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In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune vaccine, formulated with an increased adjuvant concentration (1mg of saponin rather than 0.5mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p<0.0001), a higher and stable IgG2 and a decreasing IgG1 response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune immunotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p<0.0001), the parasitological evidence (p=0.038) and a decrease in Leishmania-specific CD4+ lymphocyte proportions (p=0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune vaccine was subjected to a safety analysis and found to be well tolerated and safe.

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BackgroundVisceral leishmaniasis (VL) is an infectious disease with a variety of clinical signs. The main form of parasite transmission to humans and other mammalian hosts is through the bite of infected arthropod females with Lutzomyia longipalpis serving as the main vector in the Americas. Dogs are the main urban domestic reservoirs of the parasite and the main source of vector infection due to their high prevalence in endemic areas and the large number of parasites in the skin of infected animals. Although miltefosine has been used in Europe since 2002 for treatment of VL infected dogs, in the Americas the treatment of dogs has not been recommended. Therefore, this study aimed to evaluate efficacy of miltefosine observing a reduction of clinical signs in infected dogs and the infectiveness to the vector by Leishmania (L.) infantum.MethodsTo our knowledge, this is the first controlled study using qPCR and xenodiagnosis to evaluate the efficacy of miltefosine (Milteforan®, Virbac) as a single treatment in Brazil. Thirty-five adult dogs with canine visceral leishmaniasis (CVL), confirmed by clinical and laboratory tests, were included in this study. They received miltefosine at a dose of 2 mg/kg every 24 h for 28 days. The dogs were observed over a three-month period, during which clinical evaluations based on a scoring system were conducted at pre-established times. Parasite load was assessed by cytology and real-time polymerase chain reaction (qPCR). Transmissibility to the vector was evaluated by xenodiagnosis.ResultsAt the end of the period, the following were observed: (i) the remission of clinical signs with a reduction in clinical scores for 94.2% of the animals; (ii) a statistically significant reduction (98.7%) in parasitic load by qPCR; and (iii) a reduction in infectivity to sand flies. After treatment, 74.2% of the animals remained or had become non-infectious.ConclusionsOur study indicates that the use of miltefosine administered orally for 4 weeks contributes to a clinical improvement and reduction in infectivity of dogs to L. infantum.

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms.