RESUMEN
To identify the geographical origins of larvae of Wohlfahrtia magnifica (Diptera: Sarcophagidae) causing myiasis of sheep in Italy, comparative DNA sequence analysis of the mitochondrial cytochrome b gene was performed, based on gene fragments amplified by PCR from genomic DNA isolated from individual specimens. DNA extractions of 19 larvae from Lazio, Molise, Puglia, and Sicilia generated 17 readable sequences homologous to 2 haplotypes, either CB_magn01 or CB_magn02; DNA extracts from 4 adult flies from Calabria (reared from larvae) produced 4 readable sequences belonging to the haplotype CB_magn01. The two haplotypes found represent both the East and West phylogenetic lineages of W. magnifica, which is consistent with the species' arrival from central/southeast Europe (East lineage) and/or from southwest Europe/northwest Africa (West lineage). This is the first report of the sympatric occurrence of the two lineages, which could have resulted from natural or human-assisted dispersal. Polymorphic nuclear loci will have to be characterized in order to explain the origins and lack of mitochondrial haplotype diversity of this pest in Italy, where it poses increasing veterinary problems.
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Citocromos b/genética , Genes Mitocondriales , Miasis/veterinaria , Sarcofágidos/genética , Enfermedades de las Ovejas/parasitología , Animales , Europa (Continente) , Geografía , Haplotipos , Italia/epidemiología , Larva , Mitocondrias , Miasis/epidemiología , Miasis/parasitología , Filogenia , Reacción en Cadena de la Polimerasa , Ovinos , Enfermedades de las Ovejas/epidemiologíaRESUMEN
Phlebotomines are the sole or principal vectors of Leishmania, Bartonella bacilliformis, and some arboviruses. The coevolution of sand flies with Leishmania species of mammals and lizards is considered in relation to the landscape epidemiology of leishmaniasis, a neglected tropical disease. Evolutionary hypotheses are unresolved, so a practical phlebotomine classification is proposed to aid biomedical information retrieval. The vectors of Leishmania are tabulated and new criteria for their incrimination are given. Research on fly-parasite-host interactions, fly saliva, and behavioral ecology is reviewed in relation to parasite manipulation of blood feeding, vaccine targets, and pheromones for lures. Much basic research is based on few transmission cycles, so generalizations should be made with caution. Integrated research and control programs have begun, but improved control of leishmaniasis and nuisance-biting requires greater emphasis on population genetics and transmission modeling. Most leishmaniasis transmission is zoonotic, affecting the poor and tourists in rural and natural areas, and therefore control should be compatible with environmental conservation.
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Control de Insectos , Insectos Vectores , Leishmaniasis , Psychodidae , Animales , Interacciones Huésped-Parásitos , Insectos Vectores/clasificación , Insectos Vectores/parasitología , Insectos Vectores/fisiología , Insectos Vectores/virología , Leishmania/fisiología , Leishmaniasis/parasitología , Leishmaniasis/prevención & control , Leishmaniasis/transmisión , Leishmaniasis/veterinaria , Lagartos/parasitología , Mamíferos/parasitología , Psychodidae/clasificación , Psychodidae/parasitología , Psychodidae/fisiología , Psychodidae/virologíaRESUMEN
The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5-20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.
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Enfermedades de los Perros/epidemiología , Leishmania/aislamiento & purificación , Leishmaniasis/veterinaria , Animales , Reservorios de Enfermedades , Enfermedades de los Perros/parasitología , Perros , Ambiente , Europa (Continente)/epidemiología , Geografía , Leishmania/inmunología , Leishmaniasis/epidemiología , Leishmaniasis/parasitología , Modelos Logísticos , Estudios SeroepidemiológicosRESUMEN
OBJECTIVE: To assess the effectiveness of community-wide deployment of insecticide-impregnated collars for dogs- the reservoir of Leishmania infantum-to reduce infantile clinical visceral leishmaniasis (VL). METHODS: A pair matched-cluster randomised controlled trial involving 40 collared and 40 uncollared control villages (161 [95% C.L.s: 136, 187] children per cluster), was designed to detect a 55% reduction in 48 month confirmed VL case incidence. The intervention study was designed by the authors, but implemented by the Leishmaniasis Control Program in NW Iran, from 2002 to 2006. RESULTS: The collars provided 50% (95% C.I. 17·8%-70·0%) protection against infantile VL incidence (0·95/1000/yr compared to 1·75/1000/yr). Reductions in incidence were observed across 76% (22/29) of collared villages compared to pair-matched control villages, with 31 fewer cases by the end of the trial period. In 11 paired villages, no further cases were recorded post-intervention, whereas in 7 collared villages there were 9 new clinical cases relative to controls. Over the trial period, 6,835 collars were fitted at the beginning of the 4 month sand fly season, of which 6.9% (95% C.I. 6.25%, 7.56%) were lost but rapidly replaced. Collar coverage (percent dogs collared) per village varied between 66% and 100%, with a mean annual coverage of 87% (95% C.I. 84·2, 89·0%). The variation in post-intervention clinical VL incidence was not associated with collar coverage, dog population size, implementation logistics, dog owner compliance, or other demographic variables tested. Larger reductions and greater persistence in incident case numbers (indicative of transmission) were observed in villages with higher pre-existing VL case incidence. CONCLUSION: Community-wide deployment of collars can provide a significant level of protection against infantile clinical VL, achieved in this study by the local VL Control Program, demonstrating attributes desirable of a sustainable public health program. The effectiveness is not dissimilar to the community-level protection provided against human and canine infection with L. infantum.
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Control de Enfermedades Transmisibles/métodos , Enfermedades de los Perros/prevención & control , Insecticidas/administración & dosificación , Leishmania infantum , Leishmaniasis Visceral/prevención & control , Zoonosis/prevención & control , Animales , Niño , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/transmisión , Perros , Femenino , Humanos , Incidencia , Irán/epidemiología , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/transmisión , Leishmaniasis Visceral/veterinaria , Masculino , Oportunidad Relativa , Zoonosis/epidemiología , Zoonosis/transmisiónRESUMEN
Malaria parasites manipulate mosquitoes to ensure transmission between mammalian hosts; painstaking experiments have now demonstrated that another medically important protozoan, Leishmania, enhances its transmission through the adaptive manipulation of one of its sandfly vectors, Lutzomyia longipalpis. Experimental Leishmania infections specifically increased sandfly biting persistence and feeding on multiple hosts, but only if the parasites produced infective forms and a gel plug of filamentous proteophosphoglycan in the anterior midgut of the sandfly. This fundamental research is relevant to vaccine development.
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Adaptación Fisiológica , Leishmania/patogenicidad , Leishmaniasis/transmisión , Psychodidae/parasitología , Animales , Modelos Animales de Enfermedad , Conducta Alimentaria/fisiología , Femenino , Interacciones Huésped-Parásitos/fisiología , Insectos Vectores/parasitología , Leishmania/metabolismo , Leishmaniasis/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Enfermedades Parasitarias en Animales/parasitología , Proteoglicanos/metabolismo , Proteínas Protozoarias/metabolismo , Psychodidae/fisiología , Enfermedades de los Roedores/parasitologíaRESUMEN
In southeast Amazon, Lutzomyia (Nyssomyia) flaviscutellata is the incriminated vector of Leishmania (Leishmania) amazonensis, a causative agent of zoonotic cutaneous leishmaniasis (CL). The optimal methods for surveying Lu. flaviscutellata were investigated in the Bragança region, northeast Pará State, Brazil, selected for the presence of Le. amazonensis. The performances of modified Disney traps and CDC light traps were compared in four ecotopes within and around four village transects during the wet and dry seasons. The physiological age of female sand flies was estimated and natural infection by flagellates was evaluated by dissection. Disney traps were better for detecting the presence of Lu. flaviscutellata, while CDC traps performed well for detecting Lutzomyia (Nyssomyia) antunesi, suspected vector of Leishmania lindenbergi. The former was more abundant during the wet season, when female flies were naturally infected with Le. amazonensis. These findings identified the environments of local transmission. In order to improve surveys of Lu. flaviscutellata as part of integrated epidemiological surveillance of CL, our recommendations include focusing vector surveys with Disney traps on forest fragments where people work, during the seasonal peak of the vector. Further field studies are required to make model-based predictions of seasonal variations in the vectorial capacity of vector populations.
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Insectos Vectores/parasitología , Insectos Vectores/patogenicidad , Leishmaniasis Cutánea/transmisión , Psychodidae/parasitología , Psychodidae/patogenicidad , Animales , Brasil , Entomología , Leishmania/patogenicidad , Leishmaniasis Cutánea/parasitología , Estaciones del AñoRESUMEN
BACKGROUND: Before 1996 the phlebotomine sand fly Lutzomyia neivai was usually treated as a synonym of the morphologically similar Lutzomyia intermedia, which has long been considered a vector of Leishmania braziliensis, the causative agent of much cutaneous leishmaniasis in South America. This report investigates the likely range changes of both sand fly species in response to a stabilisation climate change scenario (RCP4.5) and a high greenhouse gas emissions one (RCP8.5). METHODS: Ecological niche modelling was used to identify areas of South America with climates currently suitable for each species, and then the future distributions of these climates were predicted based on climate change scenarios. Compared with the previous ecological niche model of L. intermedia (sensu lato) produced using the GARP algorithm in 2003, the current investigation modelled the two species separately, making use of verified presence records and additional records after 2001. Also, the new ensemble approach employed ecological niche modelling algorithms (including Maximum Entropy, Random Forests and Support Vector Machines) that have been widely adopted since 2003 and perform better than GARP, as well as using a more recent climate change model (HadGEM2) considered to have better performance at higher resolution than the earlier one (HadCM2). RESULTS: Lutzomyia intermedia was shown to be the more tropical of the two species, with its climatic niche defined by higher annual mean temperatures and lower temperature seasonality, in contrast to the more subtropical L. neivai. These different latitudinal ranges explain the two species' predicted responses to climate change by 2050, with L. intermedia mostly contracting its range (except perhaps in northeast Brazil) and L. neivai mostly shifting its range southwards in Brazil and Argentina. This contradicts the findings of the 2003 report, which predicted more range expansion. The different findings can be explained by the improved data sets and modelling methods. CONCLUSIONS: Our findings indicate that climate change will not always lead to range expansion of disease vectors such as sand flies. Ecological niche models should be species specific, carefully selected and combined in an ensemble approach.
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Cambio Climático , Insectos Vectores , Psychodidae/crecimiento & desarrollo , Animales , Modelos Teóricos , América del Sur , Análisis Espacial , Especificidad de la EspecieRESUMEN
Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia) flaviscutellata and the parasite it transmits, Leishmania (Leishmania) amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector's climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest). Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: "stabilization" and "high increase". Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador, Colombia and Venezuela. These areas will only become endemic for L. amazonensis, however, if they have competent reservoir hosts and transmission dynamics matching those in the Amazon region.
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Distribución Animal , Cambio Climático , Ecosistema , Modelos Estadísticos , Psychodidae/fisiología , Animales , Vectores de Enfermedades , América del SurRESUMEN
Phlebotomine sand flies are the subject of much research because of the role of their females as the only proven natural vectors of Leishmania species, the parasitic protozoans that are the causative agents of the neglected tropical disease leishmaniasis. Activity in this field was highlighted by the eighth International Symposium on Phlebotomine Sand flies (ISOPS) held in September 2014, which prompted this review focusing on vector control. Topics reviewed include: Taxonomy and phylogenetics, Vector competence, Genetics, genomics and transcriptomics, Eco-epidemiology, and Vector control. Research on sand flies as leishmaniasis vectors has revealed a diverse array of zoonotic and anthroponotic transmission cycles, mostly in subtropical and tropical regions of Africa, Asia and Latin America, but also in Mediterranean Europe. The challenge is to progress beyond descriptive eco-epidemiology, in order to separate vectors of biomedical importance from the sand fly species that are competent vectors but lack the vectorial capacity to cause much human disease. Transmission modelling is required to identify the vectors that are a public health priority, the ones that must be controlled as part of the integrated control of leishmaniasis. Effective modelling of transmission will require the use of entomological indices more precise than those usually reported in the leishmaniasis literature.
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Transmisión de Enfermedad Infecciosa/prevención & control , Entomología/tendencias , Control de Insectos/métodos , Control de Insectos/tendencias , Leishmaniasis/epidemiología , Leishmaniasis/prevención & control , Psychodidae/fisiología , África/epidemiología , Animales , Asia/epidemiología , Europa (Continente)/epidemiología , Humanos , Insectos Vectores , América Latina/epidemiología , Clima TropicalRESUMEN
Arguably one of the most important effects of climate change is the potential impact on human health. While this is likely to take many forms, the implications for future transmission of vector-borne diseases (VBDs), given their ongoing contribution to global disease burden, are both extremely important and highly uncertain. In part, this is owing not only to data limitations and methodological challenges when integrating climate-driven VBD models and climate change projections, but also, perhaps most crucially, to the multitude of epidemiological, ecological and socio-economic factors that drive VBD transmission, and this complexity has generated considerable debate over the past 10-15 years. In this review, we seek to elucidate current knowledge around this topic, identify key themes and uncertainties, evaluate ongoing challenges and open research questions and, crucially, offer some solutions for the field. Although many of these challenges are ubiquitous across multiple VBDs, more specific issues also arise in different vector-pathogen systems.
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Cambio Climático , Clima , Enfermedades Transmisibles/transmisión , Vectores de Enfermedades , Animales , Humanos , Factores SocioeconómicosRESUMEN
Leishmania species are the causative agents of leishmaniasis, a neglected tropical disease. These parasitic protozoans are usually transmitted between vertebrate hosts by the bite of blood sucking female phlebotomine sand flies. This review focuses on the two parasites causing most human visceral leishmaniasis (VL), which leads to substantial health problems or death for up to 400,000 people per year. Except for travel cases, Leishmania donovani infections are restricted to the (sub-)tropics of Asia and Africa, where transmission is mostly anthroponotic, while Leishmania infantum occurs in the drier parts of Latin America as well as in the Mediterranean climate regions of the Old World, with the domestic dog serving as the main reservoir host. The prevalence of VL caused by L. infantum has been declining where living standards have improved. In contrast, infections of L. donovani continue to cause VL epidemics in rural areas on the Indian subcontinent and in East Africa. The current review compares and contrasts these continental differences and suggests priorities for basic and applied research that might improve VL control. Transmission cycles, pathogenesis, diagnosis, treatment and prognosis, prevention (including vector control), surveillance, transmission modeling, and international control efforts are all reviewed. Most case detection is passive, and so routine surveillance does not usually permit accurate assessments of any changes in the incidence of VL. Also, it is not usually possible to estimate the human inoculation rate of parasites by the sand fly vectors because of the limitations of survey methods. Consequently, transmission modeling rarely passes beyond the proof of principle stage, and yet it is required to help develop risk factor analysis for control programs. Anthroponotic VL should be susceptible to elimination by rapid case detection and treatment combined with local vector control, and one of the most important interventions may well be socioeconomic development.
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Vaccine development is informed by a knowledge of genetic variation among antigen alleles, especially the distribution of positive and balancing selection in populations and species. A combined approach using population genetic and phylogenetic methods to detect selective signatures can therefore be informative for identifying vaccine candidates. Parasitic Leishmania species cause the disease leishmaniasis in humans and mammalian reservoir hosts after inoculation by female phlebotomine sandflies. Like other arthropod vectors of disease agents, sandflies use salivary peptides to counteract host haemostatic and immunomodulatory responses during bloodfeeding, and these peptides are vaccine candidates because they can protect against Leishmania infection. We detected no contemporary adaptive selection on one salivary peptide, apyrase, in 20 populations of Phlebotomus ariasi, a European vector of Leishmania infantum. Maximum likelihood branch models on a gene phylogeny showed apyrase to be a single copy in P. ariasi but an ancient duplication event associated with temporary positive selection was observed in its sister group, which contains most Mediterranean vectors of L. infantum. The absence of contemporary adaptive selection on the apyrase of P. ariasi may result from this sandfly's opportunistic feeding behaviour. Our study illustrates how the molecular population genetics of arthropods can help investigate the potential of salivary peptides for disease control and for understanding geographical variation in vector competence.
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I review species concepts, the taxonomy of phlebotomine sand flies, and some transmission cycles of leishmaniasis in order to illustrate the difficulties of classifying these vectors in a way that will be ideal both for medical parasitologists and sand fly specialists. Choices will have to be made between different classifications, either maintaining a practical one containing few vectorial genera (mostly Phlebotomus for the Old World and Lutzomyia for the Neotropics) or changing the generic names of many vectors so that the classification represents an evolutionary hypothesis. However, sand flies also transmit arboviruses and members of other sand fly genera bite humans, and so vectorial status alone might not provide the criteria for recognizing only a few genera. Vectorial roles are often determined by species-level co-evolution of susceptibility to Leishmania species, with selection being initiated and maintained by ecological contacts. There is only imperfect co-cladogenesis of genus-level groups or subgeneric complexes of sand flies and Leishmania species. Natural hybridization between sand fly species has been recorded in several species complexes, and this highlights the need to focus on gene flow and the distribution of phenotypes of biomedical importance, not on taxa.
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Insectos Vectores/clasificación , Psychodidae/clasificación , Animales , Ecología , Insectos Vectores/crecimiento & desarrollo , Insectos Vectores/parasitología , Leishmaniasis/transmisión , Psychodidae/crecimiento & desarrollo , Psychodidae/parasitologíaRESUMEN
Hungary is regarded as free of leishmaniasis because only a few imported cases have been reported. However, southern Hungary has a sub-Mediterranean climate, and so it was included in the EU FP6 EDEN project, which aimed to map the northern limits of canine leishmaniasis (CanL) in Europe. The numbers of traveling and imported dogs have increased in the last decade, raising concerns about the introduction of CanL caused by Leishmania infantum. Serum samples were collected from 725 dogs (22 localities, 6 counties) that had never traveled to endemic countries, as well as from other potential reservoir hosts (185 red foxes and 13 golden jackals). All sera were tested by the indirect fluorescent antibody test, but they were sero-negative using the OIE cut-off of 1:80 serum dilution except for those of two dogs resident since birth in southern Hungary. These had not received a blood transfusion, but the mode of transmission is unclear because no sandfly vectors were caught locally. From 2006 to 2009, phlebotomine sandflies were sampled in the summer months at 47 localities of 8 counties. They were trapped with castor-oil-impregnated sticky-paper, light, and CO(2)-baited traps. Small numbers of two vectors of Leishmania infantum were found. Phlebotomus neglectus occurred in three villages near to Croatia and one in north Hungary at latitude 47 °N, and Phlebotomus perfiliewi perfiliewi was trapped at two sites in a southeastern county close to the sites where it was first found in 1931-1932. Our report provides baseline data for future investigations into the northward spread of CanL into Hungary, which we conclude has yet to occur.
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Enfermedades de los Perros/parasitología , Enfermedades de los Perros/transmisión , Insectos Vectores/parasitología , Leishmaniasis/veterinaria , Phlebotomus/parasitología , Tunga/parasitología , Animales , Clima , Enfermedades de los Perros/sangre , Enfermedades de los Perros/epidemiología , Perros , Femenino , Geografía , Hungría/epidemiología , Leishmania infantum/aislamiento & purificación , Leishmaniasis/epidemiología , Leishmaniasis/transmisión , Masculino , Encuestas y CuestionariosRESUMEN
Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL) in which gerbils are the reservoir host. ZCL is of great public health importance in Iran. In the current investigation, nested polymerase chain reaction (PCR) protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene). The PCR assays detected L. major in three rodent species: Rhombomis opimus, Meriones lybicus and, for first time, Meriones persicus. L. major parasite was found in Natanz, Isfahan Province in the center of Iran in a focus of rural zoonotic cutaneous leishmaniasis. Four L. major infections were detected in R. opimus species, three in M. Lybicus, and two in M. persicus. All nine rodent infections of L. major were found to be the same haplotype based on the PCR detection and sequencing of parasite ITS-ribosomal DNA gene. In addition, also for the first time, the nested PCR assays detected Leishmania tropica only in one M. persicus. Allied to studies in country, the new findings mean that past conclusions about the reservoir of L. major in Iran must be treated with caution. Finding two Leishmania species in different rodent species as reservoir in Iran, therefore, careful molecular eco-epidemiological investigations will be an essential part of modeling the roles of different gerbil species in maintaining and spreading ZCL foci.
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ADN Espaciador Ribosómico/análisis , Leishmania major/aislamiento & purificación , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Enfermedades de los Roedores/parasitología , Zoonosis/parasitología , Animales , Secuencia de Bases , ADN Protozoario/análisis , ADN Espaciador Ribosómico/genética , Reservorios de Enfermedades , Gerbillinae/parasitología , Irán/epidemiología , Leishmania major/clasificación , Leishmania major/genética , Leishmaniasis Cutánea/epidemiología , Datos de Secuencia Molecular , Muridae/parasitología , Filogenia , Prevalencia , ARN Ribosómico 5.8S/genética , Enfermedades de los Roedores/epidemiología , Análisis de Secuencia de ADN , Zoonosis/epidemiologíaRESUMEN
In Spain, only two of the 12 recorded species of sandflies, Phlebotomus (Larroussius) ariasi Tonnoir and P. (L.) perniciosus Newstead, are proven vectors of Leishmania infantum Nicolle, the causative agent of endemic leishmaniasis. Studies of the distributions of phlebotomine sandflies are important for evaluating the possible effects of climate warming on any northward or altitudinal range shifts of leishmaniasis or the other diseases they transmit. We describe a recent sandfly survey in Spain, starting in the northern Ebro Valley and continuing southeast into the Levante region of the Mediterranean coast. Sandflies (P. ariasi only) were found for the first time in the northern province of Alava, in the upper Ebro Valley, where cases of canine leishmaniasis have been described during the last decade. Throughout the provinces sampled, P. ariasi predominated over P. perniciosus in cooler bioclimatic zones, and this statistically significant pattern was more marked than that with higher altitudes.