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The population genetic structure of Lutzomyia verrucarum (Townsend), a sand fly disease vector of Carrion's disease and cutaneous leishmaniasis in the Peruvian Andes, was characterized by sequencing 653 bp of cytochrome b and 1,125 bp of the NADH dehydrogenase subunit 4 genes of its mitochondrial genome. DNA sequence variation within and between valleys was compared in a sample of 220 sand flies from three valleys (Purisima, Huaylas, and Conchucos) and five departments (Amazonas, Cajamarca, Piura, Lima, and Huancavelica). Gene network and phylogenetic analyses indicated a high similarity of haplotypes collected within a single valley (0-0.52% nucleotide divergence). Flies from each valley had unique genotypes not shared with specimens from other valleys or from more distant regions (0.8-3.1% nucleotide divergence). Mountain ranges and geographic distance appear to have impeded migration (N(m) = < 0.18) between valleys and separated populations into discrete genetic units.

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Phlebotomine vector ecology was studied in the largest recorded outbreak of American cutaneous leishmaniasis in Colombia in 2004. In two rural townships that had experienced contrasting patterns of case incidence, this study evaluated phlebotomine species composition, seasonal abundance, nocturnal activity, blood source, prevalence of Leishmania infection, and species identification. CDC miniature light traps were used to trap the phlebotomines. Traps were set indoors, peridomestically, and in woodlands. Natural infection was determined in pools by polymerase chain reaction-Southern blot, and blood sources and species identification were determined by sequencing. Large differences were observed in population abundance between the two townships evaluated. Lutzomyia longiflocosa was the most abundant species (83.1%). Abundance was higher during months with lower precipitation. Nocturnal activity was associated with human domestic activity. Blood sources identified were mainly human (85%). A high prevalence of infection was found in L. longiflocosa indoors (2.7%) and the peridomestic setting (2.5%). L. longiflocosa was responsible for domestic transmission in Chaparral.

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Nineteen Aedes aegypti larvae were collected in rural Antigua, West Indies, from an 18-liter plastic bucket. The location was in a rural area at the northern end of Antigua bordering the coast of Dickenson Bay and approximately 50 m south of Halcyon Cove Beach (17 degrees 09'42.54"N, 61 degrees 50'44.50"W; elevation 16 m). Atypical morphology was noted in larvae and 3 reared adult females. Fourth instars showed a reduction in length of the lateral hair on the saddle (seta 1-X) with measurements ranging from 0.36 to 0.57 the length of the saddle. Two atypical female specimens displayed an abundance of dull white to gold scales that blanketed the abdomen. A 3rd specimen bore fine, golden scales on the mesonotum and bronze scales on the vertices of the head. These adult specimens demonstrated morphological characteristics that closely parallel described mutations, although the genetic basis for these characters was not confirmed. The remaining adults in the collection were morphologically typical. Adults and larvae were compared to field populations from Florida, Bahamas, and Antigua, as well as to the Rockefeller strain maintained at Rutgers University.

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Within the sand fly genus Lutzomyia, the Verrucarum species group contains several of the principal vectors of American cutaneous leishmaniasis and human bartonellosis in the Andean region of South America. The group encompasses 40 species for which the taxonomic status, phylogenetic relationships, and role of each species in disease transmission remain unresolved. Mitochondrial cytochrome c oxidase I (COI) phylogenetic analysis of a 667-bp fragment supported the morphological classification of the Verrucarum group into series. Genetic sequences from seven species were grouped in well-supported monophyletic lineages. Four species, however, clustered in two paraphyletic lineages that indicate conspecificity--the Lutzomyia longiflocosa-Lutzomyia sauroida pair and the Lutzomyia quasitownsendi-Lutzomyia torvida pair. COI sequences were also evaluated as a taxonomic tool based on interspecific genetic variability within the Verrucarum group and the intraspecific variability of one of its members, Lutzomyia verrucarum, across its known distribution.

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Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.

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Environmental risk factors for cutaneous leishmaniasis were investigated for the largest outbreak recorded in Colombia. The outbreak began in 2003 in Chaparral, and in the following five years produced 2,313 cases in a population of 56,228. Candidate predictor variables were land use, elevation, and climatic variables such as mean temperature and precipitation. Spatial analysis showed that incidence of cutaneous leishmaniasis was higher in townships with mean temperatures in the middle of the county's range. Incidence was independently associated with higher coverage with forest or shrubs (2.6% greater for each additional percent coverage, 95% credible interval [CI] = 0.5-4.9%), and lower population density (22% lower for each additional 100 persons/km(2), 95% CI = 7-41%). The extent of forest or shrub coverage did not show major changes over time. These findings confirmed the roles of climate and land use in leishmaniasis transmission. However, environmental variables were not sufficient to explain the spatial variation in incidence.

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The number of recorded phlebotomine sand fly species in Ecuador has nearly doubled during the past 20 years as a result of surveys. In 2005, a sand fly survey of two localities, Tiputini in the Amazon rain forest and Paraiso Escondido in the Pacific coastal lowland forest, resulted in the capture of 25 species. New records for Ecuador consisted of five species from the Amazonian region and one from Paraiso Escondido. The Amazonian species were Nyssomyia richardwardi (Ready and Fraiha), Psathyromyia dreisbachi (Causey and Damasceno), Psathyromyia runoides (Fairchild and Hertig), Trichophoromyia pabloi (Barretto, Burbano and Young), and Trichopygomyia witoto (Young and Morales). The Pacific coastal lowland species was Psathyromyia punctigeniculata (Floch and Abonnenc).

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Introducción. Debido a la importancia que tiene la vigilancia entomológica como principal medida de control en el manejo de la leishmaniasis visceral, es necesario contar con información actualizada acerca de la distribución y ecología de los insectos involucrados en la transmisión para optimizar las estrategias de prevención. Objetivo. Presentar la distribución actualizada geo-referenciada de L. longipalpis y L. evansi, vectores de los parásitos que causan leishmaniasis visceral en Colombia, teniendo en cuenta la asociación de los insectos con su hábitat. Materiales y métodos. Los registros de distribución se obtuvieron a partir de los ejemplares recolectados en Colombia desde 1967. La información obtenida se organizó en una base de datos a partir de la cual se tomaron las localidades que, posteriormente, fueron sometidas a análisis geográficos por medio de Arc View que se utilizaron para realizar los mapas de distribución. Resultados. Para L. longipalpis se obtuvieron 40 localidades todas distribuidas a lo largo del valle del río Magdalena: Alto (24), Medio (11) y Bajo (5) Magdalena. L. evansi fue registrado en 19 localidades también ubicadas en el mismo valle: cinco en el Magdalena Medio y 14 el Magdalena Bajo. Conclusiones. Ambas especies demostraron una consistente asociación con regiones clasificadas principalmente como bosque seco tropical según las zonas de vida de Holdridge lo que confirma el riesgo epidemiológico de leishmaniasis visceral en estas áreas

Introduction. Since entomological surveillance is the main control strategy for visceral leishmaniasis, updated information on the distribution and ecology of involved vector species is necessary for planning preventive measures. Objective. To present the updated and geo-referenced distribution of L. longipalpis and L. evansi, vectors of visceral leishmaniasis in Colombia, considering their relationship with their habitat. Materials and methods. Distribution was estimated from records of the sand fly specimens collected since 1967.The information was organized in a database from which the localities were selected and geographically analyzed with Arc view in order to develop the distribution maps. Results. 40 localities were established for L. longipalpis along the upper (24), middle (11) and lower (5) Magdalena river valley . L. evansi was recorded in 19 localities of the middle (5) and lower (14) Magdalena valley. Conclusions. Both species showed consistent association with dry tropical forest ( sensu Holdridge 1967), confirming the epidemiological risk for visceral leishmaniasis in these areas.

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INTRODUCTION: Since entomological surveillance is the main control strategy for visceral leishmaniasis, updated information on the distribution and ecology of involved vector species is necessary for planning preventive measures. OBJECTIVE: To present the updated and geo-referenced distribution of L. longipalpis and L. evansi, vectors of visceral leishmaniasis in Colombia, considering their relationship with their habitat. MATERIALS AND METHODS: Distribution was estimated from records of the sand fly specimens collected since 1967. The information was organized in a database from which the localities were selected and geographically analyzed with Arc view in order to develop the distribution maps. RESULTS: 40 localities were established for L. longipalpis along the upper (24), middle (11) and lower (5) Magdalena river valley. L. evansi was recorded in 19 localities of the middle (5) and lower (14) Magdalena valley. CONCLUSIONS: Both species showed consistent association with dry tropical forest (sensu Holdridge 1967), confirming the epidemiological risk for visceral leishmaniasis in these areas.

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Lutzomyia spp. are New World phlebotomine sand flies, many of which are involved in the transmission of human diseases, such as leishmaniases and bartonellosis. The systematic classification of the approximately 400 species in the genus has been based on morphological characters, but the relationships within the genus are still very much in question. We have inferred phylogenies of 32 species of phlebotomine sand flies belonging to seven sub-genera and two species groups, by using fragments of the mitochondrial small subunit (12SrRNA) and of the nuclear large subunit (28SrRNA) ribosomal gene sequences. The subgenus Helcocyrtomyia and the Verrucarum species group, prominent representatives of the Peruvian sand fly fauna, were represented by 11 and 7 species, respectively. Although based on a limited number of taxa, the resulting phylogenies, based on 837 characters, provide an initial phylogenetic backbone for the progressive reconstruction of infrageneric relationships within Lutzomyia.

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A population analysis of peridomestic, light-trapped, field specimens of the phlebotomine sand fly Lutzomyia longipalpis was targeted to six locations representing a geographic transect across eastern Brazil. Mitochondrial cytochrome b gene sequences established the pattern of genetic variation among the populations. Alignment of a 261-basepair region at the 3' end of cytochrome b identified 30 haplotypes and 21 segregating sites from 78 sand flies. Pairwise comparisons indicated statistically significant population structuring between northern and southern populations, as well as structuring among the southern populations. Prominent spatial clustering was evident for two of the populations in a minimum spanning network of the haplotypes, but sequence divergence was not sufficient to indicate cryptic species.

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Wolbachia are cytoplasmically inherited, endosymbiotic bacteria known to infect a wide variety of arthropods. Polymerase chain reaction (PCR) amplification of the Wolbachia surface protein (wsp) gene was used to assay the infection of geographically disparate populations of Aedes albopictus (Skuse) by Wolbachia. Nine North American, four South American, one Hawaiian, and four Old World populations of A. albopictus were all doubly infected with both the wAlbA and wAlbB strains of Wolbachia. A 365-bp region of the wAlbA wsp gene was sequenced from seven geographically disparate host populations, and all sequences were identical. Similarly, a 474-bp region of the wAlbB wsp gene was sequenced from the same populations, and all sequences were identical. These results suggest a role for Wolbachia infection in causing the previously established pattern of low mitochondrial DNA variability, but average nuclear gene diversity, within and among populations of A. albopictus.

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The use of PCR (polymerase chain reaction) was evaluated for its effectiveness as a tool in the detection of transmission of Leishmania chagasi to a hamster host, Mesocricetus auratus, by insect vector bite. Two pairs of uninfected and anesthetized hamsters were introduced into cages containing infected females of the typical phlebotomine sand fly vector, Lutzomyia longipalpis. The flies were experimentally infected with Leishmania chagasi and the infection was verified by dissection of subsamples. At 37 and 51 days after exposure to the infected flies, biopsies of each hamster's liver and spleen were subjected to direct histopathological and PCR examination. DNA was extracted with Chelex 100; for PCR amplification, primers specific to Leishmania minicircle DNA were used. PCR product was separated on agarose gels and visualized with UV. A band of approximately 120 base pairs was observed in 3 of the 4 biopsies, corresponding to the expected minicircle size. PCR was the only method that detected presence of the parasite. The results demonstrated that the sensitivity of PCR greatly expedites the confirmation process of a particular phlebotomine species as a vector of leishmaniasis.

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Se evaluó la efectividad de la PCR como herramienta en la detección de la transmisión experimental de Leishmania chagasi a hámster, Mesocricetus auratus, por picadura del insecto vector. Dos pares de hámsteres sanos y anestesiados fueron colocados en jaulas que contenían hembras de Lutzomyia longipalpis. Previamente, las hembras se infectaron experimentalmente con Leishmania chagasi y la infección se confirmó por disección en una submuestra. A los 37 y 51 días después de la exposición a los insectos infectados, las biopsias de hígado y bazo de cada hámster se sometieron a examen directo al microscopio, histopatología y PCR. El ADN se extrajo con Chelex 100(c); en la amplificación se utilizó un par de iniciadores específicos para la región conservada de los minicírculos del ADN de Leishmania. El producto amplificado se separó en geles de agarosa y se visualizó bajo luz UV. En tres de las cuatro biopsias se observó una banda de 120 pares de bases, aproximadamente, correspondiente al tamaño esperado de la fracción del minicírculo. La técnica de PCR fue el único método que detectó la presencia del par sito. Estos resultados demostraron que la sensibilidad de la PCR acelera los procesos de incriminación vectorial de las especies vectoras de leishmaniasis.

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The Centers for Disease Control (CDC, USA) has proposed a simplified method for the determinations of insecticide resistance in adult mosquitoes, using 250 ml Wheaton bottles containing measured dosages. Insects are transferred into the bottle for 1 hour and monitored for mortality at regular intervals. In standardizing the CDC method for use with phlebotomine sand flies, effects of the solvent without insecticide were evaluated. Two colonized sand fly vector species were used: Lutzomyia longipalpis (F50 and F54) and Lutzomyia serrana (F17). Groups of 10 to 24 unfed females 1-3 days old were transferred for 1 h to Wheaton bottles with the following pretreatment: (1) without additive, (2) 0.5 ml of acetone, or (3) 1.0 ml of acetone. Three to 5 replicates were undertaken for each condition and each species. In the control bottles, the insects rested quietly and after 1 h appeared normal. In bottles with 0.5 and 1.0 ml acetone, a repellent effect was observed in L. longipalpis and L. serrana within the first 10 min. A small proportion of the L. serrana became prostrate, but recovered quickly after removal from the bottle. Field test performed with Lutzomyia quasitownsendi produced results simialar to those of the L. serrana colony flies. The insecticide bioassays were performed with L. longipalpis (F60) flies. Females were exposed to three graded doses of lambdacyhalothrin (10, 50 and 100 micrograms/bottle), and mortality was recorded at five-minute intervals. Regression lines for the 3 concentrations were compared within the context of the CDC method. The advantages of the CDC method over the WHO protocols were four: lower cost, fewer insects required, an entire group of insects exposed to the same surface, and ease of field use.

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The WHO method for determining insecticide resistance was standardized for several species of Lutzomyia sand flies under laboratory and field conditions. The biological assays were applied solely to optimize the conditions for the control, i.e., without insecticide, and to estimate mortality due to handling or other unfavorable conditions. Adult female flies from 3 laboratory colonies and one field strain were tested: two laboratory strains of Lutzomyia longipalpis, one laboratory strain of Lutzomyia serrana and one field-collected strain of Lutzomyia quasitownsendi. The WHO method was compared with one modified in which, during the post-exposure period, the recommended plain tube apparatus was replaced with a plastic container layered with damp plaster of Paris. Three paper substrate types were compared under each condition: olive oil additive, silicon oil additive and plain paper. The measured variable was percent mortality in 24 h. For the WHO protocol, the L. longipalpis strains indicated a 0-10% mortality, L. serrana 20-80% and L. quasitownsendi 10-50%. With the modified WHO apparatus, the average mortality was < 4% for all species. No significant differences were observed among the paper treatments. These results indicate a strong species-specific effect of post-exposure conditions on sand flies. To establish baseline levels of insecticide resistance in Lutzomyia sand flies, the WHO method is recommended only for L. longipalpis, and the modified method for L. serrana, L. quasitownsendi and closely related species.

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El objetivo de este estudio fue estandarizar el método OMS para determinar susceptibilidad a insecticidas en algunas especies de Lutzomyia en condiciones de laboratorio y de campo. Los ensayos biológicos se realizaron sin insecticidas, únicamente con tratamientos controles para determinar mortalidad por manipulación y otras condiciones desfavorables para cada especie. Se emplearon hembras de tres colonias de laboratorio: dos cepas de Lutzomyia longipalpis y una de Lutzomyia serrana. También se incluyeron en el estudio hembras silvestres de Lutzomyia quasitownsendi. Se emplearon dos tipos de métodos: el método OMS y el método OMS-adaptado; este último consistió en reemplazar el tubo OMS para el período posexposición por un contenedor plástico con yeso humedecido. Para cada método se ensayaron tres tipos de papeles: papel impregnado con aceite de oliva, papel impregnado con aceite de silicona y papel sin aditivos. La variable por medir fue el porcentaje de mortalidad a las 24 h. El método OMS resultó conveniente para las dos cepas de L. logipalpis con una mortalidad entre 0 y 10 por ciento, pero registró una mortalidad elevada en L. serrana (20-80 por ciento) y L. quasitownsendi silvestres (10-50 por ciento). Con el médtodo OMS - modificado, la mortalidad promedio fue inferior a 4 por ciento en todas las especies ensayadas. Con respecto al tipo de papel, no se encontraron variaciones importantes. Según los resultados, el recipiente utilizado en el periodo posexposición afecta la supervivencia de los flebótomos y de manera variable para cada especie. Al establecer líneas base de susceptibilidad a insecticidas en especies del género Lutzomyia, se recomienda el uso del método OMS para L. longipalpis y del método OMS - modificado para L. serrana, L. Quasitownsendi y especies cercanas

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The phlebotomine sand fly Lutzomyia serrana (Damasceno & Arouck) was mass-reared tinder conditions of varying densities in an effort to improve colony production efficiency. To do this, the experimental carrying capacity of a standard rearing chamber was determined, i.e., the optimum population size in relation to density (individuals per unit of space). Rearing chambers of 100 cm3 were populated with 1-50 L. serrana engorged females and an equal number of males. Laboratory conditions were maintained at 23-26 degrees C and 85-95% RH. The following parameters were recorded for each experimental chamber (three replicates): (1) female mortality without oviposition, (2) number of eggs oviposited and (3) number of adults emerging from the egg cohort. Female mortality began to increase substantially in the 26-female chamber, from 5.7% to 15% and finally reaching 60.2% in the 46-50 female chambers. In the chambers containing 1-20 females, egg number and realized adult progeny increased linearly to reach an asymptote. In the 20-50 female chambers, the number of eggs ranged from 420 to 699, and adult production from 306 to 432. The optimum carrying capacity for the 100-cm3 chambers was 22 +/- 2 females. Beyond this number, auto-regulation was initiated, i.e., female mortality without oviposition increased as the number of females per chamber increased. Total number of eggs and adult production was similar in all chambers containing 20-50 females. In conclusion, for optimizing production of mass reared sand flies, determination of the carrying capacity is essential to optimize use of insectary resources, to avoid loss of valuable potentially ovipositing females, and to increase overall production efficiency.

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Polyacrylamide gel electrophoresis was used to elucidate genetic variation at 13 isozyme loci among forest populations of Lutzomyia shannoni from three widely separated locations in Colombia: Palambí (Nariño Department), Cimitarra (Santander Department) and Chinácota (Norte de Santander Department). These samples were compared with a laboratory colony originating from the Magdalena Valley in Central Colombia. The mean heterozygosity ranged from 16 to 22 percent, with 2.1 to 2.6 alleles detected per locus. Nei's genetic distances among populations were low, ranging from 0.011 to 0.049. The estimated number of migrants (Nm=3.8) based on Wright's F-Statistic, F ST, indicated low levels of gene flow among Lu. shannoni forest populations. This low level of migration indicates that the spread of stomatitis virus occurs via infected host, not by infected insect. In the colony sample of 79 individuals, the Gpi locus was homozygotic (0.62/0.62) in all females and heterozygotic (0.62/0.72) in all males. Although this phenomenon is probably a consequence of colonization, it indicates that Gpi is linked to a sex determining locus

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