine against canine visceral leishmaniasis in Brazil

A group of 600 healthy and asymptomatic dogs from Brazilian canine visceral leishmaniasis endemic areas was vaccinated with three sc doses of Leishmune ® which is the industrialized formulation of the FML-saponin, recently licensed for commercialization in Brazil, which previously showed 76–80% vaccine efficacy against canine visceral leishmaniasis. Safety evaluation was performed for 14 days after each vaccine injection and disclosed transient reactions of local pain (40.87%), anorexia (20.48%), apathy (24.17%), local swelling reactions (15.90%), vomit (2.4%) and diarrhoea (1.5%). All effects showed significantly correlating declines, from the first to the third dose ( p < 0.0001). Most of the noticed reactions of pain (73%), anorexia (79%) and local swelling (84.7%) were mild. No significant differences between puppies and adults dogs were found in the number of adverse reactions. Adult dogs developed however, 94.5% of the small swelling reactions (<3 cm), and indicating that they are more resistant to the inflammatory response promoted by the saponins. No dead by anaphylaxis occurred, and only two dogs (0.1%) showed allergic reactions (facial oedema and itching) after the third dose. Transient alopecia on injection site occurred in only five poodles (0.28%) with total recovery and no need of treatment. All the mild adverse events in response to Leishmune ® injection were transient and disappeared before the injection of the following vaccine dose, confirming the tolerability of the vaccine. The Leishmune ® preparation was less haemolytic (HD 50 = 180 μg/ml) than expected for a QS21 saponin-containing vaccine, indicating that its formulation with the FML antigen diminished the potential in vitro toxicity.

Are insecticide-impregnated dog collars a feasible alternative to dog culling as a strategy for controlling canine visceral leishmaniasis in Brazil?

In a zoonotic visceral leishmaniasis (ZVL)-endemic area in Brazil, deltamethrin-impregnated collars (DMC) were fitted to 136 dogs for 5 months and significantly reduced the odds of increasing their anti- Leishmania antibody titer during this period by 50% (95% confidence interval 29–87%, P=0.01), as compared with a population of 97 uncollared dogs with pre-intervention prevalence within the same town. Mathematical modeling suggests that under typical Brazilian ZVL-endemic conditions, the epidemiological impact of community-wide DMC application should be greater than the currently practiced dog culling strategy, but that its impact will be dependent on collar coverage and loss rate. Both interventions should have a higher proportional impact in regions of lower endemicity, but the relative advantage of DMC over culling increases with transmission rate. Sensitivity analyses indicate that the impact of either intervention is not significantly affected by variation in the biology of the sandfly vector, but is greatly influenced by variation in dog mortality and serorecovery rates.

The fucose-mannose ligand-ELISA in the diagnosis and prognosis of canine visceral leishmaniasis in Brazil.

The fucose-mannose ligand (FML)-ELISA assay showed a sensitivity of 100% and a specificity of 100% in diagnosis of canine visceral leishmaniasis (CVL) (kala-azar) in sera from naturally infected dogs from São Gonçalo do Amaranto, Rio Grande de Norte, Brazil. The overall prevalence of antibodies to Leishmania in the endemic area was 23% (79 of 343). Seroreactivity detected by a Leishmania chagasi immunofluorescent (IF) assay was much lower (2.9%) and similar to the percentage of dogs with kala-azar symptoms (2.6%). Twenty-one of 21 asymptomatic, FML-seropositive animals died of kala-azar in a period ranging from 0 to 6 months after diagnosis. The predictive value was 100% for the FML-ELISA, 43% for an L. mexicana ELISA, and 24% for the L. mexicana and L. chagasi IF assays, respectively. In experimentally infected dogs, all assays detected seropositivity between 90 and 120 days after infection. Since the current strategy for control of CVL is based on detection and destruction of infected dogs, the highly predictive, sensitive, and specific FML-ELISA represents a useful tool for field control of the disease.