Natural Aedes-Borne Virus Infection Detected in Male Adult Aedes aegypti (Diptera: Culicidae) Collected From Urban Settings in Mérida, Yucatán, México.

Aedes-borne viruses (ABVs) such as dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) contribute significantly to the global burden of infectious diseases, disproportionately affecting disadvantaged populations from tropical and subtropical urban areas. ABVs can be transmitted from female mosquitoes to their progeny by vertical transmission via transovarial and/or trans-egg vertical transmission and contribute to the maintenance of infected-mosquito populations year-round in endemic regions. This study describes the natural infection rate of DENV, CHIKV, and ZIKV in field-caught male Aedes (Sergentomyia) aegypti (Linnaeus) mosquitoes from Mérida, Yucatán, México, as a proxy for the occurrence of vertical virus transmission. We used indoor sequential sampling with Prokopack aspirators to collect all mosquitoes inside houses from ABV hotspots areas. Collections were performed in a DENV and CHIKV post-epidemic phase and during a period of active ZIKV transmission. We individually RT-qPCR tested all indoor collected Ae. aegypti males (1,278) followed by Sanger sequencing analysis for final confirmation. A total of 6.7% male mosquitoes were positive for ABV (CHIKV = 5.7%; DENV = 0.9%; ZIKV = 0.1%) and came from 21.0% (30/143) houses infested with males. Most ABV-positive male mosquitoes were positive for CHIKV (84.8%). The distribution of ABV-positive Ae. aegypti males was aggregated in a few households, with two houses having 11 ABV-positive males each. We found a positive association between ABV-positive males and females per house. These findings suggested the occurrence of vertical arbovirus transmission within the mosquito populations in an ABV-endemic area and, a mechanism contributing to viral maintenance and virus re-emergence among humans in post-epidemic periods.

Natural arbovirus infection rate and detectability of indoor female Aedes aegypti from Mérida, Yucatán, Mexico.

Arbovirus infection in Aedes aegypti has historically been quantified from a sample of the adult population by pooling collected mosquitoes to increase detectability. However, there is a significant knowledge gap about the magnitude of natural arbovirus infection within areas of active transmission, as well as the sensitivity of detection of such an approach. We used indoor Ae. aegypti sequential sampling with Prokopack aspirators to collect all mosquitoes inside 200 houses with suspected active ABV transmission from the city of Mérida, Mexico, and tested all collected specimens by RT-PCR to quantify: a) the absolute arbovirus infection rate in individually tested Ae. aegypti females; b) the sensitivity of using Prokopack aspirators in detecting ABV-infected mosquitoes; and c) the sensitivity of entomological inoculation rate (EIR) and vectorial capacity (VC), two measures ABV transmission potential, to different estimates of indoor Ae. aegypti abundance. The total number of Ae. aegypti (total catch, the sum of all Ae. aegypti across all collection intervals) as well as the number on the first 10-min of collection (sample, equivalent to a routine adult aspiration session) were calculated. We individually tested by RT-PCR 2,161 Aedes aegypti females and found that 7.7% of them were positive to any ABV. Most infections were CHIKV (77.7%), followed by DENV (11.4%) and ZIKV (9.0%). The distribution of infected Aedes aegypti was overdispersed; 33% houses contributed 81% of the infected mosquitoes. A significant association between ABV infection and Ae. aegypti total catch indoors was found (binomial GLMM, Odds Ratio > 1). A 10-min indoor Prokopack collection led to a low sensitivity of detecting ABV infection (16.3% for detecting infected mosquitoes and 23.4% for detecting infected houses). When averaged across all infested houses, mean EIR ranged between 0.04 and 0.06 infective bites per person per day, and mean VC was 0.6 infectious vectors generated from a population feeding on a single infected host per house/day. Both measures were significantly and positively associated with Ae. aegypti total catch indoors. Our findings provide evidence that the accurate estimation and quantification of arbovirus infection rate and transmission risk is a function of the sampling effort, the local abundance of Aedes aegypti and the intensity of arbovirus circulation.

Rickettsia spp. en garrapatas (Acari: Ixodidae) que infestan perros de una comunidad rural con antecedentes de rickettsiosis, Yucatán, México / Rickettsia spp. in ticks (Acari: Ixodidae) infesting dogs from a rural community with rickettsial diseases reports, Yucatan, Mexico

RESUMEN Introducción: Las bacterias del género Rickettsia son agentes causales de las rickettsiosis, enfermedades zoonóticas transmitidas por ectoparásitos. En Yucatán, ocurrió un brote en habitantes de Bolmay, Valladolid; sin embargo, no se identificó el probable artrópodo vector. Objetivo: Analizar la presencia de Rickettsia spp. en garrapatas que infestan perros domésticos de Bolmay. Material y métodos: Se trabajaron 105 viales con hasta ocho garrapatas. Se extrajo ADN de los ectoparásitos, posterior a la identificación del estadio de desarrollo vital, sexo, género y especie. Se realizaron dos PCR para aislar fragmentos de los genes htrA (17-kDa) y rOmpB, pertenecientes a Rickettsia spp. Los productos positivos fueron secuenciados y analizados con la herramienta BLAST y el algoritmo Megablast. Resultados: Se utilizaron 291 garrapatas de los géneros Amblyomma (55.7%, 162/291), Rhipicephalus (34%, 99/291) e Ixodes (10.3%, 30/291), y las especies Rhipicephalus sanguineus sensu lato, Ixodes affinis y Amblyomma mixtum. La positividad por PCR fue de 11.4% (12/105) para los géneros Amblyomma (66.7%, 8/12), Rhipicephalus (25%, 3/12) e Ixodes (8.3%, 1/12), y las especies A. mixtum, Rh. sanguineus s. l. e I. affinis. Se identificó ADN rickettsial en ninfas, larvas, adultos, machos y hembras. Cada análisis BLAST arrojó un 96% de cobertura e identidad con varias secuencias de Rickettsia spp. como Rickettsia typhi, Rickettsia felis y Rickettsia prowazekii, por lo que no fue posible determinar la especie infectante. Conclusión: Las garrapatas que infestan perros de Bolmay, probablemente están involucrados en el ciclo de transmisión de Rickettsia spp. Se presenta la primera evidencia molecular de Rickettsia spp. en garrapatas I. affinis de Yucatán, México.

ABSTRACT Introduction: Bacteria of the Rickettsia genus are causal agents of the rickettsial diseases, zoonoses transmitted by ectoparasites. In Yucatan, in inhabitants from Bolmay an outbreak occurred; however, the probable vector was not identified. Objective: To analyze the Rickettsia spp. presence in ticks infesting domestic dogs from Bolmay. Material and methods: We used 105 vials with up to eight ticks. Total DNA from the ectoparasites was extracted, after the identification of the life stages, sex, gender, and species. Two PCR to isolate fragments of the htrA (17-kDa) and rOmpB genes belonging to Rickettsia spp., were performed. The positive products were sequenced and analyzed using the BLAST tool and the Megablast algorithm. Results: 291 ticks of the genera Amblyomma (55.7%, 162/291), Rhipicephalus (34%, 99/291), and Ixodes (10.3%, 30/291), and the species Rhipicephalus sanguineus sensu lato, Ixodes affinis, and Amblyomma mixtum, were used. The PCR positivity was 11.4% (12/105) for the genera Amblyomma (66.7%, 8/12), Rhipicephalus (25%, 3/12), and Ixodes (8.3%, 1/12), and the species A. mixtum, Rh. sanguineus s. l., and I. affinis. Rickettsial DNA in nymph, larva, adult, males, and females, were identified. Each BLAST analysis showed an identity and coverage of 96% with several Rickettsia spp. sequences, such as Rickettsia typhi, Rickettsia felis and Rickettsia prowazekii, so it was not possible to determine the infecting species. Conclusion: Ticks infesting dogs from Bolmay are probably involved in the transmission cycle of Rickettsia spp. The first molecular evidence of Rickettsia spp. in I. affinis ticks from Yucatan, Mexico, is presented.

Estimating absolute indoor density of Aedes aegypti using removal sampling.

BACKGROUND: Quantification of adult Aedes aegypti abundance indoors has relied on estimates of relative density (e.g. number of adults per unit of sampling or time), most commonly using traps or timed collections using aspirators. The lack of estimates of the sensitivity of collections and lack of a numerical association between relative and the absolute density of adult Ae. aegypti represent a significant gap in vector surveillance. Here, we describe the use of sequential removal sampling to estimate absolute numbers of indoor resting Ae. aegypti and to calculate calibration coefficients for timed Prokopack aspirator collections in the city of Merida, Yucatan State, Mexico. The study was performed in 200 houses that were selected based on recent occurrence of Aedes-borne viral illness in residents. Removal sampling occurred in 10-minute sampling rounds performed sequentially until no Ae. aegypti adult was collected for 3 hours or over 2 consecutive 10-minute periods. RESULTS: A total of 3439 Ae. aegypti were collected. The sensitivity of detection of positive houses in the first sampling round was 82.5% for any adult Ae. aegypti, 78.5% for females, 75.5% for males and 73.3% for blood-fed females. The total number of Ae. aegypti per house was on average ~5 times higher than numbers collected for the first sampling round. There was a positive linear relationship between the relative density of Ae. aegypti collected during the first 10-min round and the absolute density for all adult metrics. Coefficients from the linear regression were used to calibrate numbers from 10-min collections into estimates of absolute indoor Ae. aegypti density for all adults, females and males. CONCLUSIONS: Exhaustive removal sampling represents a promising method for quantification of absolute indoor Ae. aegypti density, leading to improved entomological estimates of mosquito distribution, a key measure in the assessments of the risk pathogen transmission, disease modeling and the evaluation of vector control interventions.

House screening with insecticide-treated netting provides sustained reductions in domestic populations of Aedes aegypti in Merida, Mexico.

BACKGROUND: There is a need for effective methods to control Aedes aegypti and prevent the transmission of dengue, chikungunya, yellow fever and Zika viruses. Insecticide treated screening (ITS) is a promising approach, particularly as it targets adult mosquitoes to reduce human-mosquito contact. METHODOLOGY/PRINCIPAL FINDINGS: A cluster-randomised controlled trial evaluated the entomological efficacy of ITS based intervention, which consisted of the installation of pyrethroid-impregnated long-lasting insecticide-treated netting material fixed as framed screens on external doors and windows. A total of 10 treatment and 10 control clusters (100 houses/cluster) were distributed throughout the city of Merida, Mexico. Cross-sectional entomological surveys quantified indoor adult mosquito infestation at baseline (pre-intervention) and throughout four post-intervention (PI) surveys spaced at 6-month intervals corresponding to dry/rainy seasons over two years (2012-2014). A total of 844 households from intervention clusters (86% coverage) were protected with ITS at the start of the trial. Significant reductions in the indoor presence and abundance of Ae. aegypti adults (OR = 0.48 and IRR = 0.45, P<0.05 respectively) and the indoor presence and abundance of Ae. aegypti female mosquitoes (OR = 0.47 and IRR = 0.44, P<0.05 respectively) were detected in intervention clusters compared to controls. This high level of protective effect was sustained for up to 24 months PI. Insecticidal activity of the ITS material declined with time, with ~70% mortality being demonstrated in susceptible mosquito cohorts up to 24 months after installation. CONCLUSIONS/SIGNIFICANCE: The strong and sustained entomological impact observed in this study demonstrates the potential of house screening as a feasible, alternative approach to a sustained long-term impact on household infestations of Ae. aegypti. Larger trials quantifying the effectiveness of ITS on epidemiological endpoints are warranted and therefore recommended.

A Survey of the Mosquito Species in Maxcanu, Yucatan, Mexico.

A survey was carried out to identify the mosquitoes inhabiting human premises in the rural locality of Maxcanú, Yucatán, Mexico. Using the Centers for Disease Control and Prevention backpack aspirators, simple random sampling was carried out inside of 101 homes during the November 2013 rainy season. A total of 1,492 specimens were collected. Three subfamilies (Anophelinae, Culicinae, and Toxorhynchitinae) and 5 species were identified: Anopheles albimanus, Aedes aegypti, Culex interrogator, Limatus durhamii, and Toxorhynchites theobaldi. The most abundant species was Cx. interrogator (74%) followed by Ae. aegypti (25%). The Chao 1 and Bootstrap species richness estimator indicated that it was possible to collect 90% of the expected species. This is the 1st time that the presence of An. albimanus, Cx. interrogator, Li. durhamii, and Tx. theobaldi has been recorded in Maxcanú.

Molecular evidence of Rickettsia typhi infection in dogs from a rural community in Yucatán, México.

INTRODUCTION: Rickettsia typhi causes murine or endemic typhus, which is transmitted to humans primarily through flea bites contaminated with feces. Synanthropic and domestic animals also contribute to the infection cycle of R. typhi. Cases of murine typhus in humans were reported in the rural community of Bolmay, Yucatán, México, between 2007 and 2010.  OBJECTIVE: To identify the presence of R. typhi and estimate the frequency of infection in dogs from Bolmay, México, a locality with previous reports of murine typhus in humans.  MATERIALS AND METHODS: Whole blood samples were taken from 128 dogs. Total DNA was extracted for use in the polymerase chain reaction (PCR) to amplify fragments of the 17 kDa and omp B genes and confirms the presence of Rickettsia spp. The reaction products were sequenced, and alignment analysis was performed using the BLAST tool.  RESULTS: The frequency of R. typhi infection in dogs was 5.5 % (7/128). The alignment identified 99% and 100% homology to the R. typhi 17 kDa and omp B genes, respectively.  CONCLUSION: We confirmed the presence of R. typhi in dogs in the studied community but at a low frequency. However, there is potential risk of transmission to humans.

Detección molecular de Rickettsia typhi en perros de una comunidad rural de Yucatán, México / Molecular evidence of Rickettsia typhi infection in dogs from a rural community in Yucatán, México

Introducción. Rickettsia typhi es la bacteria causante del tifus múrido o endémico, el cual es transmitido al ser humano principalmente por medio de las heces infectadas de pulgas y en cuyo ciclo de infección se encuentran involucrados distintos animales sinantrópicos y domésticos. En la comunidad rural de Bolmay, Yucatán, México, se reportaron casos de tifus múrido en seres humanos durante el periodo 2007-2010. Objetivo. Identificar la presencia de R . typhi y estimar la frecuencia de infección en perros de Bolmay, México. Materiales y métodos. Se tomaron muestras de sangre completa de 128 perros, se les extrajo el ADN total y se analizaron mediante reacción en cadena de la polimerasa (PCR) para amplificar los fragmentos del gen de 17 kDa y omp B , y confirmar la presencia de Rickettsia spp. Los productos de las reacciones se enviaron a secuenciación y se les hizo un análisis de alineamiento con Basic Local Alignment Search Tool (BLAST). Resultados. Se encontró una frecuencia de infección de 5,5 % (7/128). El alineamiento demostró 99 % de homologación para el gen de 17 kDa y 100 % para el gen omp B en R . typhi . Conclusión. Se detectó la presencia de R . typhi pero una baja frecuencia de infección en perros de la comunidad de estudio; sin embargo, la especie podría representar un riesgo de transmisión para los seres humanos.

Introduction: Rickettsia typhi causes murine or endemic typhus, which is transmitted to humans primarily through flea bites contaminated with feces. Synanthropic and domestic animals also contribute to the infection cycle of R. typhi . Cases of murine typhus in humans were reported in the rural community of Bolmay, Yucatán, México, between 2007 and 2010. Objective: To identify the presence of R . typhi and estimate the frequency of infection in dogs from Bolmay, México, a locality with previous reports of murine typhus in humans. Materials and methods: Whole blood samples were taken from 128 dogs. Total DNA was extracted for use in the polymerase chain reaction (PCR) to amplify fragments of the 17 kDa and omp B genes and confirms the presence of Rickettsia spp. The reaction products were sequenced, and alignment analysis was performed using the BLAST tool. Results: The frequency of R. typhi infection in dogs was 5.5 % (7/128). The alignment identified 99% and 100% homology to the R . typhi 17 kDa and omp B genes, respectively. Conclusion: We confirmed the presence of R . typhi in dogs in the studied community but at a low frequency. However, there is potential risk of transmission to humans.

[Control of Aedes aegypti breeding sites with the program Recicla por tu bienestar in Merida, Mexico]. / Control de criaderos de Aedes aegypti con el programa Recicla por tu bienestar en Mérida, México.

OBJECTIVES: To determine the importance of Ae. aegypti breeding-sites in Merida;to evaluate the impact of Recicla por tu bienestar (RxB, a recycling program) on the reduction of breeding sites and the perception of participants. MATERIALS AND METHODS: The relative importance for pupae production of the different types of breeding-sites was determined. Pre-and post-RxB entomological surveys were performed in participant neighborhoods to evaluate the impact on total containers and positive breeding-sites. A survey on the perception of participating people about dengue prevention and control and RxB was applied. RESULTS: Buckets/pots and "small diverse items" were the most important breeding-sites. RxB had a significant impact in the reduction of total containers (IRR = 0.74), positive containers (IRR = 0.33) and the risk of a house being positive for Ae. aegypti (OR = 0.41). All the interviewed participants referred RxB as needed and most consider it useful. CONCLUSIONS: RxB should be considered as a good practice for the dengue vector control.

CHICKEN COOPS, Triatoma dimidiata INFESTATION AND ITS INFECTION WITH Trypanosoma cruzi IN A RURAL VILLAGE OF YUCATAN, MEXICO.

This study longitudinally investigated the association between Triatoma dimidiata infestation, triatomine infection with Trypanosoma cruzi and household/backyard environmental characteristics in 101 homesteads in Molas and Yucatan, Mexico, between November 2009 (rainy season) and May 2010 (dry season). Logistic regression models tested the associations between insect infestation/infection and potential household-level risk factors. A total of 200 T. dimidiata were collected from 35.6% of the homesteads, mostly (73%) from the peridomicile. Of all the insects collected, 48% were infected with T. cruzi. Infected insects were collected in 31.6% of the homesteads (54.1% and 45.9% intra- and peridomiciliary, respectively). Approximately 30% of all triatomines collected were found in chicken coops. The presence of a chicken coop in the backyard of a homestead was significantly associated with both the odds of finding T. dimidiata (OR = 4.10, CI 95% = 1.61-10.43, p = 0.003) and the presence of triatomines infected with T. cruzi (OR = 3.37, CI 95% = 1.36-8.33, p = 0.006). The results of this study emphasize the relevance of chicken coops as a putative source of T. dimidiata populations and a potential risk for T. cruzi transmission.

Control de criaderos de Aedes aegypti con el programa Recicla por tu bienestar en Mérida, México / Control of Aedes aegypti breeding sites with the program Recicla por tu bienestar in Merida, Mexico

Objetivos. Determinar la importancia de los criaderos de Ae. aegypti en Mérida; evaluar el impacto del programa Recicla por tu bienestar (RxB) sobre la presencia/abundancia de éstos y la percepción de los habitantes. Material y métodos. Se calculó la importancia de los criaderos por su productividad pupal. Se realizaron muestreos pre y post RxB en colonias para cuantificar el total de recipientes/criaderos. Se aplicó una encuesta a participantes sobre la percepción sobre RxB en colonias seleccionadas. Resultados. Los botes, cubetas y diversos objetos chicos fueron los criaderos más importantes. RxB tuvo un impacto significativo en la reducción del número de recipientes (IRR=0.74), en los recipientes positivos (IRR=0.33) y en la positividad de las viviendas para Ae.aegypti (OR=0.41 j.Todos los entrevistados opinaron que RxB es necesario y la gran mayoría piensa que es útil. Conclusiones. RxB debe ser considerada una buena práctica para el control del vector del dengue.

Objectives. To determine the importance of Ae. aegypti breeding-sites in Merida;to evaluate the impact of Recicla por tu bienestar (RxB, a recycling program) on the reduction of breeding sites and the perception of participants. Materials and methods. The relative importance for pupae production of the different types of breeding-sites was determined. Pre-and post-RxB entomological surveys were performed in participant neighborhoods to evaluate the impact on total containers and positive breeding-sites. A survey on the perception of participating people about dengue prevention and control and RxB was applied. Results. Buckets/pots and "small diverse items" were the most important breeding-sites. RxB had a significant impact in the reduction of total containers (IRR = 0.74), positive containers (IRR = 0.33) and the risk of a house being positive for Ae. aegypti (OR = 0.41). All the interviewed participants referred RxB as needed and most consider it useful. Conclusions. RxB should be considered as a good practice for the dengue vector control.