A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic lactone resistance worldwide in the nematode parasite Haemonchus contortus.

Heavy reliance on macrocyclic lactones to treat parasitic nematodes has resulted in the evolution of widespread drug resistance that threatens human and animal health. Management strategies have been proposed that would slow the rise of resistance, however testing these strategies has been hampered by the lack of identified strong-effect resistance markers in parasites. We show that the Caenorhabditis elegans gene Cel_dyf-7, necessary for amphid sensory neuron development, also confers macrocyclic lactone sensitivity. In the sheep parasite Haemonchus contortus: (i) strains selected for macrocyclic lactone resistance were enriched in a Hco_dyf-7 haplotype that was rare in the drug-naïve population, (ii) the resistant haplotype correlated with the sensory neuron defects, and (iii) the resistant haplotype was associated with decreased Hco_dyf-7 expression. Resistant field isolates of H. contortus from five continents were enriched for the resistant haplotype, demonstrating the relevance of the Hco_dyf-7 haplotype to practise and indicating that it is a locus of strong effect. Hemizygosity resulting from sex linkage of dyf-7 likely contributes to the rise of resistance in treated populations.

The role of several ABC transporter genes in ivermectin resistance in Caenorhabditis elegans.

The functions of nine ATP-binding cassette (ABC) transporter genes, mrp-1, mrp-4, mrp-6, pgp-2, pgp-3, pgp-4, pgp-5, haf-2 and haf-9, in an ivermectin (IVM) resistant strain of Caenorhabditis elegans were screened by comparing transcription levels between the resistant (IVR10) and wild-type (Bristol N2) strains, and by measuring the effects of RNA interference (RNAi) on the IVM resistant strain, on motility, pharyngeal pumping, egg production and death in the presence or varying concentrations of IVM (0-20 ng/ml). mRNA levels of mrp-1, 2, 4, 5, 6, 7, pgp-1, 2, 4, 12, 14, haf-1, 2 and 3 were significantly increased in IVR10 compared with the N2 strain. At 15 or 20 ng/ml IVM, down regulation of mrp-1, pgp-4, haf-2 and haf-9 significantly increased the effect of IVM to reduce egg production. At low to moderate IVM concentrations, down regulation of mrp-1 and haf-2 reduced the motility of C. elegans. However, at high IVM concentrations motility was increased by down regulation of transcription of pgp-3, pgp-4 and haf-9. Down regulation of expression of mrp-1, pgp-2 and pgp-5 resulted in reduced pharyngeal pumping in the presence of varying concentrations of IVM, while down regulation of mrp-6 and haf-2 increased pharyngeal pumping of the resistant strain irrespective of the IVM concentration used. Although the IVR10 strain was markedly resistant to IVM, compared with the unselected N2 strain, IVM led to the death of the C. elegans in a concentration dependent manner. However, differences in the IVM induced death rate, following RNAi, were not significantly different from the IVR10 strain without RNAi. The study shows that different ABC transporter genes may play a role in modulating the effects of IVM on pharyngeal pumping, motility and egg production, with down regulation of mrp-1 and haf-2 perhaps having the greatest effects. However, down regulation of expression of no individual ABC transporter gene profoundly affected the effect of IVM on mortality in the IVR10 strain. This suggests that some of these ABC transporter genes and their products may play a role in modulating the effects of IVM, but are not, individually, the critical gene responsible for IVM resistance. This study provides a model that may help to understand drug resistance in parasitic nematodes.

Influencia de las variables climáticas en la casuística de dengue y la abundancia de Aedes aegypti (Diptera: Culicidae) en Maracay, Venezuela / Influence of climatic variables on dengue cases and abundance of Aedes aegypti (Diptera: Culicidae) in Maracay, Venezuela

Se realizó un estudio retrospectivo y longitudinal en el área metropolitana de Maracay para el período 1997-2005, a fin de determinar si las variables climáticas y entomológicas influyen sobre la casuística de dengue. Se obtuvieron los datos climatológicos diarios y mensuales de la Estación Climatológica de la Universidad Central de Venezuela y los datos epidemiológicos basados en casos confirmados y muy sugestivos reportados por el Laboratorio Regional de Diagnóstico e Investigación del Dengue y otras Enfermedades Virales (LARDIDEV). Los datos entomológicos se obtuvieron de colectas de adultos de Aedes aegypti (L.) realizadas durante el período Noviembre 2000-Diciembre 2001. La abundancia del vector varió de 3,6 Aedes/casa durante el mes de Abril (período de sequía) a 14,7 Aedes/casa durante el mes de Agosto (período de lluvia). El análisis de correlación de Pearson mostró correlación positiva entre el número de casos reportados con la precipitación (r= 0,7183, P = 0,0038) y la abundancia de Ae. aegypti (r= 0,677, P = 0,0078), pero no con la temperatura ni la humedad relativa para el período Noviembre 2000-Diciembre 2001. El análisis de regresión mostró que para dos meses de rezago existe una regresión lineal altamente significativa (P < 0,0001) con un ajuste de R2= 88,7%, indicando que en promedio, la mayor casuística de dengue ocurre dos meses después de ocurrido el pico de precipitación. Los resultados obtenidos podrían ser utilizados para diseñar e implementar programas para la vigilancia epidemiológica y entomológica del dengue, así como establecer un sistema de alerta temprana para la prevención de brotes y/o epidemias de la enfermedad.

In order to determine the influence of climatic and entomological variables on the number of dengue cases, a retrospective and longitudinal study was conducted in the metropolitan area of Maracay during the period 1997-2005. Daily and monthly climatological data was obtained from the Climatological Station at the Universidad Central de Venezuela and epidemiological data based on confirmed and highly suggestive cases reported by the Regional Laboratory for Diagnosis and Research on Dengue and other Viral Infections (LARDIDEV). Aedes aegypti (L.) adults were collected monthly between November 2000 and December 2001. Vector abundance varied from 3,6 Aedes/house during April (dry season) to 14,7 Aedes/house (rainy season). Pearson correlation analysis showed a positive correlation between the number of dengue cases and rainfall (r= 0,7182, P =0,0038) and abundance of Ae. aegypti (r= 0,667, P =0,0078), but no correlation was found with temperature or relative humidity. Regression analysis showed that there was a highly significant relation (R2 = 88,7%, P< 0,0001) between rainfall and dengue cases at a lag of 2 months. The present results might be used to design and implement programs for the epidemiological and entomological dengue surveillance as well as to establish an early warning system to prevent epidemics.

Worldwide patterns of genetic differentiation imply multiple 'domestications' of Aedes aegypti, a major vector of human diseases.

Understanding the processes by which species colonize and adapt to human habitats is particularly important in the case of disease-vectoring arthropods. The mosquito species Aedes aegypti, a major vector of dengue and yellow fever viruses, probably originated as a wild, zoophilic species in sub-Saharan Africa, where some populations still breed in tree holes in forested habitats. Many populations of the species, however, have evolved to thrive in human habitats and to bite humans. This includes some populations within Africa as well as almost all those outside Africa. It is not clear whether all domestic populations are genetically related and represent a single 'domestication' event, or whether association with human habitats has developed multiple times independently within the species. To test the hypotheses above, we screened 24 worldwide population samples of Ae. aegypti at 12 polymorphic microsatellite loci. We identified two distinct genetic clusters: one included all domestic populations outside of Africa and the other included both domestic and forest populations within Africa. This suggests that human association in Africa occurred independently from that in domestic populations across the rest of the world. Additionally, measures of genetic diversity support Ae. aegypti in Africa as the ancestral form of the species. Individuals from domestic populations outside Africa can reliably be assigned back to their population of origin, which will help determine the origins of new introductions of Ae. aegypti.

Population genetic structure of Aedes aegypti, the principal vector of dengue viruses.

Knowledge of vector population genetic structure is critical for vector-borne disease control and prevention strategies. Advances in both molecular genotyping technology and theoretical developments have contributed to the growing impact of such approaches on medical entomology. The pattern of genetic structure may affect the design of control strategies in determining appropriate control limits necessary to disrupt pathogen transmission. In this review, we focus on the mosquito Aedes aegypti, the primary vector of dengue viruses. We describe and discuss numerous population genetic studies illustrating the local genetic variation and gene flow of Ae. aegypti populations.

Gene flow, subspecies composition, and dengue virus-2 susceptibility among Aedes aegypti collections in Senegal.

BACKGROUND: Aedes aegypti, the "yellow fever mosquito", is the primary vector to humans of the four serotypes of dengue viruses (DENV1-4) and yellow fever virus (YFV) and is a known vector of Chikungunya virus. There are two recognized subspecies of Ae. aegypti sensu latu (s.l.): the presumed ancestral form, Ae. aegypti formosus (Aaf), a primarily sylvan mosquito in sub-Saharan Africa, and Ae. aegypti aegypti (Aaa), found globally in tropical and subtropical regions typically in association with humans. The designation of Ae. aegypti s.l. subspecies arose from observations made in East Africa in the late 1950s that the frequency of pale "forms" of Ae. aegypti was higher in populations in and around human dwellings than in those of the nearby bush. But few studies have been made of Ae. aegypti s.l. in West Africa. To address this deficiency we have been studying the population genetics, subspecies composition and vector competence for DENV-2 of Ae. aegypti s.l. in Senegal. METHODS AND FINDINGS: A population genetic analysis of gene flow was conducted among 1,040 Aedes aegypti s.l. from 19 collections distributed across the five phytogeographic regions of Senegal. Adults lacking pale scales on their first abdominal tergite were classified as Aedes aegypti formosus (Aaf) following the original description of the subspecies and the remainder were classified as Aedes aegypti aegypti (Aaa). There was a clear northwest-southeast cline in the abundance of Aaa and Aaf. Collections from the northern Sahelian region contained only Aaa while southern Forest gallery collections contained only Aaf. The two subspecies occurred in sympatry in four collections north of the Gambia in the central Savannah region and Aaa was a minor component of two collections from the Forest gallery area. Mosquitoes from 11 collections were orally challenged with DENV-2 virus. In agreement with the early literature, Aaf had significantly lower vector competence than Aaa. Among pure Aaa collections, the disseminated infection rate (DIR) was 73.9% with a midgut infection barrier (MIB) rate of 6.8%, and a midgut escape barrier (MEB) rate of 19.3%, while among pure Aaf collections, DIR = 34.2%, MIB rate = 7.4%, and MEB rate = 58.4%. Allele and genotype frequencies were analyzed at 11 nuclear single nucleotide polymorphism (SNP) loci using allele specific PCR and melting curve analysis. In agreement with a published isozyme gene flow study in Senegal, only a small and statistically insignificant percentage of the variance in allele frequencies was associated with subspecies. CONCLUSIONS: These results add to our understanding of the global phylogeny of Aedes aegypti s.l., suggesting that West African Aaa and Aaf are monophyletic and that Aaa evolved in West Africa from an Aaf ancestor.

Genetic relationships among Aedes aegypti collections in Venezuela as determined by mitochondrial DNA variation and nuclear single nucleotide polymorphisms.

A population genetic analysis of gene flow was conducted among 619 Aedes aegypti from nine collections distributed among six geographic regions of Venezuela. Genetic markers included a 387-basepair region of the mitochondrial NADH dehydrogenase 4 (ND4) gene and single nucleotide polymorphisms (SNPs) at 11 nuclear loci. Genotypes at SNP loci were identified using melting curve analysis. Six different ND4 haplotypes were detected and patterns of variation suggested that collections were isolated by distance. The variance in SNP allele frequencies was much less than the variance in haplotype frequencies and a pattern of isolation by distance was not detected. Aedes aegypti from eight collections were orally challenged with dengue 2 virus. Disseminated infection rates ranged from 77% to 95%. The percentage of mosquitoes exhibiting a midgut infection barrier ranged from 2% to 15%, and those exhibiting a midgut escape barrier ranged from 2% to 18%. Venezuelan Ae. aegypti appear to be susceptible to dengue virus infection.

Evaluación de metodologías del ADN recombinante para diagnóstico de P. falciparum, en comparación con los examenes microscópico / Evaluation of DNA recombinant methodologics for the diagnostic of plasmodium falciparum and their comparatien with the microscopic assays

Desde 1984, se ha reportado el uso diagnóstico con una alta sensibilidad de una secuencia altamente repetitiva de 21 pb específica para P. falciparum. Considerando la organización genómica de esta secuencia, que se encuentra en repetidos de 21 pb en tanda, en regiones sub-teloméricas con bloques invertidos, ha sido diseñado un ensayo de PCR (I) Pf-21, con un oligonucleótido único, altamente sensible y específico, acoplado a una hibridación y revelado por métodos no radiactivos para la detección de P. falciparum, en muestras de sangre de pacientes de Tumerebo-Bolívar y en ADN purificado de diferentes aislados provenientes de Brasil y Colombia. Con este sistema de PCR y la optimización de un protocolo para el tratamiento de la muestra utilizando Chelex-100-iron, se ha podido diagnósticar, correctamente P. falciparum. Cuando el ensayo fue comparado con el diagnóstico de gota gruesa y extendido coloreado com Giemsa, se mejoró la sensibilidad del método tradicional en una r=2,7:1 y sobre infecciones mixtas en una r=5:1. Esta aproximación además de proporcionar una alternativa al diagnóstico epidemiológico de P. falciparum, ha permitido obtener patrones de fragmentos amplificados distintos, para aislados de diferente origen geográfico. Estos resultados constituyen una importante herramienta en la epidemiología de la malaria, ya que permitirá masificar el diagnóstico y hacer un reconocimiento de las distintas cepas circulantes en las áreas endémicas venezolanas