Detection of Metalloproteases and Cysteine Proteases RNA Transcripts of Leishmania (Leishmania) infantum in Ear Edge Skin of Naturally Infected Dogs.

Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive success these parasites to the mammalian hosts. Since these enzymes are poorly studied in naturally infected dogs, this work aims to show the differences in metalloprotease and cysteine proteases gene expression in ear edge skin of dogs naturally infected by Leishmania (Leishmania) infantum. A cohort of dogs (n = 20) naturally infected by L. (L.) infantum was clinically classified as asymptomatic, oligosymptomatic, and polysymptomatic and the parasite load range estimated. The analysis of proteases expression by RT-PCR in the ear edge skin was also assessed, suggesting more transcripts of proteases in cDNA samples from polysymptomatic dogs than oligosymptomatic and asymptomatic ones. Metalloprotease RT-PCR assays yielded products (202 bp) in all assessed cDNA dog samples. In contrast, cysteine proteases transcripts (227 bp) had shown to be better detected in cDNA samples of polysymptomatic dogs, compared with cDNA samples from asymptomatic and oligosymptomatic dogs. Predictive in silico assays suggested that secondary structures of metalloproteasee mRNAs can be more stable than cysteine proteases at the skin temperature of dogs. Evidence is presented that during natural infection of dogs by L. (L.) infantum, this parasite produces transcripts of metalloprotease and cysteine protease RNA in the skin from asymptomatic, oligosymptomatic, and polysymptomatic dogs.

The combination therapy of meglumine antimoniate and oxiranes (epoxy-α-lapachone and epoxymethyl-lawsone) enhance the leishmanicidal effect in mice infected by Leishmania (Leishmania) amazonensis.

Current treatment of cutaneous leishmaniasis includes pentavalent antimonials as first-line drugs, but this therapy has shown severe adverse effects. An alternative to minimize this issue is based on combination therapy scheme with other drugs. In this study we analyzed the potential of the association of meglumine antimoniate (MA) with the oxiranes epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW). Results demonstrated that association between these drugs enhanced leishmanicidal activity on Leishmania (Leishmania) amazonensis infection. The compounds were tested in monotherapy or in combinations (3:1; 1:1 and 1:3) and reduced intracellular parasite numbers, measured by the endocytic index, in all tested conditions. The most effective combination regimens were MA/LAP or MA/LAW in 3:1 ratio, which achieved a reduction of 98.3% and 93.6% in the endocytic index, respectively. BALB/c mice challenged with L. (L.) amazonensis showed significant reduction in lesion size and parasite load in both footpad and lymph nodes, after four weeks of treatment. Although, MA, LAP or LAW monotherapy were able to control the evolution of lesions when compared to untreated animals (30%, 40% and 40% of reduction, respectively), the combination of MA/LAP and LAW in 3:1 ratio showed better results reducing 61.7 and 54.4%, respectively. The results indicate that the association of meglumine antimoniate to oxiranes lead to an increment in the antileishmanial activity and represent a promising approach for the cutaneous leishmaniasis treatment.

Performance of an indigenous ß-mercaptoethanol-modified antigen in comparison with a commercial reference in direct agglutination test for detection of canine visceral leishmaniasis.

We compared the performance of a locally produced ß-mercaptoethanol-modified promastigote antigen (ß-ME-Ag) of an indigenous Leishmania infantum strain against that of a trypsinized Leishmania donovani reference (REF-Ag) in the direct agglutination test (DAT) for detection of canine visceral leishmaniasis (CVL). One hundred and fifty-one serum samples collected from dogs belonging to four groups with different conditions were included. At a DAT titre of 1 : 320, statistically determined as optimal cut-off value for ß-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for ß-ME-Ag in comparison with 96.6 % and 100 %, respectively, for REF-Ag. Overall, levels of agglutination titres recorded for the two antigens were highly concordant (Cohen's κ = 0.879) in both the CVL and non-CVL groups. Based on current results, and ease experienced in processing the antigen and reading the test outcome, we recommend incorporation of ß-ME-Ag in DAT for confirmation or exclusion of suspected CVL in dogs.