Clara B. Ocampo

Insecticide resistance status of Aedes aegypti in 10 localities in Colombia.

Insecticide resistance is one of the major threats to the effectiveness of vector control programs. In order to establish a baseline susceptibility profile of Aedes aegypti in the southwest of Colombia, 10 localities in four Departments (States) were evaluated. Standardized WHO bioassay, CDC bottle bioassay and microplate biochemical assays of non-specific β-esterase (NSE), mixed function oxidases (MFO) and acetylcholinesterase were used. Cross resistance was evaluated with field collected mosquitoes that underwent selection pressure in the laboratory from DDT, propoxur and lambdacyhalothrin during three alternate generations. Mosquitoes with mortality rates below 80% in bioassays were considered resistant. Insecticide resistance varied geographically. Insecticide resistance was observed in 100% of localities in which mosquitoes were exposed to DDT, bendiocarb and temephos using both assays. WHO bioassays showed susceptibility to pyrethroids in all the localities evaluated, however CDC bottle bioassays showed decreases in susceptibility especially with lambdacyhalothrin. All localities showed susceptibility to the organophosphate malathion. Mosquitoes from eight regions with evidence of resistance to any of the insecticide evaluated were also evaluated biochemically. Mosquitoes from five of these regions had increased levels of NSE and two regions had increased levels of MFO. Increase levels of NSE explain partially the low susceptibility to temephos found in all the localities. However, the biochemical mechanisms evaluated do not explain all the resistance observed. Cross resistance was observed between the DDT-selected strain and lambdacyhalothrin, and between the lambdacyhalothrin-selected strain and propoxur and vice versa. The selected strains do not show changes in the biochemical assays evaluated, therefore the observed cross-resistance suggests different biochemical mechanisms. This study shows that Ae. aegypti from Colombia can develop resistance to most of the insecticide classes in the market. Periodic surveillance of insecticide resistance is necessary in order to maintain effective interventions. This study helped to establish the National Network for the surveillance of the insecticide resistance in Colombia.

Environmental Risk Factors for the Incidence of American Cutaneous Leishmaniasis in a Sub-Andean Zone of Colombia (Chaparral, Tolima)

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 countys 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.

Reduction in dengue cases observed during mass control of Aedes (Stegomyia) in street catch basins in an endemic urban area in Colombia

Dengue incidence continues to increase globally and, in the absence of an efficacious vaccine, prevention strategies are limited to vector control. It has been suggested that targeting the most productive breeding sites instead of all water-holding containers could be a cost-effective vector control strategy. We sought to identify and continuously control the most productive Aedes (Stegomyia) breeding site in an endemic urban area in Colombia and followed the subsequent incidence of dengue. In the urban area of Guadalajara de Buga, southwestern Colombia, potential breeding sites inside and outside houses were first characterized, and local personnel trained to assess their productivity based on the pupae/person index. Simultaneously, training and monitoring were implemented to improve the dengue case surveillance system. Entomological data and insecticide resistance studies were used to define the targeted intervention. Then, a quasi-experimental design was used to assess the efficacy of the intervention in terms of the positivity index of the targeted and non- targeted breeding sites, and the impact on dengue cases. Street catch basins (storm drains) were the potential breeding site most frequently found containing Aedes immature stages in the baseline (58.3% of 108). Due to the high resistance to temephos (0% mortality after 24h), the intervention consisted of monthly application of pyriproxyfen in all the street catch basins (n=4800). A significant decrease in catch basins positivity for Aedes larvae was observed after each monthly treatment (p<0.001). Over the intervention period, a reduction in the dengue incidence in Buga was observed (rate ratio 0.19, 95% CI 0.12-0.30, p<0.0001) after adjusting for autocorrelation and controlling with a neighboring town, Palmira, This study highlights the importance of street catch basins as Aedes breeding sites and suggests that their targeted control could help to decrease dengue transmission in such areas.

Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain.

Differential Gene Expression from Midguts of Refractory and Susceptible Lines of the Mosquito, Aedes aegypti, Infected with Dengue-2 Virus

Abstract Suppressive subtractive hybridization was used to evaluate the differential expression of midgut genes of feral populations of Aedes aegypti (Diptera: Culicidae) from Colombia that are naturally refractory or susceptible to Dengue-2 virus infection. A total of 165 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries. The analysis showed a higher number of differentially expressed genes in the susceptible Ae. aegypti individuals than the refractory mosquitoes. The functional annotation of ESTs revealed a broad response in the susceptible library that included immune molecules, metabolic molecules and transcription factors. In the refractory strain, there was the presence of a trypsin inhibitor gene, which could play a role in the infection. These results serve as a template for more detailed studies aiming to characterize the genetic components of refractoriness, which in turn can be used to devise new approaches to combat transmission of dengue fever.

Phlebotomine Vector Ecology in the Domestic Transmission of American Cutaneous Leishmaniasis in Chaparral, Colombia

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.

Leishmania (Viannia) Infection in the Domestic Dog in Chaparral, Colombia

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.

Environmental factors associated with American cutaneous leishmaniasis in a new Andean focus in Colombia

Objective  To evaluate the environmental and ecological factors associated with Leishmania transmission and vector abundance in Chaparral, Tolima‐Colombia.

DIFFERENTIAL IDENTIFICATION OF ASCOGREGARINA SPECIES (APICOMPLEXA: LECUDINIDAE) IN AEDES AEGYPTI AND AEDES ALBOPICTUS (DIPTERA: CULICIDAE) BY POLYMERASE CHAIN REACTION

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straightforward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections.

Description of Lutzomyia (Helcocyrtomyia) tolimensis, a new species of phlebotomine sandfly (Diptera: Psychodidae) from Colombia

A description is presented of Lutzomyia tolimensis sp. nov., a new species of the subgenus Helcocyrtomyia, series sanguinaria. It was collected in dwellings, peridomestic environment and in nearby forest patches located in the foothills of the Andean Central Cordillera, where in 2004-2006 occurred the largest epidemic ever recorded of leishmaniasis in Colombia. The male of this species is differentiated from other members of the series sanguinaria based on the following combination of characters: (i) base of coxite with 0-3 subequal setae, (ii) spines of gonostyle organized in positions 2.1.2, (iii) spines inserted on distal half of gonostyle and (iv) relationship of alar indices. The female is recognized principally by the following characters: (i) palpomere V longer than III, (ii) length of labro-epipharynx and (iii) relationship of the alar indices.