Distribution and identification of sand flies naturally infected with Leishmania from the Southeastern Peruvian Amazon.
ABSTRACT: Cutaneous leishmaniasis (CL) is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission.Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR) and nested Real time PCR for species identification.A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species) and Brumptomyia (2 species) were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia) spp. (24%), Lu. whitmani (19.4%) and Lu. yucumensis (15.8%) in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools), Mavila (10 pools) and Arca Pacahuara (1 pool) and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia) spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu. llanosmartinsi had the highest infection rates (8%, 7% and 6%, respectively). We identified Leishmania guyanensis in two Lu. whitmani pools, and L. braziliensis in two Lu. llanosmartinsi pools and one Lu. davisi pool.Based on our collections there is high sand fly diversity in Madre de Dios, with differences in sand fly abundance and species composition across sites. We identified 14 sand fly species naturally infected with Leishmania spp., having detected natural infection with L. (V.) guyanensis and L. (V.) braziliensis in three sand fly species. These results suggest the presence of several potential vectors that vary in their spatial and geographical distribution, which could explain the high prevalence of CL cases in this region.
Project description:Previous works showed that immunization with saliva from Lutzomyia intermedia, a vector of Leishmania braziliensis, does not protect against experimental infection. However, L. braziliensis is also transmitted by Lutzomyia whitmani, a sand fly species closely related to Lu. intermedia. Herein we describe the immune response following immunization with Lu. whitmani saliva and the outcome of this response after L. braziliensis infection.BALB/c mice immunized with Lu. whitmani saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-? and IL-10, by both CD4+ and CD8+ cells. Mice immunized as above and challenged with L. braziliensis plus Lu. whitmani saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-? production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with Lu. whitmani saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to Lu. whitmani saliva. However CL patients, with active lesions, displayed a lower humoral response to Lu. whitmani saliva compared to individuals with subclinical Leishmania infection.Pre-exposure to Lu. whitmani saliva induces protection against L. braziliensis in a murine model. We also show that Lu. whitmani salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies.
Project description:This study aimed to describe the sand fly fauna and detect trypanosomatids in these insects from Casa Branca, state of Minas Gerais, Brazil, an endemic area of both visceral (VL) and tegumentary leishmaniasis (TL). Sand flies were collected bimonthly from May 2013 to July 2014, using automatic light traps exposed for three consecutive nights in peridomiciliary areas of nine houses with previous reports of VL and TL. ITS1-PCR and DNA sequencing were performed for trypanosomatids identification. A total of 16,771 sand flies were collected belonging to 23 species. The most abundant species was Nyssomyia whitmani (Antunes & Coutinho, 1939) (70.9%), followed by Lutzomyia longipalpis (Lutz & Neiva, 1912) (15.2%) and Migonemyia migonei (França, 1920) (9.1%). Leishmania amazonensis DNA was detected in Ny. whitmani (four pools) and Le. braziliensis DNA was detected in Psychodopygus lloydi (one pool). In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for Leishmania DNA, the parasite species was not determined due to the low quality of the sequences. Moreover, DNA of Herpetomonas spp. was detected in Ny. whitmani (two pools) and Cortelezzii complex (one pool). DNA of Crithidia spp. was detected in Ny. whitmani and Ps. lloydi (both one pool). Our results suggest that Ny. whitmani may be involved in the transmission of Le. amazonensis in the study area. The molecular detection of Le. amazonensis suggests the presence of this species in a sylvatic cycle between vertebrate and invertebrate hosts in the region of Casa Branca. Our data also reveal the occurrence of other non-Leishmania trypanosomatids in sand flies in Casa Branca District.
Project description:Every year about 3 million tourists from around the world visit Brazil, Argentina and Paraguay´s triple border region where the Iguaçu Falls are located. Unfortunately, in recent years an increasing number of autochthonous canine and human visceral leishmaniasis (VL) cases have been reported. The parasite is Leishmania (Leishmania) infantum and it is transmitted by sand flies (Phlebotominae). To assess the risk factors favorable for the establishment and spread of potential vectors the Centers for Disease Control and Prevention light trap (CDC-light trap) collections were made in the Foz do Iguaçu (FI) and Santa Terezinha de Itaipu (STI) townships and along two transects between them. Our study determined the Phlebotominae fauna, the factors that affect the presence and abundance of Lutzomyia longipalpis and Nyssomyia whitmani, the presence of L. infantum in different sand fly species and which Leishmania species are present in this region. Lutzomyia longipalpis was the prevalent species and its distribution was related to the abundance of dogs. Leishmania infantum was found in Lu. longipalpis, Ny. whitmani, Ny. neivai and a Lutzomyia sp. All the results are discussed within the Stockholm Paradigm and focus on their importance in the elaboration of public health policies in international border areas. This region has all the properties of stable VL endemic foci that can serve as a source of the disease for neighboring municipalities, states and countries. Most of the urban areas of tropical America are propitious for Lu. longipalpis establishment and have large dog populations. Pan American Health Organization´s initiative in supporting the public health policies in the border areas of this study is crucial and laudable. However, if stakeholders do not act quickly in controlling VL in this region, the scenario will inevitable become worse. Moreover, L. (Viannia) braziliensis found in this study supports the need to develop public health policies to avoid the spread of cutaneous leishmaniasis. The consequences of socioeconomic attributes, boundaries and frontiers on the spread of diseases cannot be neglected. For an efficient control, it is essential that urban planning is articulated with the neighboring cities.
Project description:American cutaneous leishmaniasis (ACL) is a common and important vector-borne parasitic zoonosis in Panamá. Here, we study Leishmania spp. infection rates and blood-feeding patterns among common sand flies in Trinidad de Las Minas, a rural community with hyperendemic ACL transmission, and where a deltamethrin fogging trial was performed. Sand flies were collected from April 2010 to June 2011 with light traps installed inside and in the peridomicile of 24 houses. We restricted our analysis to the most abundant species at the study site: Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia panamensis, Lutzomyia triramula, and Lutzomyia dysponeta. We detected Leishmania spp. infection in sand flies by polymerase chain reaction (PCR) amplification of the internal transcribed spacer region 1 (ITS-1) in pooled females (1-10 females per pool). Host species of engorged sand flies were identified using a cytochrome b PCR. From 455 sand fly pools analyzed, 255 pools were positive for Leishmania spp., with an estimated infection rate (confidence interval) of 0.096 [0.080-0.115] before the deltamethrin fogging which slightly, but not significantly (P > 0.05), increased to 0.116 [0.098-0.136] after the deltamethrin fogging. Blood meal analysis suggested that pigs, goats, and birds were the most common sand fly blood sources, followed by humans and domestic dogs. DNA sequencing from a subsample of ITS-1 positive pools suggests that Leishmania panamensis, Leishmania naiffi, and other Leishmania spp. were the parasite species infecting the most common vectors at the study site. Our data confirm an association between sand fly species, humans, domestic dogs, and pigs and Leishmania spp. parasites in rural Panamá.
Project description:Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined.We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes.We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.
Project description:BACKGROUND:Sand flies (Diptera, Psychodidae, Phlebotominae) in the genus Lutzomyia are the predominant vectors of the protozoan disease leishmaniasis in the New World. Within the watershed of the Panama Canal, the cutaneous form of leishmaniasis is a continuous health threat for residents, tourists and members of an international research community. Here we report the results of screening a tropical forest assemblage of sand fly species for infection by both Leishmania and a microbe that can potentially serve in vector population control, the cytoplasmically transmitted rickettsia, Wolbachia pipientis. Knowing accurately which Lutzomyia species are present, what their evolutionary relationships are, and how they are infected by strains of both Leishmania and Wolbachia is of critical value for building strategies to mitigate the impact of this disease in humans. METHODOLOGY AND FINDINGS:We collected, sorted and then used DNA sequences to determine the diversity and probable phylogenetic relationships of the Phlebotominae occurring in the understory of Barro Colorado Island in the Republic of Panama. Sequence from CO1, the DNA barcoding gene, supported 18 morphology-based species determinations while revealing the presence of two possible "cryptic" species, one (Lu. sp. nr vespertilionis) within the Vespertilionis group, the other (Lu. gomezi) within the Lutzomyia-cruciata series. Using ITS-1 and "minicircle" primers we detected Leishmania DNA in 43.3% of Lu. trapidoi, 26.3% of Lu. gomezi individuals and in 0% of the other 18 sand fly species. Identical ITS-1 sequence was obtained from the Leishmania infecting Lu. trapidoi and Lu. gomezi, sequence which was 93% similar to Leishmania (viannia) naiffi in GenBank, a species previously unknown in Panama, but recognized as a type of cutaneous leishmaniasis vectored broadly across northern and central South America. Distinct strains of the intracellular bacterium Wolbachia were detected in three of 20 sand fly species, including Lu. trapidoi, in which it frequently co-occurred with Leishmania. CONCLUSIONS:Both morphological and molecular methods were used to examine an assemblage of 20 sand fly species occurring in the forests of the Panama Canal area. Two of these species, members of separate clades, were found to carry Leishmania at high frequency and hence are likely vectors of leishmaniasis to humans or other mammal species. A single Leishmania species, identified with high confidence as Le. naiffi, was carried by both species. That Le. naiffi is known to cause cutaneous lesions in South America but has hitherto not been reported or implicated in Panama opens the possibility that its range has recently expanded to include the Isthmus or that it occurs as a recent introduction. The occurrence of Leishmania and Wolbachia in Lu. trapidoi identifies one important vector of the disease as a potential target for gene introductions using Wolbachia population sweeps.
Project description:The aims of the study were to determine the blood feeding preferences of sandflies and to identify species of Leishmania that infected phlebotomines in Caxias, Maranhão, Brazil, an area that is highly endemic for leishmaniasis. Sandflies were captured in light traps located in the peridomiciliary environments of randomly selected houses in urban and rural settings between 1800 and 0600 hours on new moon days between March 2013 and February 2015. DNA extracts from 982 engorged female sandflies were submitted to fragment length polymorphism analysis to identify infecting species of Leishmania, and blood sources were identified for 778 of these specimens. Infection by Leishmania infantum was detected in Lutzomyia longipalpis, Lu. whitmani and Lu. termitophila; L. infantum/L. braziliensis in Lu. longipalpis, Lu. whitmani and Lu. trinidadensis; L. shawi in Lu. longipalpis; L. mexicana in Lu. longipalpis; L. braziliensis in Lu. longipalpis and Lu. whitmani; L. guyanensis in Lu. longipalpis and Lu. termitophila; L. amazonensis in Lu. longipalpis and L. lainsoni or L. naiffi in Lu. longipalpis, while Lu. longipalpis and Lu. trinidadensis were infected with unidentified Leishmania sp. Blood sources were identified in 573 individual phlebotomines and the preferred hosts were, in decreasing order, chicken, dog, rodent and human with lower preferences for pig, horse, opossum and cattle. Lu. longipalpis and Lu. whitmani performed mixed feeding on man, dog and rodent, while Lu. longipalpis was the most opportunistic species, feeding on the blood of all hosts surveyed, but preferably on dog/chicken, dog/rodent and rodent/chicken. Our findings reveal the concomitant circulation of Leishmania species that cause visceral leishmaniasis and tegumentary leishmaniasis in the study area, and explain the occurrence of autochthonous human cases of both clinical forms of leishmaniasis in Caxias, Maranhão. The results support our hypothesis that, in the municipality of Caxias, transmission of Leishmania occurs in close proximity to humans.
Project description:Knowledge on synanthropic phlebotomines and their natural infection by Leishmania is necessary for the identification of potential areas for leishmaniasis occurrence.To analyse the occurrence of Phlebotominae in gallery forests and household units (HUs) in the city of Palmas and to determine the rate of natural infection by trypanosomatids.Gallery forests and adjacent household areas were sampled on July (dry season) and November (rainy season) in 2014. The total sampling effort was 960 HP light traps and eight Shannon traps. Trypanosomatids were detected in Phlebotominae females through the amplification of the SSU rDNA region, and the positive samples were used in ITS1-PCR. Trypanosomatid species were identified using sequencing.A total of 1,527 sand flies representing 30 species were captured in which 949 (28 spp.) and 578 (22 spp.) were registered in July and November, respectively. In July, more specimens were captured in the gallery forests than in the HUs, and Nyssomyia whitmani was particularly frequent. In November, most of the specimens were found in the HUs, and again, Ny. whitmani was the predominant species. Lutzomyia longipalpis was commonly found in domestic areas, while Bichromomyia flaviscutellata was most frequent in gallery forests. Molecular analysis of 154 pools of females (752 specimens) identified Leishmania amazonensis, L. infantum, and Crithidia fasciculata in Ny. whitmani, as well as L. amazonensis in Lu. longipalpis, Trypanosoma sp. and L. amazonensis in Pintomyia christenseni, and L. amazonensis in both Psathyromyia hermanlenti and Evandromyia walkeri.These results show the importance of gallery forests in maintaining Phlebotominae populations in the dry month, as well as their frequent occurrence in household units in the rainy month. This is the first study to identify Leishmania, Trypanosoma, and Crithidia species in Phlebotominae collected in Palmas, Tocantins, Brazil.
Project description:Visceral leishmaniasis (VL) is an important public health challenge in Brazil because of the high number of human and canine cases reported annually. Leishmania infantum is the etiological agent of VL and Lutzomyia longipalpis is its main vector. However, evidence suggests that this taxon constitutes a species complex. In Sao Paulo state, there are two populations of Lu. longipalpis, each secreting distinct pheromones, (S)-9-methylgermacrene-B and Cembrene 1; both have been associated with different patterns of VL transmission. The aim of the present study was to investigate the temporal distribution and natural infection of the (S)-9-methylgermacrene-B population of the Lu. longipalpis complex in a highly VL endemic area of Sao Paulo state to obtain information that may contribute to the surveillance of this zoonosis and to the planning of preventive and control measures.The study was carried out in Panorama municipality, Sao Paulo State. Captures were made during 24 months in seven domiciles. The relation between sand fly abundance and climatic variables, temperature and humidity, was analyzed and natural infection by Leishmania spp. in sand fly females was investigated by nested PCR.A total of 4120 sand flies, with predominance of Lu. longipalpis (97.2%) were captured. The highest averages of sand flies/night/trap occurred in the rainy season (November-March) and a positive, significant correlation between sand fly abundance and the temperature and humidity 20 days before the capture days was found. Leishmania infantum DNA was detected in three out of 250 pools of females analyzed, giving an estimated minimum infection rate of 1.2%.The identification of the climatic association between the high abundance of the vector in this highly endemic VL focus constitutes a fundamental point for evaluating future vector and dog control measures and this information increases the data of VL foci in Sao Paulo state that could contribute to the public health authorities in planning prevention and control measures. The identification of natural infection by Le. infantum in Lu. longipalpis specimens reinforces the importance of entomological surveillance activities in this municipality.
Project description:Sand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive.We screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia.Salivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.