Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge.

Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the "LbSapSal" vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with "LbSapSal" is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-ß), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after "LbSapSal" immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the "LbSapSal" vaccination is a potential tool to control the Leishmania chagasi infection.

Effect of the preservative and temperature conditions on the stability of Leishmania infantum promastigotes antigens applied in a flow cytometry diagnostic method for canine visceral leishmaniasis.

The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.