Detection of Rickettsia amblyommatis and Rickettsia bellii in ticks collected from pet dogs in peri-urban and rural areas in Yucatan, Mexico.

Rickettsia species are bacteria that may cause multiple diseases in animals and humans, via transmission through multiple arthropod vectors. Routine surveillance of Rickettsia spp. within vectors is critical to determine their presence and risk to mammalian hosts within human populations. Therefore, to better characterize the circulating Rickettsia species in an understudied region we targeted pet dogs to survey. Ticks were collected from pet dogs in three populations of the Yucatan where we tested for the presence of Rickettsia spp. by PCR in metagenomic DNA. In these ticks removed from pet dogs we detected Rickettsia amblyommatis and Rickettsia bellii in Amblyomma auriculatum, Amblyomma ovale and Amblyomma mixtum ticks obtained in a rural community in the Mexican state of Yucatan. This is the first report detecting both species for this state in Mexico, underpinning the importance of more routine surveillance.

Evaluation of an Immunoglobulin E Capture Enzyme-Linked Immunosorbent Assay for the Early Diagnosis of Dengue.

Dengue virus (DENV) is the etiological agent of dengue, the most important mosquito-transmitted viral disease of humans worldwide. Enzyme-linked immunosorbent assays (ELISAs) designed to detect DENV IgM are commonly used for dengue diagnosis. However, DENV IgM is not reliably detected until ≥4 days after illness onset. Reverse transcription-polymerase chain reaction (RT-PCR) can diagnose early dengue but requires specialized equipment, reagents, and trained personnel. Additional diagnostic tools are needed. Limited work has been performed to determine whether IgE-based assays can be used for the early detection of vector-borne viral diseases, including dengue. In this study, we determined the efficacy of a DENV IgE capture ELISA for the detection of early dengue. Sera were collected within the first 4 days of illness onset from 117 patients with laboratory-confirmed dengue, as determined by DENV-specific RT-PCR. The serotypes responsible for the infections were DENV-1 and DENV-2 (57 and 60 patients, respectively). Sera were also collected from 113 dengue-negative individuals with febrile illness of undetermined etiology and 30 healthy controls. The capture ELISA detected DENV IgE in 97 (82.9%) confirmed dengue patients and none of the healthy controls. There was a high false positivity rate (22.1%) among the febrile non-dengue patients. In conclusion, we provide evidence that IgE capture assays have the potential to be explored for early diagnosis of dengue, but further research is necessary to address the possible false positivity rate among patients with other febrile illnesses.

Molecular survey of Zika virus in the animal-human interface in traditional farming.

Backyard animal husbandry is common in rural communities in developing countries and, given the conditions in which it occurs, it can increase the risk of disease transmission, such as arboviruses. To determine the presence of the Zika virus (ZIKV) and abundance of its arthropod vectors we evaluated the socioeconomic implications involved in its transmission in two highly vulnerable Mayan communities in the state of Yucatan that practice backyard farming. An analytical cross-sectional study was carried out throughout 2016 to understand socioeconomic variables and seasonal patterns in mosquito populations. We selected 20 households from each community. Social exclusion indicators were analyzed, human and domestic animals were sampled, and mosquitoes were collected and identified. Four out of eight indicators of social exclusion were higher than the reported national averages. We captured 5,825 mosquitoes from 16 species being Culex quinquefasciatus and Aedes aegypti the most abundant. The presence of chickens and human overcrowding in dwellings were the most significant factors (P = 0.026) associated with the presence of Ae. aegypti. Septic tanks (odds ratio = 6.64) and chickens (odds ratio = 27.41) in backyards were the main risk factors associated with the presence of immature states of Ae. aegypti in both communities. Molecular analysis to detect ZIKV was performed in blood samples from 416 humans, 1,068 backyard animals and 381 mosquito pools. Eighteen humans and 10 pig pools tested positive for ZIKV. Forty-three mosquito pools tested positive for flavivirus. Ten of the 43 pools of positive mosquitoes were sequenced, corresponding 3/10 to ZIKV and 1/10 to Dengue virus type 2. The findings obtained indicate the continuous circulation of Flavivirus (including ZIKV) in backyard environments in vulnerable communities, highlighting the importance of studying their transmission and maintenance in these systems, due that backyard animal husbandry is a common practice in these vulnerable communities with limited access to health services.

Evidence of Zika Virus Infection in Pigs and Mosquitoes, Mexico.

Evidence suggests that pigs seroconvert after experimental exposure to Zika virus and are potential sentinels. We demonstrate that pigs are also susceptible to natural Zika virus infection, shown by the presence of antibodies in domestic pigs in Yucatan, Mexico. Zika virus RNA was detected in 5 species of mosquitoes collected inside pigpens.

Frequency of the L1014F Mutation in the Sodium Channel Gene, in Culex quinquefasciatus (Diptera: Culicidae) Populations From Rural and Urban Areas of Yucatan State, Mexico.

Culex quinquefasciatus Say (Diptera: Culicidae) is a mosquito species that has attracted a lot of attention from a medical and veterinary point of view; however, little is known about the frequency of L1014F mutations that have been found in the sodium channel gene, with this being a target for DDT and pyrethroid insecticides. The distribution and frequency of the L1014F mutation in Cx. quinquefasciatus populations was determined in rural and urban areas of Yucatan, Mexico from January 2015 to March 2016. Nine hundred fifty adult females out of 17,727 immature states were collected and analyzed in all sites sampled (n = 10). Susceptible homozygotes were identified (L1014/L1014) in 12% (114/950), heterozygous individuals (F1014/L1014) in 34% (323/950), and mutated homozygotes (F1014/F1014) in 54% (513/950) during the dry and rainy seasons. In this work, study areas with a high frequency of L1014F mutation were identified. These findings may help guarantee a more effective and efficient use of the resources available for the control of this vector.

The Use of Insecticide-Treated Curtains for Control of Aedes aegypti and Dengue Virus Transmission in "Fraccionamiento" Style Houses in México.

Dengue, chikungunya, yellow fever, and Zika viruses transmitted by Aedes aegypti mosquitoes are major public health threats in the tropical and subtropical world. In México, construction of large tracts of "fraccionamientos" high density housing to accommodate population growth and urbanization has provided fertile ground for Ae. aegypti-transmitted viruses. We investigated the utility of pyrethroid-treated window curtains to reduce both the abundance of Ae. aegypti and to prevent dengue virus (DENV) transmission in fraccionamiento housing. Windows and doors of fraccionamiento homes in urban/suburban areas, where Ae. aegypti pyrethroid resistance associated with the Ile1016 knock down resistance (kdr) mutation in the voltage gated sodium channel gene was high, and in rural areas, where kdr resistance was low, were fitted with either insecticide-treated curtains (ITCs) or non-treated curtains (NTCs). The homes were monitored for mosquito abundance and DENV infection. ITCs reduced the indoor abundance of Ae. aegypti and the number of DENV-infected mosquitoes in homes in rural but not in urban/suburban study sites. The presence of non-treated screens also was associated with reduced numbers of mosquitoes in homes. "Super-infested" homes, yielding more than 50 mosquitoes, including DENV-infected mosquitoes, provide a significant public health risk to occupants, visitors, and people in neighboring homes.

Ecological Niche Model for Predicting Distribution of Disease-Vector Mosquitoes in Yucatán State, México.

The majority of the Yucatán State, México, presents subtropical climate that is suitable for many species of mosquitoes that are known to be vectors of diseases, including those from the genera Aedes and Culex. The objective of this study is to identify the geographic distribution of five species from these two genera and estimate the human population at risk of coming in contact with them. We compiled distributional data for Aedes aegypti (L.), Aedes (Howardina) cozumelensis (Diaz Najera), Culex coronator Dyar and Knab, Culex quinquefasciatus Say, and Culex thriambus Dyar from several entomological studies in Yucatán between March 2010 and September 2014. Based on these data, we constructed ecological niche models to predict the spatial distribution of each species using the MaxEnt algorithm. Our models identified areas with suitable environments for Ae. aegypti in most of Yucatán. A similar percentage of urban (97.1%) and rural (96.5%) populations were contained in areas of highest suitability for Ae. aegypti, and no spatial pattern was found (Moran's I = 0.33, P = 0.38); however, we found an association of abundance of immature forms of this species with annual mean temperature (r = 0.19, P ≤ 0.001) and annual precipitation (r = 0.21, P ≤ 0.001). Aedes cozumelensis is also distributed in most areas of the Yucatán State; Cx. quinquefasciatus, Cx. coronator, and Cx. thriambus are restricted to the northwest. The information generated in this study can inform decision-making to address control measures in priority areas with presence of these vectors.

Chikungunya Virus in Febrile Humans and Aedes aegypti Mosquitoes, Yucatan, Mexico.

Chikungunya virus (CHIKV) was isolated from 12 febrile humans in Yucatan, Mexico, in 2015. One patient was co-infected with dengue virus type 1. Two additional CHIKV isolates were obtained from Aedes aegypti mosquitoes collected in the homes of patients. Phylogenetic analysis showed that the CHIKV isolates belong to the Asian lineage.

Local evolution of pyrethroid resistance offsets gene flow among Aedes aegypti collections in Yucatan State, Mexico.

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.

Urban Mosquito Fauna in Mérida City, México: Immatures Collected from Containers and Storm-water Drains/Catch Basins.

We examined the species composition and temporal occurrence of immature mosquitoes in containers and storm-water drains/catch basins from November 2011 to June 2013 in Mérida City, México. A wide range of urban settings were examined, including residential premises, vacant lots, parking lots, and streets or sidewalks with storm-water drains/catch basins. In total, 111,776 specimens of 15 species were recorded. The most commonly collected species were Aedes (Stegomyia) aegypti (L.) (n = 60,961) and Culex quinquefasciatus Say (45,702), which together accounted for 95.4% of the immatures collected. These species were commonly encountered during both rainy and dry seasons, whereas most other mosquito species were collected primarily during the rainy season. Other species collected were Aedes (Howardina) cozumelensis Diaz Najera, Aedes (Ochlerotatus) taeniorhynchus (Wiedemann), Aedes (Ochlerotatus) trivittatus (Coquillett), Culex coronator Dyar and Knab, Culex interrogator Dyar and Knab, Culex lactator Dyar and Knab, Culex nigripalpus Theobald, Culex salinarius Coquillett, Culex tarsalis Coquillett, Culex thriambus Dyar, Haemagogus equinus Theobald, Limatus durhamii Theobald, and Toxorhynchites rutilus (Coquillett). The greatest number of species was recorded from vacant lots (n = 11), followed by storm-water drains/catch basins (nine) and residential premises (six). Our study demonstrated that the heterogeneous urban environment in Mérida City supports a wide range of mosquito species, many of which are nuisance biters of humans and/or capable of serving as vectors of pathogens affecting humans or domestic animals. We also briefly reviewed the medical importance of the encountered mosquito species.

QTL mapping of genome regions controlling temephos resistance in larvae of the mosquito Aedes aegypti.

INTRODUCTION: The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Temephos is an organophosphate insecticide used globally to suppress Ae. aegypti larval populations but resistance has evolved in many locations. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative Trait Loci (QTL) controlling temephos survival in Ae. aegypti larvae were mapped in a pair of F3 advanced intercross lines arising from temephos resistant parents from Solidaridad, México and temephos susceptible parents from Iquitos, Peru. Two sets of 200 F3 larvae were exposed to a discriminating dose of temephos and then dead larvae were collected and preserved for DNA isolation every two hours up to 16 hours. Larvae surviving longer than 16 hours were considered resistant. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 23 single copy genes and 26 microsatellite loci of known physical positions in the Ae. aegypti genome. In both reciprocal crosses, Multiple Interval Mapping identified eleven QTL associated with time until death. In the Solidaridad×Iquitos (SLD×Iq) cross twelve were associated with survival but in the reciprocal IqxSLD cross, only six QTL were survival associated. Polymorphisms at acetylcholine esterase (AchE) loci 1 and 2 were not associated with either resistance phenotype suggesting that target site insensitivity is not an organophosphate resistance mechanism in this region of México. CONCLUSIONS/SIGNIFICANCE: Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome.

Vacant lots: productive sites for Aedes (Stegomyia) aegypti (Diptera: Culicidae) in Mérida City, México.

We assessed the potential for vacant lots and other nonresidential settings to serve as source environments for Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) in Mérida City, México. Mosquito immatures were collected, during November 2011-June 2013, from residential premises (n = 156 site visits) and nonresidential settings represented by vacant lots (50), parking lots (18), and streets or sidewalks (28). Collections totaled 46,025 mosquito immatures of 13 species. Ae. aegypti was the most commonly encountered species accounting for 81.0% of total immatures, followed by Culex quinquefasciatus Say (12.1%). Site visits to vacant lots (74.0%) were more likely to result in collection of Ae. aegypti immatures than residential premises (35.9%). Tires accounted for 75.5% of Ae. aegypti immatures collected from vacant lots. Our data suggest that vacant lots should be considered for inclusion in mosquito surveillance and control efforts in Mérida City, as they often are located near homes, commonly have abundant vegetation, and frequently harbor accumulations of small and large discarded water-holding containers that we now have demonstrated to serve as development sites for immature mosquitoes. In addition, we present data for associations of immature production with various container characteristics, such as storage capacity, water quality, and physical location in the environment.

Detection of hand, foot and mouth disease in the yucatan peninsula of Mexico.

We report a case of hand, foot and mouth disease (HFMD) in a 5-year-old male from Merida City in the Yucatan Peninsula of Mexico. A clinical and physical examination revealed that the patient had symptoms typical of HFMD, including fever, fatigue, odynophagia, throat edema, hyperemia, lesions on the hands and feet, and blisters in the oral cavity. The patient fully recovered after a convalescence period of almost three weeks. Reverse transcription-polymerase chain reaction and nucleotide sequencing revealed that the etiological agent was enterovirus 71 (EV71). The sequence has greatest (90.4%) nucleotide identity to the corresponding regions of EV71 isolates from the Netherlands and Singapore. Although HFMD is presumably common in Mexico, surprisingly there are no data in the PubMed database to support this. This case report provides the first peer-reviewed evidence of HFMD in Mexico.

Aedes (Stegomyia) aegypti and Aedes (Howardina) cozumelensis in Yucatán State, México, with a summary of published collection records for Ae. cozumelensis.

We collected mosquito immatures from artificial containers during 2010-2011 from 26 communities, ranging in size from small rural communities to large urban centers, located in different parts of Yucatán State in southeastern México. The arbovirus vector Aedes (Stegomyia) aegypti was collected from all 26 examined communities, and nine of the communities also yielded another container-inhabiting Aedes mosquito: Aedes (Howardina) cozumelensis. The communities from which Ae. cozumelensis were collected were all small rural communities (<6,000 inhabitants) in the north-central part of Yucatán State. These new collection records for Ae. cozumelensis demonstrate that this mosquito has a far broader geographic range in the Yucatán Peninsula than previously known. Ae. cozumelensis immatures were collected from both residential premises and cemeteries, with specimens recovered from rock holes as well as various artificial containers including metal cans, flower vases, buckets, tires, and a water storage tank. The co-occurrence with Ae. aegypti in small rural communities poses intriguing questions regarding linkages between these mosquitoes, including the potential for direct competition for larval development sites. Additional studies are needed to determine how commonly Ae. cozumelensis feeds on human blood and whether it is naturally infected with arboviruses or other pathogens of medical or veterinary importance. We also summarize the published records for Ae. cozumelensis, which are restricted to collections from México's Yucatán Peninsula and Belize, and uniformly represent geographic locations where Ae. aegypti can be expected to occur.

Rhythms and synchronization patterns in gene expression in the Aedes aegypti mosquito.

BACKGROUND: Aedes aegypti is arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock. RESULTS: Herein we analyze the transcription profiles of 21,494 messenger RNAs using an Ae. aegypti Agilent® microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways. CONCLUSION: These results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

Recent rapid rise of a permethrin knock down resistance allele in Aedes aegypti in México.

BACKGROUND: Aedes aegypti, the 'yellow fever mosquito', is the primary vector to humans of dengue and yellow fever flaviviruses (DENV, YFV), and is a known vector of the chikungunya alphavirus (CV). Because vaccines are not yet available for DENV or CV or are inadequately distributed in developing countries (YFV), management of Ae. aegypti remains the primary option to prevent and control outbreaks of the diseases caused by these arboviruses. Permethrin is one of the most widely used active ingredients in insecticides for suppression of adult Ae. aegypti. In 2007, we documented a replacement mutation in codon 1,016 of the voltage-gated sodium channel gene (para) of Ae. aegypti that encodes an isoleucine rather than a valine and confers resistance to permethrin. Ile1,016 segregates as a recessive allele conferring knockdown resistance to homozygous mosquitoes at 5-10 microg of permethrin in bottle bioassays. METHODS AND FINDINGS: A total of 81 field collections containing 3,951 Ae. aegypti were made throughout México from 1996 to 2009. These mosquitoes were analyzed for the frequency of the Ile1,016 mutation using a melting-curve PCR assay. Dramatic increases in frequencies of Ile1,016 were recorded from the late 1990's to 2006-2009 in several states including Nuevo León in the north, Veracruz on the central Atlantic coast, and Yucatán, Quintana Roo and Chiapas in the south. From 1996 to 2000, the overall frequency of Ile1,016 was 0.04% (95% confidence interval (CI95) = 0.12%; n = 1,359 mosquitoes examined). The earliest detection of Ile1,016 was in Nuevo Laredo on the U.S. border in 1997. By 2003-2004 the overall frequency of Ile1,016 had increased approximately 100-fold to 2.7% (+ or - 0.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (+ or - 1.15% CI95; n = 473). This was followed in 2007-2009 by a sudden jump in Ile1,016 frequency to 33.2% (+ or - 1.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity in Ile1,016 frequencies among 2007-2008 collections, which ranged from 45.7% (+ or - 2.00% CI95) in the state of Veracruz to 51.2% (+ or - 4.36% CI95) in the Yucatán peninsula and 14.5% (+ or - 2.23% CI95) in and around Tapachula in the state of Chiapas. Spatial heterogeneity was also evident at smaller geographic scales. For example within the city of Chetumal, Quintana Roo, Ile1,016 frequencies varied from 38.3%-88.3%. A linear regression analysis based on seven collections from 2007 revealed that the frequency of Ile1,016 homozygotes accurately predicted knockdown rate for mosquitoes exposed to permethrin in a bioassay (R(2) = 0.98). CONCLUSIONS: We have recorded a dramatic increase in the frequency of the Ile1,016 mutation in the voltage-gated sodium channel gene of Ae. aegypti in México from 1996 to 2009. This may be related to heavy use of permethrin-based insecticides in mosquito control programs. Spatial heterogeneity in Ile1,016 frequencies in 2007 and 2008 collections may reflect differences in selection pressure or in the initial frequency of Ile1,016. The rapid recent increase in Ile1,016 is predicted by a simple model of positive directional selection on a recessive allele. Unfortunately this model also predicts rapid fixation of Ile1,016 unless there is negative fitness associated with Ile1,016 in the absence of permethrin. If so, then spatial refugia of susceptible Ae. aegypti or rotational schedules of different classes of adulticides could be established to slow or prevent fixation of Ile1,016.