Laboratory Evaluation and Field Feasibility of Micro-Encapsulated Insecticide Effect on Rhodnius prolixus and Triatoma dimidiata Mortality in Rural Households in Boyacá, Colombia

Abstract: Chagas disease is a neglected vector-borne zoonosis caused by the parasite Trypanosoma cruzi that is primarily transmitted by insects of the subfamily Triatominae. Although control efforts targeting domestic infestations of Rhodnius prolixus have been largely successful, with several regions in Boyacá department certified free of T. cruzi transmission by intradomicile R. prolixus, novel native species are emerging, increasing the risk of disease. Triatoma dimidiata is the second most important species in Colombia, and conventional control methods seem to be less effective. In this study we evaluated the efficacy and usefulness of micro-encapsulated insecticide paints in laboratory conditions and its applicability in rural communities to avoid triatomine domiciliation. Laboratory conditions measured mortality at 6 months and 12 months, with an average mortality between 93­100% for T. dimidiata and 100% for R. prolixus. Evaluation of triatomine infestation in rural households was measured after one year, with an overall perception of effectiveness in reducing household domiciliation. Although triatomines were still spotted inside and around the homes, our findings demonstrate the ability of micro-encapsulated insecticide to prevent colonization inside the households when comparing infestation rates from previous years. Current control measures suggest insecticide spraying every six months, which implies great economic cost and logistical effort. Complementary triatomine control measures with insecticide spraying and micro-encapsulated insecticide paint would make public health efforts more efficient and reduce the frequency of treatment.

INFECCIÓN NATURAL POR Trypanosoma cruzi (TRYPANOSOMATIDAE) EN TRIATOMINOS INTRADOMÉSTICOS DEL DEPARTAMENTO DE GUAINÍA / Natural infection with Trypanosoma cruzi (TRYPANOSOMATIDAE) in intradomestic triatomine bugs from Guainía department

RESUMEN La búsqueda continua de triatominos forma parte de los programas de vigilancia de la enfermedad de Chagas en zonas con características ecológicas apropiadas para la presencia del vector, permitiendo priorizar y definir las acciones de intervención. El objetivo del presente trabajo fue determinar la presencia y estado de infección de triatominos en viviendas del municipio de Inírida (Guainía). El estudio se realizó entre 2018 y 2019 mediante vigilancia comunitaria y búsqueda activa. Los vectores se identificaron y evaluaron por PCR. Se recolectaron cinco triatominos (un Panstrongylus lignarius y cuatro P. geniculatus). Se evidenció la infección natural en ambas especies. La genotipificación mostró la presencia de TcI Dom. Se actualiza así la presencia e infección P. lignarius, así como la infección de P. geniculatus para Inírida.

ABSTRACT The continuous search and characterization of triatomine bugs is essential for Chagas disease surveillance programs in areas with ideal ecological conditions for the distribution of these vectors. These activities are necessary to define and optimize intervention strategies. The objective of this work was to determine the presence of triatomine and its infection status in households located in the municipality of Inírida (Guainía). Between 2018 and 2019, we developed a community participation strategy where an active search was essential for the capturing of triatomine bugs. The collected bugs were evaluated by PCR allowing to identify one as Panstrongylus lignarius and four as P. geniculatus. Genotyping showed the presence of TcI Dom confirming infection in both species. Thus, this study presents an update of the infection status of P. geniculatus and the presence of infected P. lignarius in the Guainía region in Colombia.

Population structure and ancestry prediction of Aedes aegypti (Diptera: Culicidae) supports a single African origin of Colombian populations

BACKGROUND A previous phylogeographic study revealed two Aedes aegypti African-related mitochondrial lineages distributed in Colombian's cities with different eco-epidemiologic characteristics with regard to dengue virus (DENV). It has been proposed these lineages might indicate independent invasion sources. OBJECTIVES Assessing to Colombian population structure and to support evidence of its probable source origin. METHODS We analysed a total of 267 individuals from cities of Bello, Riohacha and Villavicencio, which 241 were related to the West and East African mitochondrial lineages (termed here as WAL and EAL, respectively). Eight polymorphic microsatellite loci were analysed aiming population structure. FINDINGS Results indicate substantial gene flow among distant and low-connected cities composing a panmictic population with incipient local differentiation of Ae. aegypti is placed in Colombia. Likewise, genetic evidence indicates no significant differences among individuals related to WAL and EAL is placed. MAIN CONCLUSIONS Minimal genetic differentiation in low-connected Ae. aegypti populations of Colombia, and lack concordance between mitochondrial and nuclear genealogies suggest that Colombian Ae. aegypti shared a common demographic history. Under this scenario, we suggest current Ae. aegypti population structure reflects a single origin instead of contemporary migration, which founding populations have a single source from a mitochondrial polymorphic African ancient.

Distribution and natural infection status of synantrophic triatomines (Hemiptera: Reduviidae), vectors of Trypanosoma cruzi, reveals new epidemiological scenarios for chagas disease in the Highlands of Colombia

Updating the distribution and natural infection status of triatomine bugs is critical for planning, prioritizing, and implementing strategies to control Chagas disease (CD), especially after vector reduction programs. After carrying out a control program, the Department of Boyaca contains the highest number of Colombian municipalities certified by PAHO to be free of intradomiciliary transmission of Trypanosoma cruzi by Rhodnius prolixus. The present work describes the spatial distribution, natural infection (NI), and molecular characterization of T. cruzi in synanthropic triatomines from the Department of Boyaca in 2017 and 2018. Materials and methods: An entomological survey was conducted in 52 municipalities in Boyaca known to have had previous infestations of triatomine bugs. Insects were collected through active searches carried out by technical personnel from the Secretary of Health and community members using Triatomine Collection Stations (PITs-acronym in Spanish). For evaluation of natural infection, triatomines were identified morphologically and grouped in pools of one to five individuals of the same species collected in the same household. DNA derived from the feces of each pool of insects was analyzed by PCR for the presence of T. cruzi using primers flanking the satellite DNA of the parasite. SL-IR primers were used to differentiate TCI from the other DTUs and to identify different genotypes. The distribution of the collected triatomines was analyzed to determine any vector hotspots using spatial recreation. Results: A total of 670 triatomine bugs was collected, belonging to five species: Triatoma dimidiata (73.2%), Triatoma venosa (16.7%), Panstrongylus geniculatus (5.7%), Rhodnius prolixus (4.4%), and Panstrongylus rufotuberculatus (0.4%), from 29 of the 52 municipalities. In total, 71.6% of the bugs were collected within houses (intradomiciliary) and the rest around the houses (peridomiciliary). Triatoma dimidiata was the most widely distributed species and had the highest natural infection index (37.8%), followed by T. venosa and P. geniculatus. TcI was the only DTU found, with the TcI Dom genotype identified in 80% of positive samples and TcI sylvatic in the other insects. Spatial analysis showed clusters of T. dimidiata and T. venosa in the northeast and southwest regions of Boyaca. Conclusions: After some municipalities were certified free of natural transmission within houses (intradomiciliary transmission) of T. cruzi by R. prolixus, T. dimidiata has become the most prevalent vector present, and represents a significant risk of resurgent CD transmission. However, T. venosa, P. geniculatus, and P. rufotuberculatus also contribute to the increased risk of transmission. The presence of residual R. prolixus may undo the successes achieved through vector elimination programs. The molecular and spatial analysis used here allows us to identify areas with an ongoing threat of parasite transmission and improve entomological surveillance strategies.

Natural infection of Aedes aegypti, Ae. albopictus and Culex spp. with Zika virus in Medellin, Colombia / Infección natural de Aedes aegypti, Ae. albopictus y Culex spp. con virus Zika en Medellín, Colombia

Abstract Introduction: The Zika virus has generated serious epidemics in the different countries where it has been reported and Colombia has not been the exception. Although in these epidemics Aedes aegypti traditionally has been the primary vector, other species could also be involved in the transmission. Methods: Mosquitoes were captured with entomological aspirators on a monthly ba sis between March and September of 2017, in four houses around each of the 250 entomological surveillance traps installed by the Secretaria de Sa lud de Medellin (Colombia). Additionally, 70 Educational Institutions and 30 Health Centers were visited each month. Results: 2 504 mosquitoes were captured and grouped into 1045 pools to be analyzed by RT-PCR for the detection of Zika virus. Twenty-six pools of Aedes aegypti, two pools of Ae. albopictus and one for Culex quinquefasciatus were positive for Zika virus. Conclusion: The presence of this virus in the three species and the abundance of these species in the different sectors of the city, suggests that the control strategies should be addressed to all species that could be potential vec tors until the vectorial role of Ae. albopictus and Culex quinquefasciatus is completely elucidated in the transmission of the Zika virus.

Resumen Introducción: El virus Zika ha generado serias epidemias en los diferentes países en donde se ha reportado, Colombia no ha sido la excepción. Aunque en estas epidemias el vector presuntamente involucrado ha sido Aedes aegypti, otras especies también podrían estar implicadas en su transmisión. Meto dología: en el marco del Programa de vigilancia virológica en mosquitos de la Secretaría de Salud de Medellín (Colombia). Resultados: entre marzo y septiembre de 2017 se capturaron 2 504 mosquitos en predios visitados durante los levantamientos de índices entomológicos. El material entomo lógico fue agrupado en 1 045 pooles para ser analizados por RT-PCR para la detección de virus Zika. Resultaron positivos para virus Zika 26 pooles de Aedes aegypti, dos pooles de Ae. albopictus y uno para Culex quinquefasciatus. Conclusión: la presencia de este virus en las tres especies y su abundancia en los distintos sectores de la ciudad sugieren que las estrategias de control deban dirigirse a todos los potenciales vectores, hasta tanto no se dilucide comple tamente el papel vectorial de Ae. albopictus y Cx. quinquefasciatus en la transmisión del virus Zika.

Tripanosomiasis bovina en ganadería lechera de trópico alto: primer informe de Haematobia irritans como principal vector de T. vivax y T. evansi en Colombia / Bovine trypanosomiasis in dairy farming in the high tropics: First report of Haematobia irritans as the main vector for T. vivax and T. evansi in Colombia / Tripanossomíase bovina em gado leiteiro de trópico alto: primeiro relatório de Haematobia irritans como principal vetor de T. vivax e T. evansi na Colômbia

Resumen: La tripanosomiasis bovina es una enfermedad hemoparasitaria transmitida en Latinoamérica principalmente por moscas picadoras de la familia Tabanidae. El objetivo del estudio fue evaluar la infección por Trypanosoma vivax y Trypanosoma evansi en ganadería bovina especializada en producción de leche en una hacienda y sus potenciales vectores. Se realizó un estudio parasitológico y entomológico directo por técnicas de microscopia y reacción en cadena de la polimerasa (PCR) con dos marcadores moleculares para diferenciar especies de Trypanosoma en muestras de sangre de bovinos y moscas. La frecuencia de infección por Trypanosoma vivax y Trypanosoma evansi en bovinos fue de 3,6 y 0 %, respectivamente. La caracterización de vectores muestra a Haematobia irritans como la mosca más frecuente en la zona de estudio (97,1 %), seguida de Stomoxys calcitrans (2,8 %). No se identificaron tabánidos. Se encontró T. vivax y T. evansi en probóscide y toráx-abdomen de las moscas picadoras Haematobia irritans y Stomoxys calcitrans, lo que representa un comportamiento epizoótico atípico al que sucede en países de Suramérica. Por su alta densidad poblacional, se sugiere la mosca Haematobia irritans como el principal potencial vector.

Abstract: Bovine trypanosomiasis is a hemoparasitic disease transmitted in Latin America mainly by biting flies of the family Tabanidae. The study aimed to evaluate infection by Trypanosoma vivax and Trypanosoma evansi in cattle specialized in milk production on a farm and their potential vectors. A direct parasitological and entomological study was performed using microscopy techniques and polymerase chain reaction (PCR) with two molecular markers to differentiate Trypanosoma species in blood samples of cattle and flies. Infection frequency with Trypanosoma vivax and Trypanosoma evansi in cattle was 3.6 and 0%, respectively. Characterization of vectors shows Haematobia irritans as the most frequent fly in the study area (97.1%), followed by Stomoxys calcitrans (2.8%). No horseflies were identified. T. vivax and T. evansi were found in proboscis and thorax-abdomen of biting flies Haematobia irritans and Stomoxys calcitrans, representing an epizootic behavior, atypical in South American countries. Due to its high population density, it is suggested that the Haematobia irritans fly is the main potential vector.

Resumo: A tripanossomíase bovina é uma doença causada por hemoparasitas e transmitida na América Latina principalmente por moscas picadoras da família Tabanidae. O objetivo do estudo foi avaliar a infecção por Trypanosoma vivax e Trypanosoma evansi em gado bovino especializada em produção de leite em uma fazenda e seus potenciais vectores. Se realizou um estudo parasitológico e entomológico direto por técnicas de microscopia e reação em cadeia da polimerase (PCR) com dois marcadores moleculares para diferenciar espécies de Typanosoma em amostras de sangue de bovinos e moscas. A frequência de infecção por Trypanosoma vivax e Trypanosoma evansi em bovinos foi de 3,6 e 0 %, respectivamente. A caracterização de vetores mostra a Haematobia irritans como a mosca mais frequente na zona de estudo (97,1 %), seguida de Stomoxys calcitrans (2,8 %). Não se identificaram tabanídeos. Se encontrou T. vivax e T. evansi em proboscídea e tórax-abdômen das moscas picadoras Haematobia irritans e Stomoxys calcitrans, o que representa um comportamento epizoótico atípico ao que sucede em países da América do Sul. Por sua alta densidade populacional, se sugere a mosca Haematobia irritans como o principal potencial vetor.

Tripanosomiasis bovina en ganadería lechera de trópico alto: primer informe de Haematobia irritans como principal vector de T. vivax y T. evansi en Colombia

La tripanosomiasis bovina es una enfermedad hemoparasitaria transmitida en Latinoamérica principalmente por moscas picadoras de la familia Tabanidae. El objetivo del estudio fue evaluar la infección por Trypanosoma vivax y Trypanosoma evansi en ganadería bovina especializada en producción de leche en una hacienda y sus potenciales vectores. Se realizó un estudio parasitológico y entomológico directo por técnicas de microscopia y reacción en cadena de la polimerasa (PCR) con dos marcadores moleculares para diferenciar especies de Trypanosoma en muestras de sangre de bovinos y moscas. La frecuencia de infección por Trypanosoma vivax y Trypanosoma evansi en bovinos fue de 3,6 y 0 %, respectivamente. La caracterización de vectores muestra a Haematobia irritans como la mosca más frecuente en la zona de estudio (97,1 %), seguida de Stomoxys calcitrans (2,8 %). No se identificaron tabánidos. Se encontró T. vivax y T. evansi en probóscide y toráx-abdomen de las moscas picadoras Haematobia irritans y Stomoxys calcitrans, lo que representa un comportamiento epizoótico atípico al que sucede en países de Suramérica. Por su alta densidad poblacional, se sugiere la mosca Haematobia irritans como el principal potencial vector.

Bovine trypanosomiasis is a hemoparasitic disease transmitted in Latin America mainly by biting flies of the family Tabanidae. The study aimed to evaluate infection by Trypanosoma vivax and Trypanosoma evansi in cattle specialized in milk production on a farm and their potential vectors. A direct parasitological and entomological study was performed using microscopy techniques and polymerase chain reaction (PCR) with two molecular markers to differentiate Trypanosoma species in blood samples of cattle and flies. Infection frequency with Trypanosoma vivax and Trypanosoma evansi in cattle was 3.6 and 0%, respectively. Characterization of vectors shows Haematobia irritans as the most frequent fly in the study area (97.1%), followed by Stomoxys calcitrans (2.8%). No horseflies were identified. T. vivax and T. evansi were found in proboscis and thorax-abdomen of biting flies Haematobia irritans and Stomoxys calcitrans, representing an epizootic behavior, atypical in South American countries. Due to its high population density, it is suggested that the Haematobia irritans fly is the main potential vector.

A tripanossomíase bovina é uma doença causada por hemoparasitas e transmitida na América Latina principalmente por moscas picadoras da família Tabanidae. O objetivo do estudo foi avaliar a infecção por Trypanosoma vivax e Trypanosoma evansi em gado bovino especializada em produção de leite em uma fazenda e seus potenciais vectores. Se realizou um estudo parasitológico e entomológico direto por técnicas de microscopia e reação em cadeia da polimerase (PCR) com dois marcadores moleculares para diferenciar espécies de Typanosoma em amostras de sangue de bovinos e moscas. A frequência de infecção por Trypanosoma vivax e Trypanosoma evansi em bovinos foi de 3,6 e 0 %, respectivamente. A caracterização de vetores mostra a Haematobia irritans como a mosca mais frequente na zona de estudo (97,1 %), seguida de Stomoxys calcitrans (2,8 %). Não se identificaram tabanídeos. Se encontrou T. vivax e T. evansi em proboscídea e tórax-abdômen das moscas picadoras Haematobia irritans e Stomoxys calcitrans, o que representa um comportamento epizoótico atípico ao que sucede em países da América do Sul. Por sua alta densidade populacional, se sugere a mosca Haematobia irritans como o principal potencial vetor.

Sensibilidad al benzonidazol de cepas de Trypanosoma cruzi sugiere la circulación de cepas naturalmente resistentes en Colombia / Trypanosoma cruzi strains resistant to benznidazole occurring in Colombia

Introducción. La enfermedad de Chagas, causada por Trypanosoma cruzi, es uno de los problemas más graves de salud pública en el continente americano. El benzonidazol es uno de los dos medicamentos utilizados para tratar la enfermedad de Chagas. Sin embargo, la variación de la sensibilidad del parásito a este medicamento es una de las principales causas del fracaso del tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benzonidazol de cepas colombianas de T. cruzi de diferentes orígenes y procedencia geográfica. Materiales y métodos. Treinta y tres cepas colombianas de T. cruzi aisladas de humanos, vectores y mamíferos, se analizaron in vitro mediante el micrométodo enzimático de MTT para determinar la concentración inhibitoria 50 (CI50) al benzonidazol. Se estudió la correlación entre la sensibilidad in vitro al medicamento y diferentes parámetros biológicos y eco-epidemiológicos. Resultados. El análisis de sensibilidad al medicamento indicó que el 36 % de las cepas eran sensibles, el 48 %, parcialmente resistentes y, el 16 %, resistentes al benzonidazol. Los análisis de correlación entre las CI50 con algunos parámetros biológicos y eco-epidemiológicos, mostraron diferencias en cuanto a la sensibilidad según el origen biológico y el área geográfica de procedencia de la cepa. Conclusiones. Existe una gran variabilidad en cuanto a la sensibilidad al benzonidazol de las cepas circulantes de T. cruzi en Colombia, lo cual sugiere la presencia de cepas naturalmente resistentes en el país.

Introduction. Chagas disease caused by Trypanosoma cruzi is one of the most serious public health problems in the Americas. Benznidazole is one of two drugs used to treat Chagas´ disease. However, the variation in susceptibility of the parasite to this drug is one of the main causes of treatment failure. Objective. The in vitro susceptibility to benznidazole was assessed in Colombian strains of T. cruzi from several sources and geographical regions. Materials and methods. Thirty-three Colombian T. cruzi strains were isolated from humans, vectors and mammals. These were analyzed in vitro by the MTT enzymatic micromethod to determine the IC50 to benznidazole. Additionally, the in vitro susceptibility was correlated with several biological and ecoepidemiological parameters. Results. Thirty-six percent of the strains were considered to be sensitive, 48% partially resistant, and 16% were resistant. Correlations between the IC50 and several biological and eco-epidemiological parameters indicated that differences in susceptibility depended on the biological source and geographical origin of the strain. Conclusions. A high degree of variability exists in the susceptibility to benznidazole of T. cruzi strains in Colombia. The distribution data indicate the presence and circulation of naturally resistant strains.

Quantification of the genetic change in the transition of Rhodnius pallescens Barber, 1932 (Hemiptera: Reduviidae) from field to laboratory

Previous studies have reported genetic differences between wild-caught sylvatic, domestic and laboratory pop-ulations of several Triatominae species. The differences between sylvatic and laboratory colonies parallel are similar to the differences observed between sylvatic and domestic populations. Laboratory colonies are frequently used as references for field populations, but the consequences of founder events on the genetic makeup of laboratory or domestic populations are rarely quantified. Our goal was to quantify the genetic change in Rhodnius pallescens populations artificially submitted to founder effects via laboratory colonization. We compared the genetic makeup of two sylvatic populations and their laboratory descendants using a panel of 10 microsatellite markers. Both sylvatic populations were initially collected from palm trees, but the colonies differed in the number of founder insects and amount of time kept in the laboratory. We evaluated allelic polymorphism, differences between expected and observed heterozygosity, estimates of population differentiation (Fst) and inbreeding (Fis, Fit) and cluster analyses based on Nei's distances. We found a unique genetic structure for each sample population, with significant differentiation between the field insects and each of the laboratory generations. These analyses showed strong founder effects and showed that genetic drift had led to a genetic equilibrium over several generations of isolation. Our results suggest that laboratory colonies of R. pallescens have a different genetic structure than their wild relatives and similar processes likely affect other Triatominae laboratory stocks.

Chromosome variability in the Chagas disease vector Rhodnius pallescens (Hemiptera, Reduviidae, Rhodniini)

Rhodnius pallescens is the main vector of Trypanosoma cruzi in Panama and one of the most relevant secondary vectors in Colombia. Despite the importance of this species, there is limited knowledge about the genetic variability along its geographical distribution. In order to evaluate the degree of karyotype variability we analyzed the meiotic behavior and banding pattern of the chromosomes of 112 males of R. pallescens coming from different regions of Colombia and Panama. Using the C-banding technique we identified two chromosomal patterns or cytotypes characterized by differences in the amount, size and distribution of constitutive heterochromatic regions in the chromosome complement (2n = 20 autosomes plus XY in males). The individuals can be easily classified in each cytotype by the analysis of the chromosomes during first meiotic prophase. The frequencies of the cytotypes are variable according to the geographic origin of the populations. This chromosomal divergence together with morphological data supports the existence of three genetically different populations of R. pallescens and provides new information to understand the distribution dynamics of this species.