Incipient colonisation of Lutzomyia longipalpis in the city of Resistencia,province of Chaco,Argentina (2010-2012)

Lutzomyia longipalpis was recorded for the first time in Argentina in 2004, in the province of Formosa. In the following years, the vector spread to the south and west in the country and was recorded in the province of Chaco in 2010. From November 2010-May 2012, captures of Phlebotominae were made in the city of Resistencia and its surroundings, to monitor the spread and possible colonisation of Lu. longipalpis in the province of Chaco. In this monitoring, Lu. longipalpis was absent in urban sampling sites and its presence was restricted to Barrio de los Pescadores. This suggests that the incipient colonisation observed in 2010 was not followed by continuous installation of vector populations and expansion of their spatial distribution as in other urban centres of Argentina.     

Metagenomic analysis of taxa associated with Lutzomyia longipalpis, vector of visceral leishmaniasis, using an unbiased high-throughput approach

Background

Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases.

Methodology/Principal Findings

An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans.

Conclusions/Significance

This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.     

Predicting the geographic distribution of Lutzomyia longipalpis (Diptera: Psychodidae) and visceral leishmaniasis in the state of Mato Grosso do Sul,Brazil

To understand the geographic distribution of visceral leishmaniasis (VL) in the state of Mato Grosso do Sul (MS), Brazil, both the climatic niches of Lutzomyia longipalpis and VL cases were analysed. Distributional data were obtained from 55 of the 79 counties of MS between 2003-2012. Ecological niche models (ENM) of Lu. longipalpis and VL cases were produced using the maximum entropy algorithm based on eight climatic variables. Lu. longipalpis showed a wide distribution in MS. The highest climatic suitability for Lu. longipalpis was observed in southern MS. Temperature seasonality and annual mean precipitation were the variables that most influenced these models. Two areas of high climatic suitability for the occurrence of VL cases were predicted: one near Aquidauana and another encompassing several municipalities in the southeast region of MS. As expected, a large overlap between the models for Lu. longipalpis and VL cases was detected. Northern and northwestern areas of MS were suitable for the occurrence of cases, but did not show high climatic suitability for Lu. longipalpis . ENM of vectors and human cases provided a greater understanding of the geographic distribution of VL in MS, which can be applied to the development of future surveillance strategies.     

Polymerase chain reaction‐based assay for the detection and identification of sand fly gregarines in Lutzomyia longipalpis,a vector of visceral leishmaniasis

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly host‐specific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina).     

Spatial and temporal changes in Lutzomyia longipalpis abundance,a Leishmania infantum vector in an urban area in northeastern Argentina

This study aimed to analyse changes in the spatial distribution of Lutzomyia longipalpis in Posadas, an urban area located in northeastern Argentina. Data were obtained during the summer of 2007 and 2009 through two entomological surveys of peridomiciles distributed around the city. The abundance distribution pattern for 2009 was computed and compared with the previous pattern obtained in 2007, when the first human visceral leishmaniasis cases were reported in the city. Vector abundance was also examined in relation to micro and macrohabitat characteristics. In 2007 and 2009, Lu. longipalpis was distributed among 41.5% and 31% of the households in the study area, respectively. In both years, the abundance rates at most of the trapping sites were below 30 Lu. longipalpis per trap per night; however, for areas exhibiting 30-60 Lu. longipalpis and more than 60 Lu. longipalpis, the areas increased in both size and number from 2007-2009. Lu. longipalpis was more abundant in areas with a higher tree and bush cover (a macrohabitat characteristic) and in peridomiciles with accumulated unused material (a microhabitat characteristic). These results will help to prioritise and focus control efforts by defining which peridomiciles display a potentially high abundance of Lu. longipalpis.     

Focus: Zoonotic Disease: Eco-epidemiological Aspects of Visceral Leishmaniasis in the Municipality of Diamantina,Jequitinhonha Valley (Minas Gerais State, Brazil)

Background: The present study was carried out in the rural and urban area of Diamantina/Minas Gerais (MG), an endemic municipality for visceral leishmaniasis (VL) in Brazil. Methods: Patient notification records, canine prevalence, and phlebotomine fauna were evaluated. Results: In the period from 2016 to 2018, eight human cases were confirmed, with three deaths, predominantly in males. In the same period, a total of 1,388 dogs resided in the rural and urban area of the municipality were submitted to the DPP® and ELISA, with a percentage of confirmed canine cases of 29.9% and 29.4%, respectively. The entomological study conducted in the municipality revealed the presence of 10 species of sand flies, with a predominance of Lutzomyia longipalpis (55.75%), mainly in the rural area. Conclusions: Unlike what is happening in urban centers, the results of this study suggest that the VL in Diamantina is in the process of urbanization, given the high percentage of confirmed canine cases and the high density of Lu. longipalpis in the rural area of the municipality. These risk factors warn about the need for continuous surveillance and the need to control actions of VL in this area.     

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

Background

Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure.

Methodology/principal findings

ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples.

Conclusion

Our results show the possibility of substituting Lu. longipalpis SGS for two recombinant proteins, LJM17 and LJM11, in order to probe for vector exposure in individuals residing in endemic areas.     

Lutzomyia longipalpis saliva or salivary protein LJM19 protects against Leishmania braziliensis and the saliva of its vector, Lutzomyia intermedia

Background

Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis.

Methodology/Principal Findings

Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-β. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression.

Conclusions/Significance

Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.     

Effect of Leishmania spp infection on the survival,life expectancy,fecundity and fertility of Lutzomyia longipalpis s.l. and Lutzomyia pseudolongipalpis

We evaluated the effects of Leishmania spp infection on several population parameters of Lutzomyia longipalpis sensu lato andLutzomyia pseudolongipalpis, vectors of visceral leishmaniasis in Venezuela, under experimental conditions during the first post-feeding period. Females of both species were allowed to feed and engorge on a suspension of fresh washed human red blood cells in foetal calf serum. These blood cells were either non-infected or infected with one of the fourLeishmania spp strains and were offered through a chicken skin membrane. The longevity, life expectancy and the fecundity of uninfected flies were similar in both species, but the fertility was significantly lower in uninfected Lu. longipalpis females. In all cases, the infection of Lu. longipalpis and Lu. pseudolongipalpis by the Leishmania strains resulted in significant detrimental effects, which exerted a fitness cost expressed by reduced survival and life expectancy, as well as decreased fertility and fecundity compared with the control groups. Nevertheless, differences in these parameters were observed between these vector species depending on whether they were infected with the autochthonous Venezuelan Leishmania infantum strain (NESA) or the Brazilian reference strain (PP75). The experimental data obtained agree with field data on the natural infection of these vector species and the significance of this scenario is discussed.     

Climatic factors and population density of Lutzomyia longipalpis (Lutz & Neiva, 1912) in an urban endemic area of visceral leishmaniasis in midwest Brazil

The life cycle of vectors and the reservoirs that participate in the chain of infectious diseases have a strong relationship with the environmental dynamics of the ecosystems in which they live. Oscillations in population abundance and seasonality of insects can be explained by factors inherent in each region and time period. Therefore, knowledge of the relationship and influence of environmental factors on the population of Lutzomyia longipalpis is necessary because of the high incidence of visceral leishmaniasis (VL) in Brazil. This study evaluates the influence of abiotic variables on the population density and seasonal behavior of L. longipalpis in an urban endemic area of VL in Brazil. The sand fly captures were performed every two months between November, 2009 and November, 2010 in the peridomicile of 13 randomly selected residences. We captured 1,367 specimens of L. longipalpis, and the ratio of male/female flies was 2.86:1. The comparison of the total male specimens in the two seasons showed a statistical difference in the wet season, but there was no significant difference when considering the total females. With respect to climatic variables, a significant negative association was observed only with wind speed. During periods of high wind speeds, the population density of this vector decreased. The presence of L. longipalpis was found in all months of the study with bimodal behavior and population peaks during the wet season.     

Phlebotomine fauna,natural infection rate and feeding habits of Lutzomyia cruzi in Jaciara,state of Mato Grosso, Brazil

Visceral leishmaniasis (VL) in Brazil is transmitted by the phlebotomine Lutzomyia longipalpis and in some midwestern regions by Lutzomyia cruzi. Studies of the phlebotomine fauna, feeding habits and natural infection rate by Leishmania contribute to increased understanding of the epidemiological chain of leishmaniases and their vectorial capacity. Collections were performed in Jaciara, state of Mato Grosso from 2010-2013, during which time 2,011 phlebotomines (23 species) were captured (68.70% Lu. cruzi and 20.52% Lutzomyia whitmani). Lu. cruzi females were identified by observing the shapes of the cibarium (a portion of the mouthpart) and spermatheca, from which samples were obtained for polymerase chain reaction to determine the rates of natural infection. Engorged phlebotomines were assessed to identify the blood-meal host by ELISA. A moderate correlation was discovered between the number of Lu. cruzi and the temperature and the minimum rate of infection was 6.10%. Twenty-two females were reactive to the antisera of bird (28%), dog (3.30%) and skunk (1.60%). We conclude that Lu. cruzi and Lu. whitmani have adapted to the urban environment in this region and that Lu. cruzi is the most likely vector of VL in Jaciara. Moreover, maintenance of Leishmania in the environment is likely aided by the presence of birds and domestic and synanthropic animals.     

Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.     

Cases and distribution of visceral leishmaniasis in western São Paulo: A neglected disease in this region of Brazil

Visceral leishmaniasis (VL) is one of the most prevalent parasitic diseases worldwide. In 2019, 97% of the total numbers of cases in Latin America were reported in Brazil. In São Paulo state, currently 17.6% of infected individuals live in the western region. To study this neglected disease on a regional scale, we describe the spread of VL in 45 municipalities of the Regional Network for Health Assistance11(RNHA11). Environmental, human VL (HVL), and canine VL (CVL) cases, Human Development Index, and Lutzomyia longipalpis databases were obtained from public agencies. Global Moran’s I index and local indicators of spatial association (LISA) statistics were used to identify spatial autocorrelation and to generate maps for the identification of VL clusters. On a local scale, we determined the spread of VL in the city of Teodoro Sampaio, part of the Pontal of Paranapanema. In Teodoro Sampaio, monthly peri-domicile sand fly collection; ELISA, IFAT and Rapid Test serological CVL; and ELISA HVL serum surveys were carried out. In RNHA11 from 2000 to 2018, Lu. longipalpis was found in 77.8%, CVL in 69%, and HVL in 42.2% of the 45 municipalities, and 537 individuals were notified with HVL. Dispersion occurred from the epicenter in the north to Teodoro Sampaio, in the south, where Lu. longipalpis and CVL were found in 2010, HVL in 2018, and critical hotspots of CVL were found in the periphery. Moran’s Global Index showed a weak but statistically significant spatial autocorrelation related to cases of CVL (I = 0.2572), and 11 municipalities were identified as priority areas for implementing surveillance and control actions. In RNHA11, a complex array of socioeconomic and environmental factors may be fueling the epidemic and sustaining endemic transmission of VL, adding to the study of a neglected disease in a region of São Paulo, Brazil.     

Phlebotominae of epidemiological importance in cutaneous leishmaniasis in northwestern Argentina: risk maps and ecological niche models

In Argentina, 58.2% out of the 8126 Cutaneous Leishmaniasis (CL) incident cases accumulated from 1954 to 2006 were reported in the provinces of Salta and Jujuy. The aim of this study was to develop an exploratory risk map and a potential distribution map of the vector, in order to offer recommendations for CL prevention. A total of 12 079 Phlebotominae (Diptera: Psychodidae) belonging to the species Lutzomyia neivai (Pinto), Lu. migonei (França), Lu. cortelezzii (Brèthes), Lu. shannoni (Dyar), Lu. quinquefer (Dyar) and Brumptomyia spp. (França & Parrot) were captured. Potential distribution models were created for two species, Lu. neivai (incriminated vector of Leishmania braziliensis) and Lu. migonei, associated with domestic animals in Argentina and that in turn could be involved as a link between zoonotic transmission cycles and anthropozoonotic. The Maximum Entropy Modeling System (MaxEnt) was used. The Jackknife test was performed, and the ‘rainfall of the driest month’ was the variable that best generalized the models. Accuracy was evaluated by the area under the curve (AUC) and validated by the Cohen's kappa index. This approximation provides a new analytical resource of high potential for the prevention of the disease, in order to allocate resources properly and to develop the most suitable strategies for action.     

New insights into the chemical biodiversity of Minthostachys mollis in Argentina

Variations in the chemical composition of the essential oils of Minthostachys mollis Griseb. (Lamiaceae) collected in their natural habitat in Argentina were determined using GC-FID-MS analysis. Seventy five samples collected out of 40 wild plant populations in the Central and Northwestern Argentina during four years sampling, were analyzed. Principal Component Analysis was made on the chemical data showing that samples from Córdoba and San Luis belonged to the typical menthone – pulegone chemotype in accordance with previous reports; meanwhile samples from Tucumán, Salta and Catamarca showed the presence of different compositions, some previously unreported, mainly those detected in Tucumán province. In addition, cluster analysis was applied using Euclidean distance to measure the dissimilarity in the relative composition of samples from Tucumán, Salta and Catamarca, using Ward's method as the agglomeration technique. Five groups were retained with less than 10% of information loss: i) dihydrocarvone-carvone, ii) pulegone with absence of menthone, iii) carvacryl acetate-carvacrol, iv) limonene and v) linalool.It is worth mentioning that the compositions of the different chemotypes remained constant throughout the four years of collection and despite the highly diverse pattern of compositions revealed in this search, the typical chemotype was not found present northwards Córdoba and San Luis provinces. This is the first report of a deep study on natural populations of peperina throughout the whole distribution area in Argentina with consecutive sampling repetitions of the same populations during several years.     

A new flea of the genus Ctenidiosomus (Siphonaptera,Pygiopsyllidae) from Salta Province,Argentina

A new species of flea of the genus Ctenidiosomus Jordan, 1931 (Siphonaptera: Pygiopsyllidae) is described from Phyllotis osilae J. A. Allen, 1901, from Salta Province, Argentina. This is the first time that Ctenidiosomus has been recorded in Argentina. A key to species of males of Ctenidiosomus is presented.     

Lutzomyia longipalpis Presence and Abundance Distribution at Different Micro-spatial Scales in an Urban Scenario

The principal objective of this study was to assess a modeling approach to Lu. longipalpis distribution in an urban scenario, discriminating micro-scale landscape variables at microhabitat and macrohabitat scales and the presence from the abundance of the vector. For this objective, we studied vectors and domestic reservoirs and evaluated different environmental variables simultaneously, so we constructed a set of 13 models to account for micro-habitats, macro-habitats and mixed-habitats. We captured a total of 853 sandflies, of which 98.35% were Lu. longipalpis. We sampled a total of 197 dogs; 177 of which were associated with households where insects were sampled. Positive rK39 dogs represented 16.75% of the total, of which 47% were asymptomatic. Distance to the border of the city and high to medium density vegetation cover ended to be the explanatory variables, all positive, for the presence of sandflies in the city. All variables in the abundance model ended to be explanatory, trees around the trap, distance to the stream and its quadratic, being the last one the only one with negative coefficient indicating that the maximum abundance was associated with medium values of distance to the stream. The spatial distribution of dogs infected with L. infantum showed a heterogeneous pattern throughout the city; however, we could not confirm an association of the distribution with the variables assessed. In relation to Lu. longipalpis distribution, the strategy to discriminate the micro-spatial scales at which the environmental variables were recorded allowed us to associate presence with macrohabitat variables and abundance with microhabitat and macrohabitat variables. Based on the variables associated with Lu. longipalpis, the model will be validated in other cities and environmental surveillance, and control interventions will be proposed and evaluated in the microscale level and integrated with socio-cultural approaches and programmatic and village (mesoscale) strategies.     

Distribution of Lutzomyia longipalpis Chemotype Populations in S?o Paulo State,Brazil

BackgroundAmerican visceral leishmaniasis (AVL) is an emerging disease in the state of São Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector.Conclusion/SignificanceThe maps in the present study show that there are two distinct epidemiological patterns of AVL in São Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures.