Home sweet home: sand flies find a refuge in remote indigenous villages in north-eastern Brazil, where leishmaniasis is endemic.

BACKGROUND: From 2012 to 2013, an outbreak of cutaneous leishmaniasis by Leishmania braziliensis was detected in indigenous villages located in a remote rural area of Pernambuco state, north-eastern Brazil. Considering that the principal activities of this indigenous community are farming and crop plantation, and also that the outbreak involved many children, we investigated the presence of sand fly vectors inside human houses and also the exposure of dogs to leishmanial parasites. Our general objective was to gather epidemiological data that could indicate the occurrence of a peri-domestic/domestic transmission cycle of L. braziliensis in these indigenous villages. METHODS: From March 2015 to March 2016, sand flies were collected using light traps in the indoor and immediate outdoor environments in the three indigenous villages that reported the most cutaneous leishmaniasis cases during the 2012-2013 outbreak. Moreover, samples obtained from 300 dogs living in the outbreak villages and two nearby villages were tested by a rapid immunochromatographic test and by a real-time PCR for detecting anti-Leishmania antibodies and Leishmania DNA, respectively. RESULTS: In total, 5640 sand flies belonging to 11 species were identified. Males (n = 3540) predominated over females (n = 2100). Migonemyia migonei (84.3%) was the most abundant species, followed by Evandromyia lenti (5.5%), Lutzomyia longipalpis (4.1%), Nyssomyia intermedia (1.6%) and Micropygomyia capixaba (1.4%), representing together ~97% of the sand flies collected. Nine out of the 11 species identified in this study were found indoors, including M. migonei, L. longipalpis and N. intermedia, which are proven vectors of Leishmania spp. Out of 300 dogs tested, 26 (8.7%) presented anti-Leishmania antibodies and six (2%) were Leishmania DNA-positive. The level of exposure in dogs living in the indigenous villages where the 2012-2013 outbreak of human CL was detected was almost 2-fold higher than in the two nearby villages (11.0 vs 6.2% for serology and 2.6 vs 1.4% for real-time PCR). CONCLUSIONS: The results suggest that different sand fly vectors may be adapted to human dwellings, thus increasing the risk of transmission in the indoor and immediate outdoor environments. The adaptation of sand flies to the indoor environment in the studied indigenous villages may be partly explained by the poor housing conditions and the proximity of the houses to crop plantations and forest fragments.

Morphological and phylogenetic analyses of Lutzomyia migonei from three Brazilian states.

Lutzomyia migonei is incriminated as a vector of Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. Recently, this phlebotomine sand fly species has been suggested as a vector for Leishmania infantum, which causes zoonotic visceral leishmaniasis. Considering the widespread distribution of Lu. migonei in South America, the existence of isolated populations has been hypothesized. Three Lu. migonei populations, two from north-eastern Brazil (Machados, Pernambuco State, and Baturité, Ceará State) and other from the south-eastern region (Niterói, Rio de Janeiro State) were analysed both morphologically and genetically. Though no significant morphological differences were found amongst the sand fly specimens analysed, discriminant analysis based on specific morphometric characters (i.e., length of wing, antennal segment 3 and coxite for males, and length of wing and antennal segment 3 for females), showed that specimens from Machados were closer to Baturité than to Niterói. The molecular analysis of cytochrome c oxidase subunit I gene sequences also supported this observation by the distinct separation of two monophyletic clades, grouping specimens from Machados and Baturité separately from those of Niterói. Our results suggest the existence of different populations within the distribution range of Lu. migonei. Whether these populations are reproductively isolated and/or present differences in terms of vector competence/capacity for L. braziliensis and L. infantum needs to be further investigated.

Sandflies (Diptera: Psychodidae) in an urban area of Northeastern Brazil.

INTRODUCTION:: The sandfly fauna is well studied globally. In Brazil, sandfly fauna is very diverse in the Northeast region, especially in states such as Maranhão, Ceará, and Bahia. However, in the State of Pernambuco, the distribution of these insects is still not well known. Therefore, the objective of this study was to identify the different species that constitute the sandfly fauna in an urban area in the Northeast region of Brazil, where an outbreak of visceral leishmaniasis (VL) was recently reported. METHODS:: The sandflies were collected from an urban area endemic for VL, at five collection points. The collection of samples was carried out from November 2014 to December 2015, using CDC light traps installed in intradomiciliary and peridomiciliary fashion. RESULTS:: The collected sandflies (n = 297) belonged to eight species: Lutzomyia lenti, Lutzomyia longipalpis, Lutzomyia sallesi, Lutzomyia migonei, Lutzomyia walkeri, Lutzomyia capixaba, Lutzomyia carmelinoi, and Lutzomyia whitmani. Most of the specimens collected were peridomiciliary (247/297, 83%). L. lenti (154/297, 52%) was the most frequently sampled species, followed by L. longipalpis (88/297, 29.6%), and L. sallesi (42/297, 14.1%), which together accounted for over 90% of the collected sandfly specimens. CONCLUSIONS:: The continued presence of L. longipalpis in urban areas, including that in intradomiciliary areas, with a predominance of females, is crucial because of the high possibility of them causing VL outbreaks, since this species is the main vector of Leishmania infantum in Brazil.

Sandflies (diptera: psychodidae) in an urban area of Northeastern Brazil

Abstract INTRODUCTION: The sandfly fauna is well studied globally. In Brazil, sandfly fauna is very diverse in the Northeast region, especially in states such as Maranhão, Ceará, and Bahia. However, in the State of Pernambuco, the distribution of these insects is still not well known. Therefore, the objective of this study was to identify the different species that constitute the sandfly fauna in an urban area in the Northeast region of Brazil, where an outbreak of visceral leishmaniasis (VL) was recently reported. METHODS: The sandflies were collected from an urban area endemic for VL, at five collection points. The collection of samples was carried out from November 2014 to December 2015, using CDC light traps installed in intradomiciliary and peridomiciliary fashion. RESULTS: The collected sandflies (n = 297) belonged to eight species: Lutzomyia lenti, Lutzomyia longipalpis, Lutzomyia sallesi, Lutzomyia migonei, Lutzomyia walkeri, Lutzomyia capixaba, Lutzomyia carmelinoi, and Lutzomyia whitmani. Most of the specimens collected were peridomiciliary (247/297, 83%). L. lenti (154/297, 52%) was the most frequently sampled species, followed by L. longipalpis (88/297, 29.6%), and L. sallesi (42/297, 14.1%), which together accounted for over 90% of the collected sandfly specimens. CONCLUSIONS: The continued presence of L. longipalpis in urban areas, including that in intradomiciliary areas, with a predominance of females, is crucial because of the high possibility of them causing VL outbreaks, since this species is the main vector of Leishmania infantum in Brazil.

Identification of phlebotomine sand fly blood meals by real-time PCR.

BACKGROUND: Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. METHODS: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 µl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. RESULTS: The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. CONCLUSIONS: The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.

Molecular detection of Leishmania in phlebotomine sand flies in a cutaneous and visceral leishmaniasis endemic area in northeastern Brazil.

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

Detection and quantification of Leishmania braziliensis in ectoparasites from dogs.

American cutaneous leishmaniasis (ACL) is a disease caused by different species of Leishmania protozoa, Leishmania braziliensis being the main species found in Brazil. In this study, two rural areas in Pernambuco, northeastern Brazil, where ACL is endemic, were selected. Genomic DNA was extracted from canine ectoparasites (ticks, fleas, and lice) and tested using a conventional PCR and a quantitative real time PCR. A total of 117 ectoparasites were collected, being 50 (42.74%) of them positive for L. braziliensis (in at least one PCR protocol), with a mean parasite load of 14.14 fg/µL. Furthermore, 46 (92.00%) positive ectoparasites were collected from positive dogs and 4 (8.00%) from negative ones. This study reports the detection of L. braziliensis DNA in ectoparasites, but does not prove their vector competence. Certainly, experimental transmission studies are necessary to assess their role, if any, in the transmission of Leishmania parasites to dogs.

Ecology of Lutzomyia longipalpis in an area of visceral leishmaniasis transmission in north-eastern Brazil.

Visceral leishmaniasis is a major public health issue in South America, where the disease is rapidly spreading. Changes in ecology and distribution of the principal vector, Lutzomyia longipalpis are among the factors accounting for the increasing incidence of the disease in this region. However, information about the ecology of L. longipalpis is still incipient, which may directly impair the implementation of effective control programs. Herein, the ecology of L. longipalpis was studied in a focus of visceral leishmaniasis in north-eastern Brazil. From August 2009 to August 2010, phlebotomine sand flies were monthly collected in four localities using CDC light traps (~37 per month) and a lantern-baited Shannon trap with mouth aspirators. A total of 24,226 phlebotomine sand flies were collected with light traps and 375 with mouth aspirators. The most abundant species was L. longipalpis, representing 97.9% of the specimens collected with light traps and 91.5% with the mouth aspirator. Other species (Lutzomyia evandroi, Lutzomyia lenti and Lutzomyia sallesi) were found in low numbers. Most phlebotomine sand flies (94.6%) were collected at chicken coops and corrals. No significant correlation was found between the monthly abundance of phlebotomine sand flies and the monthly averages of temperature, relative humidity or rainfall. However, interestingly enough, 82.4% of L. longipalpis specimens were collected in months when relative humidity surpassed 75%. This study points out that this vector is well adapted to live in different habitats and to different climate conditions. It also suggests that some north-eastern populations of L. longipalpis may be more xerotolerant than southern populations. Further studies to assess the relationship between microclimate and L. longipalpis density in different Brazilian regions are advised.

Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

Phlebotomine sandflies (Diptera: Psychodidae) in São Vicente Férrer, a sympatric area to cutaneous and visceral leishmaniasis in the state of Pernambuco, Brazil.

INTRODUCTION: In the last decades, a considerable geographic expansion of the leishmaniases in all regions of Brazil has been observed. The present study was carried out to identify the composition of the phlebotomine sandfly fauna and verify the seasonal variation of the main species after environmental changes occurred in São Vicente Férrer Municipality, State of Pernambuco, Brazil. METHODS: Captures were carried out during four consecutive nights of each month using Centers for Disease Control and Prevention light traps from September 2009 to September 2010. The correlation between the number of phlebotomine sandflies captured and climatic factors (temperature and rainfall) was evaluated. RESULTS: A total of 13,872 specimens belonging to 20 species were captured, of which, 6,247 (45%) were females, and 7,625 (55%) were males. Lutzomyia migonei was the most abundant species with 9,964 (71.8%) specimens, being predominant in the intradomicile and peridomicile areas with 108 (86.4%) and 9,746 (97%), respectively. In the forest remnants, Lutzomyia complexa 2,395 (65%) and Lutzomyia sordellii 770 (20.8%) predominated. The correlation analysis between the total number of sandflies captured and climatic factors did not show a significant influence on population density. CONCLUSIONS: The high abundance of Lutzomyia migonei and Lutzomyia complexa indicates the possibility of new cases of cutaneous leishmaniasis (CL).

Microspatial distributional patterns of vectors of cutaneous leishmaniasis in pernambuco, northeastern Brazil.

The purpose of this study is to analyze the spatial distribution and population trends through time of Lutzomyia species in a long-term focus of cutaneous leishmaniasis transmission in an Atlantic Forest area, northeastern Brazil. Sand fly populations of different ecological niches were monitored spatiotemporally in 2009. To summarize vegetation characteristics and phenology, we calculated the Normalized Difference Vegetation Index from Landsat images. Using niche modeling approaches, we assessed suites of environmental factors to identify areas of transmission risk. Although 12 species were detected, L. whitmani was the most abundant and broadly distributed across the area, particularly in peridomiciliary locations, and associated negatively with denser vegetation areas. On the other hand, L. complexa, L. sordelli, and L. tupynambai were found almost exclusively in forested areas (P < 0.05), and associated positively with denser vegetation. Lutzomyia species' occurrences are related to specific environmental combinations (with contrast among species) in the region.

Phlebotomine sandflies (Diptera: Psychodidae) in São Vicente Férrer, a sympatric area to cutaneous and visceral leishmaniasis in the state of Pernambuco, Brazil / Flebotomíneos (Diptera: Psychodidae) em São Vicente Férrer, uma área simpátrica para leishmaniose tegumentar e visceral, no estado de Pernambuco, Brasil

INTRODUCTION: In the last decades, a considerable geographic expansion of the leishmaniases in all regions of Brazil has been observed. The present study was carried out to identify the composition of the phlebotomine sandfly fauna and verify the seasonal variation of the main species after environmental changes occurred in São Vicente Férrer Municipality, State of Pernambuco, Brazil. METHODS: Captures were carried out during four consecutive nights of each month using Centers for Disease Control and Prevention light traps from September 2009 to September 2010. The correlation between the number of phlebotomine sandflies captured and climatic factors (temperature and rainfall) was evaluated. RESULTS: A total of 13,872 specimens belonging to 20 species were captured, of which, 6,247 (45 percent) were females, and 7,625 (55 percent) were males. Lutzomyia migonei was the most abundant species with 9,964 (71.8 percent) specimens, being predominant in the intradomicile and peridomicile areas with 108 (86.4 percent) and 9,746 (97 percent), respectively. In the forest remnants, Lutzomyia complexa 2,395 (65 percent) and Lutzomyia sordellii 770 (20.8 percent) predominated. The correlation analysis between the total number of sandflies captured and climatic factors did not show a significant influence on population density. CONCLUSIONS: The high abundance of Lutzomyia migonei and Lutzomyia complexa indicates the possibility of new cases of cutaneous leishmaniasis (CL).

INTRODUÇÃO: Nas últimas décadas tem se observado uma considerável expansão geográfica das leishmanioses em todas as regiões do Brasil. O presente estudo foi realizado para identificar a fauna de flebotomíneos e verificar a sazonalidade das principais espécies após mudanças ambientais ocorridas no município de São Vicente Férrer, Estado de Pernambuco, Brasil. MÉTODOS: As capturas foram realizadas durante quatro noites consecutivas por mês usando armadilhas luminosas CDC, no período de setembro de 2009 a setembro de 2010. A correlação entre o número de flebotomíneos capturados e fatores climáticos (temperatura e umidade) foi avaliada. RESULTADOS: Foi capturado um total de 13.872 espécimes pertencentes a 20 espécies, sendo 6.247 (45 por cento) fêmeas e 7.625 (55 por cento) machos. Lutzomyia migonei foi a espécie mais abundante com 9.964 (71,8 por cento) espécimes, sendo predominante no intradomicílio 108 (86,4 por cento) e peridomicílio 9.746 (97 por cento). Nos resquícios de mata primária, predominaram Lutzomyia complexa 2.395 (65 por cento) e Lutzomyia sordellii 770 (20,8 por cento). A análise de correlação entre o número total de flebotomíneos capturados e os fatores climáticos não mostrou uma influência significativa na densidade da população. CONCLUSÕES: A elevada abundância de Lutzomyia migonei e Lutzomyia complexa indica a possibilidade de novos casos de leishmaniose cutânea (LC).

Spatial and temporal patterns of occurrence of Lutzomyia sand fly species in an endemic area for cutaneous leishmaniasis in the Atlantic Forest region of northeast Brazil.

Sand fly populations of different ecological niches in the Amaraji endemic American Cutaneous Leishmaniasis (ACL) focus of the Pernambuco Atlantic Forest region of northeastern Brazil were monitored spatiotemporally. Lutzomyia whitmani was dominant in all niches but occurred in smaller numbers in forested locations. L. whitmani was significantly less seasonal than the other species, being present throughout the year while other species were more abundant between February and April. These results suggest that L. whitmani may potentially be the principal vector of ACL in the region, even though the sand fly fauna was diverse: 88% were L.whitmani and 12% belonged to 11 other species. Two other species, L. complexa (1.3%) and L. migonei (0.8%), considered to be ACL vectors in other regions, were also present. This detailed picture of the sand fly population's abundance and spatiotemporal distribution provides a basis for future modeling studies of forecasting sand fly activity patterns and ACL occurrence.

Comportamento da fauna de flebotomíneos, com ênfase em Lutzomyia Longpalpis, em área endêmica para Leishmaniose Visceral no Município de Passira, Agreste de Pernambuco / Behavior phlebotomine fauna, with emphasis on Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in municipal Passirra, Agreste of Pernambuco

As leishmanioses são doenças infecciosas, causadas por protozoários do gênero Leishmania, trasmistidos por insetos da família Phlebotominae (Diptera: Psychodidae). Em Pernambuco, a LV vem apresentando importante expansão geográfica. Passira registrou 06 casos de LV humana entre 2002 e 2007 e, 35 casos de LVC entre 2005 e 2009. Este estudo objetivou descrever o comportamento da fauna de flebotomíneos, com ênfase em Lutzomyia longipalpis, em área endêmica para leishmaniose visceral no município de Passira, Agreste de Pernambuco. Quatro localidades foram selecionadas para o estudo (Apara, Poço do Pau, Sítio Borba e Varjada de Cima). Utilizou-se armadilhas CDC, armadilha de Shannon e armadilha Disney modificada com isca animal (Galea spixii e Rattus rattus) para coletar os flebotomíneos em diferentes ecótopos. Dos flebotomíneos coletados alguns foram dissecados e o restante foi feito pools com 5 ou 10 exemplares para extração e detecção do parasito através do teste de PCR. Entre agosto/2009 a agosto/2010 foram capturados 24.226 flebotomíneos com armadilha CDC, em diferentes ecótopos. Destes, 23.716 exemplares são de Lutzomyia longipalpis. Ademais, foram realizadas capturas com Armadilha de Shannon, na qual se coletaram 373 espécimens de Lutzomyia longipalpis, sendo então dissecados 30 exemplares. Verificou-se que de todos os ecótopos trabalhados, o E1 referente ao galinheiro foi o que mais atraiu os flebotomíneos. Três animais (dois Galea spixii e um Rattus rattus) foram utilizados para captura de flebotomíneos com armadilha Disney modificada, sendo 228 espécimens coletados de Lutzomyia longipalpis. Adicionalmente, verificou-se a flutuação da populaçao de flebotomíneos com a variação climática na região, no qual se podem observar picos de flebotomíenos em períodos após a chuva, que correspondeu ao mês de fevereiro. Para detecção de L. infantum foram analisadas 628 pools de flebotomíneos, sendo 01 exemplar de Lutzomyia longipalpis apresentando positividade...

Leishmaniasis are infectious diseases caused by protozoa of the genus Leishmania, transmited by insects of Phlebotominae family (Diptera: Psychodidae). In Pernambuco, the VL has shown significant geographic expansion. Passira recorded 06 cases of human VL between 2002 and 2007, and 35 cases of VCL between 2005 and 2009. This study aimed describe thebehavior of the sandfly fauna, with emphasis on Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in the municipality of Passira, Agreste of Pernambuco. Four locations were selected for the study (Apara, Poço do Pau, Sítio Borba and Varjada de Cima). We used CDC light traps, Shannon traps and traps baited with Disney modified animal (Galea spixii and Rattus rattus) to collect sand flies in different ecotypes. Some of the sandflies collected were dissected and the remainder was made pools of 5 or 10 copies for extraction anddetection of parasites by PCR test. Between August/2009 and August/2010 were captured 24.226 san flies with CDC light traps, in differents ecotypes. Of these, 23.716 specimens are Lutzomyia longipalpis. Moreover, there were captured with Shannon trap, in wich collected 373 specimens of Lutzomyia longipalpis, and then dissected 30 specimens. It was found that all workd ecotypes, E1 corresponding to henhouse was the most attracted sand flies. Threeanimals (two Galea spixii and one Rattus rattus) were used to capture sand flies with Disney trap changed, being 228 specimens of Lutzomyia longipalpis collected. Additionally, there was a fluctuation of the population of sand flies with climate variability in the region in which we can observe peaks of sand flies after rain periods, which corresponded to the month of February. For detection of L. infantum were analyzed 628 pools of sand flies, and 01 copie of Lutzomyia longipalpis was positive.