In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality.

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to the role that some species play in the transmission of leishmaniasis. This work aimed to study some ecological aspects among sand flies fauna inhabiting two different environments: the várzea (lowland Amazonian forest) and terra firme (upland Amazonian forest), both located in Tefé Municipality, Amazonas State, Braziland to detect Leishmania infection in those phlebotomine populations.MethodsSand flies were collected using HP light traps. Collection took place over the course of six months: January, February, April, August, September, and October of 2013. To detect natural infection by Leishmania, DNA samples were extracted from female sand flies and submitted to Polymerase Chain Reaction (PCR) targeting the kDNA gene; Leishmania species were identified by PCR-RFLP targeting the hsp70 gene and genetic sequencing.ResultsIn all, 5,716 individuals were collected, and 46 species were identified. Trichophoromyia ubiquitalis (3,330 – 58.26%) and Nyssomyia antunesi (661 – 11.26%) were the most abundant species. Species richness was greater in terra firme environments (42 species) than in the várzea environments (22 species), and forests ecotopes (43 species) were richer than peridomiciles (28 species). DNA of Leishmania was found in Th. ubiquitalis and Psychodopygus davisi, both of which inhabit the terra firme environment and sequencing analysis confirmed the presence of Leishmania (Viannia) lainsoni DNA in Th. ubiquitalis in Tefé Municipality.ConclusionsThe high abundance of Th. ubiquitalis and Ps. davisi and detection of DNA of Leishmania sp. may indicate that both species could be putative vectors for American Cutaneous Leishmaniasis (ACL) in the terra firme environment of Tefé. The sand fly fauna found in várzea is rich and diverse, exhibiting several species, nevertheless the seasonal hydric stress during part of the year that could influence the local diversity, if compared with other studies. This is the first report in Amazonas State of Th. ubiquitalis with presence of L. (V.) lainsoni DNA.

SUMMARYIn this study, Leishmaniaspecies were identified by Polymerase Chain Reaction (PCR). The epidemiology of patients suspected of having American Cutaneous Leishmaniasis in the municipality of Assis Brasil, Acre State, located in the Brazil/Peru/Bolivia triborder was also investigated. By PCR, the DNA of Leishmaniawas detected in 100% of the cases (37 samples) and a PCR-Restriction Fragment Length Polymorphism (RFLP) of the hsp 70gene identified the species in 32 samples: Leishmania (Viannia) braziliensis (65.6%) , L. (V.) shawi (28.1%) , L. (V.) guyanensis (3.1%) and mixed infection L. (V.) guyanensis and L. (Leishmania) amazonensis (3.1%)This is the first report of L. (V.) shawiand L. (L.) amazonensis in Acre. The two predominant species were found in patients living in urban and rural areas. Most cases were found in males living in rural areas for at least three years and involved in rural work. This suggests, in most cases, a possible transmission of the disease from a rural/forest source, although some patients had not engaged in activities associated with permanence in forestall areas, which indicate a possible sandflies adaptation to the periurban setting.

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This study evaluated the species composition of sand flies and identified potential vectors of Leishmania Ross species in rural areas of Porto Velho, Rondônia State, Brazil. American cutaneous leishmaniasis (ACL) is one of the gravest threats to public health in this state. Sand flies were collected over the course of 2014 and 2015 using HP light traps. Polymerase chain reaction was performed by targeting the Leishmania mkDNA region. In total, 2,344 sand flies were collected, from which 45 species, nine subgenera, and five species group were identified. The most abundant species were Lutzomyia antunesi (Coutinho) (n = 597, 25.47%), Lutzomyia ubiquitalis (Mangabeira) (n = 496, 21.16%), and Lutzomyia octavioi (Vargas) (n = 199, 8.49%). The greatest diversity occurred in the forest environment where the most abundant species were L. antunesi (n = 588, 25.07%), L. ubiquitalis (n = 493, 21.02%), L. octavioi (n = 199, 8.49%), and Lutzomyia flaviscutellata (Mangabeira) (n = 132, 5.63%). Two pools of L. ubiquitalis were positive for Leishmania DNA, which suggests that L. ubiquitalis is a putative vector of leishmaniasis in the municipality of Porto Velho.

Canine leishmaniosis caused by L. infantum is a severe zoonotic disease. Although macrophages are the definitive host cells, neutrophils are the first cells to encounter the parasite soon after its inoculation in the dermis by the phlebotomine vector. To study the interaction of dog neutrophils and L. infantum promastigotes, blood neutrophils were isolated from healthy donors and the infection was established in vitro. In the majority of the dogs, L. infantum was efficiently phagocytized by neutrophils, and oxidative (superoxide production) and non-oxidative (neutrophil elastase exocytosis) intracellular effector mechanisms were activated, but the release of neutrophil extracellular traps was minimized. Furthermore, promastigotes and culture supernatants induced neutrophil migration, but the prior contact with Leishmania inhibits chemotaxis, which might contribute to neutrophil retention at the inoculation site. Neutrophil-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survive and maintain their proliferative capacity. These findings indicate that dog neutrophils are competent effector cells able to control the initial L. infantum infection. However, some parasites evade intracellular effector mechanisms and can be transferred to the definitive host cell, the macrophage, contributing to the development of canine leishmaniosis.

Snake venoms contain various proteins, especially phospholipases A (PLAs), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA-II as a basic Lys49 PLA homologue, compatible with other basic snake venom PLAs (svPLA), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100μg/mL. The venom and BmajPLA-II presented IC of 0.14±0.08 and 6.41±0.64μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC cytotoxicity values against HepG2 cells of 43.64±7.94 and >150μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

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