Revisiting the heterogeneous global genomic population structure of Leishmania infantum.

Leishmania infantum is the main causative agent responsible for visceral leishmaniasis (VL), a disease with global distribution. The genomic structure and genetic variation of this species have been widely studied in different parts of the world. However, in some countries, this information is still yet unknown, as is the genomic behaviour of the main antigens used in VL diagnosis (rK39 and rK28), which have demonstrated variable sensitivity and specificity in a manner dependent on the geographic region analysed. The objective of this study was to explore the genomic architecture and diversity of four Colombian L. infantum isolates obtained in this study and to compare these results with the genetic analysis of 183 L. infantum isolates from across the world (obtained from public databases), as well as to analyse the whole rK39 and rK28 antigen sequences in our dataset. The results showed that, at the global level, L. infantum has high genetic homogeneity and extensive aneuploidy. Furthermore, we demonstrated that there are distinct populations of L. infantum circulating in various countries throughout the globe and that populations of distant countries have close genomic relationships. Additionally, this study demonstrated the high genetic variability of the rK28 antigen worldwide. In conclusion, our study allowed us to (i) expand our knowledge of the genomic structure of global L. infantum; (ii) describe the intra-specific genomic variability of this species; and (iii) understand the genomic characteristics of the main antigens used in the diagnosis of VL. Additionally, this is the first study to report whole-genome sequences of Colombian L. infantum isolates.

DNA barcoding to identify species of phlebotomine sand fly (Diptera: Psychodidae) in the mixed leishmaniasis focus of the Colombian Caribbean.

Identification of the species of phlebotomine sand flies present in each focus of leishmaniasis is necessary to incriminate vectors and implement vector control strategies. Although the cytochrome oxidase I (COI) gene has been proposed as a barcode for the identification of animal species, less than 20% of New World phlebotomines have been characterized to date. In this study DNA barcoding was used to identify phlebotomine species of the mixed leishmaniasis focus in the Colombian Caribbean by means of three evolutionary models: Kimura's two parameter (K2P) nucleotide substitution model, that of (Tamura and Nei, 1993) (TN93) and proportional sequence divergence (p-distances). A 681bp sequence of the COI gene was obtained from 66 individuals belonging to 19 species of the genus Lutzomyia (Lu. abonnenci, Lu. atroclavata, Lu. bicolor, Lu. carpenteri, Lu. cayennensis cayennensis, Lu. dubitans, Lu. evansi, Lu. gomezi, Lu. gorbitzi, Lu. longipalpis, Lu. micropyga, Lu. migonei, Lu. panamensis, Lu. (Psathyromyia) sp., Lu. rangeliana, Lu. serrana, Lu. shannoni, Lu. trinidadensis and Lu. venezuelensis) and one of Brumptomyia (B. mesai). The genetic divergence values for TN93 among individuals of the same species fluctuated up to 3.2% (vs. 2.9% for K2P and 2.8% for p-distances), while the values between species ranged from 8.8-43.7% (vs. 6.8-19.6% for K2P and 6.6-17.4% for p-distances). A dendrogram constructed by means of the Neighbor-Joining method grouped phlebotomines into 20 clusters according to species, with bootstrap values of up to 100% in those with more than one individual. However, loss of the phylogenetic signal of the gene COI was observed at the supraspecific level as a consequence of substitutional saturation. In conclusion, irrespective of the evolutionary model selected, all phlebotomines were correctly assigned to species, showing 100% concordance with morphological identification.

Evidence for anthropophily in five species of phlebotomine sand flies (Diptera: Psychodidae) from northern Colombia, revealed by molecular identification of bloodmeals.

Identification of the bloodmeal sources of phlebotomine sand flies is fundamental to determining which species are anthropophilic and understanding the transmission of Leishmania parasites in natural epidemiological settings. The objective of this study was to identify sand fly bloodmeals in the mixed leishmaniasis focus of the department of Sucre, northern Colombia. In all 141 engorged female sand flies were analyzed, after being captured in intradomiciliary, peridomiciliary and extradomiciliary habitats with Shannon and CDC traps and by active searching in diurnal resting sites. Bloodmeals were identified by sequencing and analysis of a 358bp fragment of the mitochondrial gene Cytochrome b (CYB) and a 330bp fragment of the nuclear gene prepronociceptin (PNOC). Using both genes 105 vertebrate bloodmeals were identified, with an efficiency of 72% for CYB but only 7% for PNOC. Ten species of vertebrates were identified as providing bloodmeal sources for 8 sand fly species: Homo sapiens (Lutzomyia evansi, Lutzomyia panamensis, Lutzomyia micropyga, Lutzomyia shannoni and Lutzomyia atroclavata), Equus caballus (L. evansi, L. panamensis and Lutzomyia cayennensis cayennensis), Equus asinus (L. evansi and L. panamensis), Bos taurus (L. evansi, L. panamensis and L. c. cayennensis), Tamandua mexicana (L. shannoni and Lutzomyia trinidadensis), Proechimys guyanensis (L. evansi, L. panamensis and L. c. cayennensis), Mabuya sp. (Lutzomyia micropyga), Anolissp. (L. micropyga), Sus scrofa (L. evansi and Lutzomyia gomezi) and Gallus gallus (L. evansi). Cattle, donkeys, humans and pigs were significantly more important than other animals (P=0.0001) as hosts of L. evansi, this being the most abundant sand fly species. The five Lutzomyia species in which blood samples of human origin were detected included L. micropyga and L. atroclavata, constituting the first evidence of anthropophily in both species.

Study on natural breeding sites of sand flies (Diptera: Phlebotominae) in areas of Leishmania transmission in Colombia.

BACKGROUND: The location of the microhabitats where immature phlebotomine sand flies of the genus Lutzomyia develop is one of the least-known aspects of this group of medically important insects. For this reason strategies of source reduction approach for their control have not been possible in contrast to other insect vectors (such as mosquitoes), because their juvenile stages in terrestrial microhabitats is difficult to detect. METHODS: Direct examination of soil samples, incubation of substrates and the use of emergence traps were the methods used to identify juvenile stages in 160 soil samples from urban and forest habitats within the foci of Leishmania transmission in Colombia. Immatures collected were identified subsequent from the rearing and emergence of adults using taxonomic keys or the analysis of the mitochondrial marker cytochrome oxidase I. Plant species associated with the natural breeding sites were identified and physicochemical properties of the soils were analyzed. RESULTS: A total of 38 (23.7%) sampling sites were identified as breeding sites, 142 phlebotomine sand flies were identified, belonging to 13 species of the genus Lutzomyia and two of Brumptomyia. The greatest numbers of immature were found within the tabular roots (51 immature sand flies from eight positive sites) and bases of trees (35 immature sand flies from 11 sites). The characterization and presence of the tree species (mainly Ceiba pentadra, Anacardium excelsum, Pseudosamanea guachapale) and the physicochemical properties (relative humidity and carbon/nitrogen ratio) of the soils associated with these breeding sites are significant factors in explaining the diversity and abundance of phlebotomine sand flies. CONCLUSIONS: Immature phlebotomine sand flies of the genus Lutzomyia in Colombia can be found in a wide variety of breeding sites rich in organic matter, high relative humidity and are associated with a typical vegetation of each locality. These results provide new perspectives for the study of the ecology of the genus Lutzomyia in Colombia and the development of vector control strategies.

tRNASer (UCN) MITOCONDRIAL DE Lutzomyia hartmanni PREDICCIÓN DE LA ESTRUCTURA SECUNDARIA DEL tRNASer (UCN) MITOCONDRIAL DEL FLEBOTOMÍNEO Lutzomyia hartmanni (DIPTERA: PSYCHODIDAE) / Prediction of the Secondary Structure of the Mitochondrial tRNASer (UCN) of Lutzomyia hartmanni (Diptera: Psychodidae)

Lutzomyia (Helcocyrtomyia) hartmanni es un flebotomíneo implicado en la transmisión de Leishmania (Viannia) colombiensis, uno de los agentes etiológicos de leishmaniasis cutánea en Colombia. El objetivo de este trabajo fue explorar la utilidad potencial del RNA de transferencia mitocondrial para Serina (UCN) (tRNASer), en la discriminación taxonómica de L. hartmanni. El DNA mitocondrial se extrajo, amplificó y secuenció a partir de material entomológico recolectado en Envigado, Antioquia, Colombia. El gen tRNASer de L. hartmanni mostró una longitud de 68 pares de bases, con un contenido AT del 80,9%. Éste se diferencia de los demás tRNASer de Lutzomyia conocidos a la fecha tanto por sustituciones en la secuencia primaria de nucleótidos como por los cambios que éstas generan en la estructura secundaria. El número de apareamientos intracatenarios fue siete en el brazo aceptor del aminoácido, tres en el brazo dihidrouridina (DHU), cinco en el brazo del anticodón y cinco en el brazo ribotimidina-pseudouridina-citosina (T C). El tamaño de las lupas DHU, anticodón, variable y T C correspondió a cinco, siete, cuatro y ocho nucleótidos, respectivamente. La ausencia notoria de pares de bases no-Watson-Crick en los cuatro brazos del tRNASer de L. hartmanni, la distingue de otras especies de Lutzomyia.

Lutzomyia (Helcocyrtomyia) hartmanni is a sand fly that has been implicated in the transmission of Leishmania (Viannia) colombiensis, an etiologic agent of cutaneous leishmaniasis in Colombia. The objective of this work was to explore the potential usefulness of the mitochondrial serine transfer RNA (UCN) (tRNASer) in the taxonomic determination of L. hartmanni. Mitochondrial DNA was extracted, amplified and sequenced from entomological material collected in Envigado, Antioquia, Colombia. The tRNASer gene length was 68 nucleotide pairs, with an average adenine-thymine content of 80,9%. The studied tRNASer differs from other sand fly tRNASer known to date, on the basis of its primary and secondary structure. The observed number of intrachain base pairing was 7 in the acceptor arm, 3 in the dihydrouridine (DHU) arm, 5 in the anticodon arm, and 5 in the ribothymidine-pseudouridine-cytosine (T C) arm. The size of the DHU, anticodon, variable and T C loops was estimated to be 5, 7, 4, and 8 nucleotides, respectively. The notorious absence of non-Watson-Crick base pairs in the four arms of the tRNASer distinguishes that of L. hartmanni from others Lutzomyia spp.

Canis familiaris, UN NUEVO HOSPEDERO DE Ornithodoros (A.) puertoricensis FOX, 1947 (ACARI: IXODIDA) EN COLOMBIA / Canis familiaris, a New Host of Ornithodoros (A.) puertoricensis Fox, 1947 (Acari: Ixodida) in Colombia

Las garrapatas revisten gran importancia en el campo biomédico por sus hábitos hematófagos y asociación con la transmisión de agentes patógenos a humanos y animales. El objetivo de esta investigación fue establecer las especies de garrapatas que parasitan perros en tres poblaciones del área rural del Caribe colombiano. Durante los meses de agosto y diciembre del año 2006 se realizó búsqueda activa de garrapatas sobre caninos domésticos de las localidades de El Campín, Sabanas del Potrero y Escobar Arriba, departamento de Sucre. Las garrapatas recolectadas fueron almacenadas en viales con etanol al 70% e identificadas empleando claves morfológicas de referencia para cada familia. Para la determinación de especie en la familia Argasidae se realizaron estimaciones morfométricas de estructuras externas. Se recolectaron 420 garrapatas a partir de 50 caninos infestados, de un total de 134 perros examinados, que corresponde a una tasa de infestación del 37,3%. Las garrapatas fueron identificadas como Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus y Amblyomma ovale pertenecientes a la familia Ixodidae, y Ornithodoros (Alectorobius) puertoricensis de la familia Argasidae. La especie predominante fue R. sanguineus (92,1%) en los estados de larva, ninfa y adulto, seguida por larvas de O. puertoricensis, que fueron halladas en menor número sobre caninos de las tres localidades. Se registra, por primera vez en América, el parasitismo de O. puertoricensis sobre caninos domésticos y se confirma su presencia en Colombia.

Ticks are very important from the biomedical point of view, by their hematophagic activity and their role in the transmission of pathogenic microorganisms to man and animals. The main goal of this work was to establish the tick species parasiting dogs in three rural localities of the Colombian Caribbean. From August to December 2006, an active search of ticks on dogs was carried out in the localities of El Campín, Sabanas del Potrero and Escobar Arriba, department of Sucre. The collected ticks were preserved into eppendorf tubes with 70% ethanol, and identified using standard morphological keys for each family. Argasid species were determined by measuring external morphological characters. Of 134 examined dogs in the three localities, 50 were found infested by ticks, representing a infestation rate of 37,3%. A total of 420 ticks were collected from dogs and identified as Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus, and Amblyomma ovale of the Ixodidae family, and Ornithodoros puertoricensis of the Argasidae family. R. sanguineus was the predominant species (92,1%) in the stages of larva, nymph and adult, following by O. puertoricensis larvae recorded in low numbers in the three regions sampled. The tick O. puertoricensis is recorded for the first time as ectoparasite of domestic dogs in America. Additionally, the presence of this tick species is confirmed in Colombia.

Primer informe de Lutzomyia França en el departamento de Guainía, Amazonia Colombiana, y de Brumptomyia mesai Sherlock (Diptera: Psychodidae) en el Litoral Caribe Colombiano / First Report of Lutzomyia França in the department of Guainía, Amazonian Colombia, and of Brumptomyia mesai Sherlock (Diptera: Psychodidae) in the Colombian Caribbean Coast

Se registra por primera vez para el Departamento de Guainía, Colombia, la presencia del género Lutzomyia França de reconocida importancia médica. Los flebotomíneos se recolectaron mientras intentaban picar al humano en los alrededores del casco urbano del municipio de Inírida. Se identificaron taxonómicamente tres especies de Lutzomyia, incluidas L. davisi (Root), L. olmeca bicolor Fairchild & Theodor, y L. antunesi (Coutinho). Adicionalmente, Brumptomyia mesai Sherlock es citada por primera vez en el litoral Caribe Colombiano a partir de ejemplares colectados con una trampa de luz CDC modificada en la Reserva Forestal Protectora Serranía de Coraza y Montes de María, Departamento de Sucre.

This is the first record of the presence of the medically important genus Lutzomyia França in the Department of Guainía, Colombia. Sand flies were collected biting humans in the surroundings of the urban area of the Municipality of Inírida. Three Lutzomyia species were taxonomically identified as L. davisi (Root), L. olmeca bicolor Fairchild & Theodor, and L. antunesi (Coutinho). Additionally, Brumptomyia mesai Sherlock is cited for the first time in the Caribbean Coast of Colombia based on specimens collected with a modified CDC light trap in the Reserva Forestal Protectora Serranía de Coraza y Montes de María, Department of Sucre.

[First Report of Lutzomyia França in the department of Guainía, Amazonian Colombia, and of Brumptomyia mesai Sherlock (Diptera: Psychodidae) in the Colombian Caribbean Coast]. / Primer informe de Lutzomyia França en el departamento de Guainía, Amazonia Colombiana, y de Brumptomyia mesai Sherlock (Diptera: Psychodidae) en el Litoral Caribe Colombiano.

This is the first record of the presence of the medically important genus Lutzomyia França in the Department of Guainía, Colombia. Sand flies were collected biting humans in the surroundings of the urban area of the Municipality of Inírida. Three Lutzomyia species were taxonomically identified as L. davisi (Root), L. olmeca bicolor Fairchild & Theodor, and L. antunesi (Coutinho). Additionally, Brumptomyia mesai Sherlock is cited for the first time in the Caribbean Coast of Colombia based on specimens collected with a modified CDC light trap in the Reserva Forestal Protectora Serranía de Coraza y Montes de María, Department of Sucre.

Mitochondrial DNA divergence between wild and laboratory populations of Anopheles albimanus Wiedemann ( Diptera: Culicidae )

Las colonias de laboratorio facilitan el estudio de los insectos vectores; sin embargo, se ha sugerido que tales colonias de insectos no son representativas de las poblaciones naturales, llevando en algunos casos, a interpretaciones erróneas respecto a la variación intraespecífica entre los individuos. En el presente estudio se evaluó la variabilidad del gen mitocondrial citocromo b entre una colonia de laboratorio de Anopheles albimanus Wiedemann fundada hace 20 años y la población del campo de la cual se derivó. Los análisis revelan la presencia de cinco y tres haplotipos en las poblaciones del campo y la colonia, respectivamente; los individuos del campo presentaron una mayor variabilidad que los de la colonia basada en el numero de sitios polimórficos, la diversidad haplotípica, la diversidad nucleotídica y el valor promedio de las diferencias nucleotídicas. El promedio y el número neto de sustituciones nucleotídicas por sitio entre las poblaciones y los valores calculados para el FST (0,37179, P = 0.05) indican que existe un considerable grado de diferenciación genética entre ellas; el árbol filogenético muestra que los haplotipos de la colonia parecen derivarse de las poblaciones del campo. Estos resultados sugieren una mayor variabilidad genética existente entre los especimenes del campo en comparación con los individuos de la colonia debido en parte, al largo tiempo de colonización.

Occurrence of the malaria Vector Anopheles albimanus Wiedemman (Diptera: Culicidae) in Isla Fuerte, Colombia / Ocorrência do vetor da malária Anopheles albimanus wiedemann (Diptera: Psychodidae) em Isla Fuerte, Colômbia

Pela primeira vez, é registrada a ocorrência do Anopheles albimanus Wiedemann em Isla Fuerte, Colômbia. Um total de doze mosquitos fêmeas foi capturado com armadilha Shannon em um ambiente peridomiciliar da ilha. Discute-se a relevância epidemiológica desta descoberta.

This is the first report of the occurrence of Anopheles albimanus Wiedemann in Isla Fuerte, Colombia. Twelve female mosquitoes were collected using an illuminated Shannon trap in a peridomestic environment of the island. The epidemiological relevance of this finding is discussed.

Nuevas herramientas para la clasificación taxonómica de los insectos vectores de Leishmaniosis: utilidad de los genes mitocondriales / New tools for the taxonomic classification of the insect vectors of leishmaniasis: usefulness of mitochondrial genes

La controversia en torno a la posición taxonómica de los flebotomíneos, debida en parte a dificultades morfológicas, ha evidenciado la necesidad de buscar nuevas herramientas que permitan no sólo establecer una clasificación más acorde con los procesos evolutivos que han experimentado estos insectos, sino también diferenciar los taxones que conforman los complejos de especies. Con la aparición de las técnicas de biología molecular, el estudio del genoma en insectos vectores de enfermedades se ha convertido en un paso importante en la resolución de muchos conflictos taxonómicos. Las características del ADN mitocondrial, entre las que se destacan su rápida evolución y la facilidad con la que se puede aislar y manipular, han favorecido su elección como marcador molecular para estudios de taxonomía. Recientemente, el polimorfismo de algunos genes mitocondriales comenzó a utilizarse para explorar las relaciones filogenéticas y evolutivas en especies de los géneros Lutzomyia y Phlebotomus. La presente revisión recopila la literatura disponible sobre los principales grupos de flebotomíneos estudiados, los genes mitocrondriales más utilizados y su contribución a la clasificación de los vectores de leishmaniosis