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The subfamily Phlebotominae comprises important insects for public health. The use of complementary tools such as molecular taxonomy is necessary for interspecific delimitation and/or discovery of cryptic species. Here, we evaluated the DNA barcoding tool to identify different species in the southwestern Brazilian Amazon. For this, we collected sand flies in forest fragments along the highway BR-317, in the municipality of Brasiléia, state of Acre, Brazil. The specimens were DNA-barcoded using a fragment of the cytochrome c oxidase subunit I (COI) gene. The sequences were analyzed to generate K2P pairwise genetic distances and a Neighbour-joining tree. The sand fly barcodes were also clustered into Molecular Operation Taxonomic Units (MOTU) using Automatic Barcode Gap Discovery (ABGD) approach. A total of 59 COI sequences comprising 22 nominal species and ten genera were generated. Of these, 11 species had not been sequenced before, thus being new COI sequences to science. Intraspecific genetic distances ranged between 0 and 4.9%, with Pintomyia serrana presenting the highest values of genetic distance, in addition to having been partitioned into three MOTUs. Regarding the distances to the nearest neighbour, all species present higher values in relation to the maximum intraspecific distance, in addition to forming well supported clusters in the neighbour-joining analysis. The DNA barcoding approach is useful for the molecular identification of sand flies from Brasiléia, state of Acre, and was efficient in detecting cryptic diversity of five species which can be confirmed in future studies using an integrative approach. We also generated new COI barcodes for Trichophoromyia auraensis, Nyssomyia shawi, and Psychodopygus paraensis, which may play a role in the transmission of Leishmania spp. in the Brazilian Amazon.

Background: Brazil faces the expansion and urbanization of American Visceral Leishmaniasis. The presence of the vector in the urban area is one of the major challenges of the Brazilian Program for Control of Visceral Leishmaniasis, which refers the need for a better understanding the behavior of Lutzomyia longipalpis, as well as the factors of its adaptation to new habitats. The aim of the study was to combine diagnostic molecular tools capable to identifying natural infection by Leishmania (L.) infantum chagasi and the origin of food sources in L. longipalpis.Methods: The specimens were captured in the municipalities of Araguaína/Tocantins, Fortaleza/Ceará, Sobral/Ceará, and Rio de Janeiro/Rio de Janeiro. Molecular diagnoses were performed through Polymerase Chain Reaction using primers that amplify kinetoplast deoxyribonucleic acid in Leishmania sp. and the cacophony gene in phlebotomines for the diagnosis of natural infection, primers that amplify the cytochrome b gene, in addition to the sequencing technique, for the study of alimentary habit.Results: Among the analyzed females, 28.6% were diagnosed in the food evaluation. Among the total of 141 samples that were analyzed, showed a higher positivity for the human food source (62.5%), followed by dogs (27.5%) and birds (10%). In Araguaína, the samples showed positivity for human blood (61.5%) and dogs (38.5%). In Fortaleza and Sobral, specimens showed a positive percentage for human blood (54.5% and 66.7%), followed by dogs (27.3% and 20.0%) and birds (18.2% and 13.3%). In the females of Rio de Janeiro was detected exclusive feeding of human blood. In the natural infection, a general index of positivity was obtained equal to 4.3%. When associated, detection of the power source and natural infection, two infected specimens were found in Araguaína, with dog and human feeding; and in Sobral, 3 specimens infected and positive for human blood.Conclusions: The results corroborate with studies that demonstrate the eclecticism and food opportunism of L. longipalpis, as well as their occurrence in several environments, which are determinant factors of the important process of domiciliary vectorization. The low positivity feeding on human blood may suggest that other mammals, possibly rodents, would be acting as a food source.

Protozoan parasites, such as Leishmania spp., are the causative agents of many insect-borne infectious diseases with medical and veterinary importance. Leishmaniasis, caused by Leishmania spp., is transmitted by female phlebotomine sand flies. In the Alentejo region of Portugal, located at the north of Algarve, cases of human and canine leishmaniasis caused by Leishmania infantum have been notified. However, no recent studies regarding the sand fly fauna in the region are available. We therefore aimed to explore the phlebotomine sand fly species found in both, Évora and Beja Districts, to gain an insight about the leishmaniasis epidemiology in these areas. After the identification of the insect species, PCR molecular tests were used to assess L. infantum infection rate in the sand fly captured females, together with the analysis of blood meal sources of the insect vectors. One Sergentomyia minuta female was positive for L. infantum infection and another for human blood as a meal source. The occurrence of this phlebotomine species infected with L. infantum may suggest that, in the Mediterranean basin, leishmaniasis epidemiology is changing. Also, if the importance of S. minuta for the zoonotic and anthroponotic cycle of leishmaniasis is later proven, the strategies to control its vector will inevitably to be rethought.

Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.

In Argentina, American Cutaneous Leishmaniasis (ACL) extends up to 29°S in the phytogeographic regions of the Yungas (west), Chaco (center) and Paranaense (east). Since the Phlebotominae vectors of this disease in the western Chaco (dry Chaco) are unknown, in the present work, we studied the natural infection in Phlebotominae by PCR-ERFLP and Dot blot in order to incriminate these organisms as potential vectors. Captures with CDC-type traps were performed monthly in the domicile, the peridomicile and the forest in the Municipio Misión Nueva Pompeya, Chaco, Argentina, in two sites with human cases of ACL: Los Pozos (24°54′S, 61°22′W) and Fortín Arenales (24°58′S, 61°21′W), from November 2006 to December 2007. A total of 1702 Phlebotominae were captured: Mygonemyia migonei (83.8%), cortelezzii complex (11.1%), Mycropigomyia peresi (3.3%), Mycropygomy quinquefer (1.2%), Pintomyia torresi (0.2%) and Nyssomyia neivai (0.2%). Although no significant differences were found in species diversity, there were significant differences in abundance between both sites studied. A total of 80 phlebotomine females were analyzed: 50 of the cortelezzii complex and 30 My. migonei. No intestinal flagellates were observed by light microscopy. Two pools of 10 individuals of the cortelezzii complex of the peridomicile and forest of Fortín Arenales were reactive by PCR and Dot blot for Leishmania (Viannia) braziliensis. In Argentina, Evandromyia cortelezzii has been incriminated as a likely vector of ACL because of its abundance in areas of sporadic outbreaks. In the present work, Ev. cortelezzii females were found naturally infected, thus reinforcing the hypothesis that the members of the cortelezzii complex act as vectors of the disease. Fil: Rosa, Juan Ramón. Universidad Nacional del Nordeste. Instituto de Medicina Regional; Argentina Fil: Pita Pereira, Daniela. Instituto Oswaldo Cruz; Brasil Fil: Peçanha Brazil, Reginaldo. Instituto Oswaldo Cruz; Brasil Fil: Andrade Filho, José Dilermando. Instituto Oswaldo Cruz; Brasil Fil: Salomón, Oscar Daniel. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Szelag, Enrique Alejandro. Universidad Nacional del Nordeste. Instituto de Medicina Regional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes.

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.

To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.

Background Notifications concerning American Cutaneous Leishmaniasis have increased in recent years in the state of Acre, Brazil. Despite identification of distinct Leishmania species isolated from cutaneous lesions, there are still no records of visceral leishmaniasis in the state. However, studies on the sand fly fauna in this region are still limited. Findings Insects were collected from April 2011 to April 2012, using HP light traps distributed in four residential areas and one recreational area in Rio Branco, Capital of the State of Acre in the Amazon region of Brazil. A total of 456 sand flies were collected, comprising 256 females and 200 males. Taxonomic identification revealed 16 Phlebotominae genera and 23 species, as follows: Trichophoromyia auraensis, Nyssomyia whitmani, Nyssomyia antunesi, Pressatia choti, Evandromyia saulensis, Evandromyia walkeri, Evandromyia begonae, Migonemyia migonei, Pintomyia serrana, Psychodopygus paraensis, Sciopemyia sordelii, Migonemyia pusilla, Pintomyia nevesi, Brumptomyia avellari, Micropygomyia acanthopharynx, Micropygomyia micropyga, Pintomyia odax, Lutzomyia sherlocki, Pressatia calcarata, Pressatia duncanae, Bichromomyia flaviscutellata, Evandromyia bourrouli and Evandromyia bacula. From this group, Tr. auraensis and Ny. whitmani were the most abundant species in both forested areas and the peridomiciliary environment. Conclusions We find that the sand fly fauna in the urban and peri urban areas of Rio Branco is very diverse comprising 23 species, as diverse as that in areas of primary forest. Some species, such as Nyssomyia whitmani, Ny. antunesi and Bichromomyia flaviscutellata are known vectors of parasites responsible for cutaneous leishmaniasis, and Trichophoromyia auraensis is a putative vector in this Amazonian region.

To identify Lutzomyia (Nyssomyia) neivai naturally infected by Leishmania a multiplex polymerase chain reaction (PCR) was used for the analysis of 450 specimens (270 females, 180 males) collected in an endemic periurban area of cutaneous leishmaniasis in Porto Alegre, Brazil. Insects were grouped into pools of 10 and positive results were achieved in 3/27 Lu. (N.) neivai female pools. Infection by L. (Viannia) braziliensis was confirmed after hybridizing PCR products with a subgenus-specific biotinylated probe. Considering the detection of three positive pools with at least one infected insect in each, an infection rate of 1.1% was estimated. Our results associated with epidemiologic data suggest a potential ability of Lu. (N.) neivai in transmitting L. braziliensis in Porto Alegre, where the first notifications of autochthonous cutaneous leishmaniasis in humans occurred in 2002, with an increase in the number of cases in recent years possibly as a consequence of deforestation and agricultural activities in the area.

This paper is the first to report visceral leishmaniasis in domestic cats (Felis catus domesticus) from an endemic area in Rio de Janeiro state, Brazil. A relatively high seroprevalence of 25% was observed although none of them have presented any symptom. Our results support the observation of previous authors, suggesting that cats may be considered as alternative domestic hosts of visceral leishmaniasis and should be included in serological investigations performed in endemic areas.

Background The south coast of Rio de Janeiro State, in Brazil, is endemic for cutaneous and visceral leishmaniases and is frequently visited by tourists from different parts of the world. Since the complex epidemiology of leishmaniases demands local studies, the goal of this study was to investigate the phlebotomine sand fly fauna and leishmaniases transmission in Ilha Grande, an ecotourism area of Angra dos Reis municipality. Methods Sand fly fauna was sampled in three monitoring stations using HP light traps in domiciles, peridomiciles and forests. Species abundance was evaluated by the Index of Species Abundance. A Leishmania natural infection survey was done using multiplex PCR and dot blot hybridization. Results During 15 consecutive months of sand fly monitoring, 1093 specimens from 16 species were captured. The potential leishmaniases vectors found were Lutzomyia (Nyssomyia) intermedia, L. migonei, L. (N.) flaviscutellata, L. (Psychodopygus) ayrozai and L. (Lutzomyia) longipalpis. Five species were new records in Ilha Grande: L. (Sciopemyia) microps, L. termitophila, L. firmatoi, L. rupicola and L. (P.) ayrozai. Higher species richness was found inside forest areas, although potential leishmaniases vectors were present in deforested areas, peridomiciles and inside houses. Lutzomyia (N.) intermedia and L. migonei were the most abundant species. Females of L. migonei showed a high rate (10.3%) of natural infection by Leishmania (Viannia) sp., probably Leishmania (V.) braziliensis. Conclusions The detection of leishmaniases transmission and potential vectors in Ilha Grande is of public health concern, especially because tourists are frequently visiting the island. Besides reinforcing the epidemiological importance of L. (N.) intermedia in Rio de Janeiro State, the role of L. migonei in cutaneous leishmaniasis transmission is highlighted with its high rate of Leishmania natural infection. The finding of L. (L.) longipalpis confirmed the human autochthonous case of visceral leishmaniasis from the island. The presence of L. (N.) flaviscutellata in peridomestic areas is also an important finding, since the species is involved in the transmission of diffuse cutaneous leishmaniasis. Health education practices directed to the local community and tourists are important control actions that can be taken in Ilha Grande to reduce the burden of leishmaniases.

In order to determine natural Leishmania (Viannia) infection in Lutzomyia (Pintomyia) fischeri, a multiplex PCR methodology coupled to non-isotopic hybridization was adopted for the analysis of sand fly samples collected by CDC light traps in an endemic area of American Cutaneous Leishmaniasis (ACL) in the periurban region of the municipality of Porto Alegre, Rio Grande do Sul State, Brazil. We analyzed by PCR methodology 560 specimens of Lutzomyia (Pintomyia) fischeri (520 females and 40 males). The wild sand flies were grouped into 56 pools (52 females and 4 males) of 10 each, and positive results were detected in 2 of the 52 female pools, representing a minimum infection rate of 0.38% based on the presence of at least 1 infected insect in the pool. This result associated with some local evidence such as anthopophily, spatial distribution in accordance with the transmission area and human case incidence, suggests that L. (P.)fischeri may be considered as a secondary vector of ACL in the studied locality.

A study of the natural infection of phlebotomine sand flies by Leishmania (Leishmania) infantum was conducted in an area of visceral leishmaniasis in Sao Vicente Ferrer, located in the northern part of the Atlantic rain forest region in the State of Pernambuco, Brazil. In a previous study, Migonemyia migonei have been found predominantly in peridomiciles and houses in this endemic area. The analysis of M. migonei, collected by CDC light trap, by multiplex PCR assay coupled to non-isotopic hybridization showed that 2 females out of 50 were infected by L. infantum. This is the first finding of natural infection of M. migonei by L. infantum suggesting that M. migonei may be the vector of L. infantum in areas of visceral leishmaniasis where Lutzomyia longipalpis, the usual vector, is absent.

We performed a serological, clinical, and parasitological follow-up of a dog population in an endemic area of American Visceral Leishmaniasis estimated by indirect immunofluorescent assay (IFA) and western blot (WB). After twelve months, the results obtained from IFA demonstrated that 50% were seropositive and two serological profiles were observed: the first one ranging from 1/40 to 1/80 and the second≥1/160. By WB, it was observed that the same percentage and sera from positive dogs presented the recognition of the peptides of 29 and 32 kDa up to 8 months before IFA serum conversion. Among the positive dogs, all the sera from symptomatic ones with tissue parasitism recognized the peptide of 68.5 kDa. Our results suggest the need of modifications in the control measures regarding the elimination of the dogs. They also corroborate the high sensitivity and specificity of western blot in the diagnosis of canine leishmaniasis, suggesting the possibility of its association with IFA.