Luis Ibarra-Juarez

UNIVERSAL PRIMERS FOR THE AMPLIFICATION AND SEQUENCE ANALYSIS OF ACTIN-1 FROM DIVERSE MOSQUITO SPECIES

Abstract We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5′-ATGGTCGGYATGGGNCAGAAGGACTC-3′) and Act-8R (5′-GATTCCATACCCAGGAAGGADGG-3′), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report.

Detection of West Nile virus-specific antibodies and nucleic acid in horses and mosquitoes, respectively, in Nuevo Leon State, northern Mexico, 2006–2007

In the last 5 years, there has been only one reported human case of West Nile virus (WNV) disease in northern Mexico. To determine if the virus was still circulating in this region, equine and entomological surveillance for WNV was conducted in the state of Nuevo Leon in northern Mexico in 2006 and 2007. A total of 203 horses were serologically assayed for antibodies to WNV using an epitope‐blocking enzyme‐linked immunosorbent assay (bELISA). Seroprevalences for WNV in horses sampled in 2006 and 2007 were 26% and 45%, respectively. Mosquito collections in 2007 produced 7365 specimens representing 15 species. Culex mosquitoes were screened for WNV RNA and other genera (Mansonia, Anopheles, Aedes, Psorophora and Uranotaenia) were screened for flaviviruses using reverse‐transcription (RT)‐PCR. Two pools consisting of Culex spp. mosquitoes contained WNV RNA. Molecular species identification revealed that neither pool included Culex quinquefasciatus (Say) (Diptera:Culicidae) complex mosquitoes. No evidence of flaviviruses was found in the other mosquito genera examined. These data provide evidence that WNV is currently circulating in northern Mexico and that non‐Cx. quinquefasciatus spp. mosquitoes may be participating in the WNV transmission cycle in this region.

Indicators for elevated risk of human exposure to host-seeking adults of the Rocky Mountain wood tick (Dermacentor andersoni) in Colorado

ABSTRACT The human-biting adult stage of the Rocky Mountain wood tick (Dermacentor andersoni) can cause tick paralysis in humans and domestic animals and is the primary tick vector in the intermountain west of the pathogens causing Colorado tick fever, Rocky Mountain spotted fever, and tularemia. We conducted drag sampling studies in Poudre Canyon and Rocky Mountain National Park of Larimer County, CO, to determine microhabitat use patterns by host-seeking D. andersoni adults and find environmental factors signaling elevated risk of tick exposure. Big sagebrush (Artemisia tridentata) was found to serve as a general indicator of areas with elevated risk of exposure to host-seeking D. andersoni adults; this likely results from a shared climate tolerance of big sagebrush and D. andersoni. Grass was the favored substrate for host-seeking ticks. Drag sampling of open grass or grass bordering rock or shrub produced abundances of D. andersoni adults significantly higher than sampling of brush. Sampling sites in Rocky Mountain National Park, relative to Poudre Canyon, were characterized by more intense usage by elk (Cervus elaphus) but decreased brush coverage, smaller brush size, and lower abundances of hostseeking D. andersoni adults. There has been a tremendous increase in the population of elk in Rocky Mountain National Park over the last decades and we speculate that this has resulted in an ecological cascade where overgrazing of vegetation by elk is followed by suppression of rodent populations, decreased tick abundance, and, ultimately, reduced risk of human exposure to D. andersoni and its associated pathogens.

Detection of Dengue Virus Serotype 2 in Aedes aegypti in Quintana Roo, Mexico, 2011

Abstract. In October 2011, the State Health Department announced that several laboratory-confirmed cases of dengue had occurred among residents in two neighborhoods of Benito Juarez, Quintana Roo State, Mexico. To identify the dengue virus serotype(s) temporally and spatially associated with the cases, entomologic-based virus surveillance was initiated in October 2011 in both neighborhoods. Adult mosquitoes were collected from 88 houses by CDC-backpack aspirator, and all female Aedes aegypti L. (n = 419) were individually homogenized and assayed in pools of as many as 10 by reverse transcriptionpolymerase chain reaction (RT-PCR) using dengue virus-specific primers. Five (12%) of 41 pools were positive for dengue virus RNA. The individual mosquitoes that comprised the pools were analyzed separately by RT-PCR using dengue virus serotype-specific primers. Six mosquitoes were positive for dengue virus serotype-2 (DENV-2) RNA, three of which were collected in the same house. The mean number of female Ae. aegypti collected in each house was 4.76 ± 6.19. The overall dengue virus-infection rate in female Ae. aegypti was 1.4%. Interestingly, most (60%) of mosquito females were collected only from 15 (17%) houses. In summary, we provide evidence of recent DENV-2 transmission in Quintana Roo State.

Detection of Aedes aegypti Mosquitoes Infected with Dengue Virus as a Complementary Method for Increasing the Sensitivity of Surveillance: Identification of Serotypes 1, 2, and 4 by RT-PCR in Quintana Roo, Mexico

Abstract. Sensitivity of monitoring Aedes aegypti (L.) populations was determined to identify the distribution of dengue virus (DENV) during epidemics in Quintana Roo. From September to November 2012, we used a motorized aspirator to collect 2,144 female Ae. aegypti from 569 homes. These were grouped into 220 to use semi-nested RT-PCR for DENV, and positive groups were analyzed individually. Five groups (2.27%) were positive for DENV. Individual analysis yielded eight groups that tested positive, six with DENV-2, one DENV-1, and one DENV-4. The latter was not reported by the surveillance system that year. The mean number of female mosquitoes per household was 3.77 ± 5.71, and the rate of viral infection of Ae. aegypti was 0.4%. Most infected mosquitoes (49%) were concentrated in 10% of the houses. Monitoring Ae. aegypti infected with DENV has the potential to complement the current system of clinical and entomological surveillance.

West Nile Virus Survey of Birds, Horses, and Mosquitoes of the Pacific Coast, Southern Mexico

Abstract. Serology of West Nile virus vectors and non-human reservoirs was surveyed at Acapulco, Jose Azueta, and Ometepec, three Pacific Coast localities of Guerrero State, Mexico. The objectives of this study were to use enzyme-linked immnosorbent assay (ELISA) to assess West Nile virus antibodies of bird and equine serum samples and use reverse transcription of polymerase chain reaction (RT-PCR) to detect the virus in field-collected resting mosquitoes. Forty birds trapped using mist nets yielded 10% seroprevalence. Similarly, 18.6% of 102 equine blood samples had West Nile virus. In addition, 4,854 mosquitoes were caught using motorized backpack aspirators and grouped into 116 pools. Of the 16 species and seven genera, no mosquito was positive for West Nile virus. Our study demonstrated West Nile virus seroprevalence on resident birds and equines in Guerrero State, Mexico.

Risks of Dengue Secondary Infective Biting Associated with Aedes aegypti in Home Environments in Monterrey, Mexico

Abstract. Secondary dengue virus infections are a major risk for developing dengue hemorrhagic fever. Recent exposure to infectious bites of Aedes aegypti (L.) females in previously diagnosed dengue cases fulfills the epidemiological model of dengue hemorrhagic fever. A study was comprised of 357 (89.2%) dengue and 43 (10.8%) dengue hemorrhagic fever cases confirmed by laboratory tests and clinical manifestations. An entomological survey was done in homes and backyards. Concurrently, a questionnaire was used to assess the impact of healthpromotion campaigns through knowledge of the vector and its epidemiological role. Seventy-six (28.4%) of the 268 (67.0%) total wet or dry oviposition sites were positive for the presence of larvae or pupae, while adult Ae. aegypti were found in 32 (8.0%). One hundred thirty-two (33%) householders who formerly had dengue fever or dengue hemorrhagic fever had knowledge of either larval or adult dengue vector stages. According to gender distribution, 145 (36.2%) and 14 (3.5%) of the males confirmed with cases of dengue and dengue hemorrhagic fever lived in houses with 17.9 and 2% of the Ae. aegypti larval and pupal habitats. Houses with females who had dengue and dengue hemorrhagic fever were 212 (53%) and 29 (7.3%), with containers with immature Ae. aegypti in 19.4 and 7%, respectively. Lack of sustainability of government-targeted health education campaigns is the major problem for involving communities in prevention and control of dengue.

Seroprevalence of equine influenza virus in northeast and southern Mexico

EQUINE influenza A virus (EIV) is a highly infectious respiratory pathogen of horses ([Hannant and Mumford 1996][1], [Palese and Shaw 2007][2]). The illness is characterised by an abrupt onset of fever, depression, coughing and nasal discharge, and is often complicated by secondary bacterial