Species abundance and temporal variation of arbovirus vectors in Brownsville, Texas/Abundancia de temporal de los vectores de arbovirus en brownsville, texas.
Brownsville and South Texas are not strangers to arboviral threats. While DENV has historically been present in South Texas, a yellow fever virus (YFV) campaign conducted in the 1940s largely exterminated its Aedes vectors (Ae. aegypti and Ae. Albopictus). However, since the 1980s there has been a resurgence in the virus and its vectors (2, 3). In a 1980 outbreak in South Texas, one of the largest ever, 25 locally acquired cases and 27 imported cases were recorded, for a total of 52 reported cases of DENV. Since then additional outbreaks have been documented with increasing frequency. Peak activity occurred in 1999, with six local and 14 imported cases, and in 2005, with three local and 23 imported cases (4). In 2013, South Texas experienced another significant DENV outbreak, with 53 laboratory-confirmed cases. Twentysix of those cases reported no recent travel, making the outbreak the largest number of clinical, locally acquired cases reported during a single year (5). More than half of all cases were identified in Cameron County, in the border city of Brownsville, Texas. This was unsurprising, given Brownsville's close proximity to Mexico, which was also experiencing a DENV outbreak at the same time.
SURVEILLANCE AND VECTOR CONTROL STRATEGIES
Brownsville has long been regarded as one of the most vulnerable regions in the United States for outbreaks of mosquito-borne diseases (6, 7). With its favorable vector ecology (e.g., environmental effects on vector incidence), this and other areas in South Texas are considered high-risk for arbovirus activity, particularly DENV (8, 9). Some of this risk is attributed to the humid, subtropical, coastal climate that results in year-round temperatures ideal for vector breeding (10). In Brownsville, the average maximum temperature from 1981 to 2010 was 83 degrees and the average minimum temperature was 65.4 degrees. Currently, there are no commercially available human vaccines or targeted drug therapies for CHIKV, DENV, WNV, or ZIKV, leaving mosquito control as the principal method of disease prevention (11-13). Yet very little research has been carried out on vector species' habitat. In the Texas-Mexico border region, most U.S.-based research has focused on identifying cases, investigating peri-domicilar areas for vector breeding containers, and establishing the likelihood of autochthonous versus travel-acquired DENV (2, 14).
Directly across the border from Brownsville, in Matamoros, Mexico, similar efforts are under way. It has long been understood that the Mexican side of the border has a significantly higher burden of arboviral disease than the U.S. side, even though the two entities are only divided by the Rio Grande River (15). Therefore, Mexico devotes a great amount of resources to proactive human case identification, and mosquito extermination, using chemicals and ovi-traps, rather than monitoring species abundance and incidence with gravid traps, like the surveillance program analyzed by this study. Regardless of the methods used, effective vector control requires a good understanding of vector ecology. The objective of this study was to describe the various species of mosquitoes found by vector surveillance in Brownsville, Texas, during 20092013; quantify their prevalence; and identify any associations with temporal or weather-related variations. This study is the first in a series of steps to examine environmental factors that potentially drive the transmission of arboviruses in the South Texas region.
Collection and testing methods
Systematic collection and testing of mosquitoes leads to better understanding of vector ecology. Therefore, the City of Brownsville Public Health Department has used a vector control and surveillance strategy since 2009. Starting in June and continuing through November, gravid traps from the U.S. Centers for Disease Control and Prevention (CDC) are placed around the city twice per week, in predetermined locations, and retrieved within 24 hours. Traps are also placed in response to community complaints. The mosquitoes are collected from the traps, transferred, alive, into a container, and shipped to TDSHS in Austin for species identification, pooling, and virus testing. Female mosquitoes are separated by species, and only those known to be human disease vectors are pooled for testing. After mosquito samples are homogenized and centrifuged, supernatants are inoculated onto two vertebrate cell lines and observed for 10 days. If cytopathic effects (CPEs) are observed, virus identification tests are performed using an indirect fluorescent antibody (IFA) assay for Western equine encephalitis virus (WEEV), Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis (VEEV), SLEV, California encephalitis virus (CEV), Highlands J virus (HJV), Tensaw virus (TV), and CHIKV. If the results are inconclusive, samples are sent to the CDC Arbovirus Diagnostic Laboratory in Fort Collins, Colorado, for further identification (16).
Using arbovirus isolation and mosquito species data from TDSHS and the local National Oceanic and Atmospheric Administration (NOAA) weather station, the Brownsville Public Health Department surveillance teams gained a better understanding of local vector ecology. First, they looked at mosquito species abundance in the region. Although the presence of Ae. aegypti and Ae. albopictus in South Texas had already been established, they wanted a better understanding of other potential disease vectors (9). A wide variety of mosquito species were caught in the surveillance traps and identified. Subsequent studies focused on the three most common species and any associations between their incidence and temporal or weather variations in Brownsville.
Species abundance and incidence
Table 1 shows a list of all species isolated in Brownsville from 2009 to 2013. Of the 23 different species caught in the gravid traps, the most common were Culex quinquefasciatus (82%), Ae. aegypti (16%), Ae. albopictus (1%), and "other" (< 1%). Vector abundance was examined by species and by month to evaluate vector species dynamics throughout the arbovirus season--an important factor in optimizing resource allocation. Because so few Ae. albopictus mosquitoes were found, the category for that species was combined with the Ae. aegypti group under a new name ("Aedes spp.") for analysis of species incidence over time (Table 2). The data showed a large decrease in the percentage of C. quinquefasciatus from 2009 to 2010, followed by a gradual increase through 2013.
In 2012, Texas experienced its largest outbreak of WNV. Brownsville had very few human cases, and until recently, that was the only year that any local mosquitoes positive for WNV were recorded. Three years later, in 2015, two more pools of mosquitoes tested positive for WNV. Both specimen pools were from the neighborhood that had WNV-positive mosquitoes in 2012, a finding with interesting public health and preventative implications.
Using the mosquito trap results, plus weather and species data from the NOAA, the surveillance teams generated descriptive statistics by year of data collection, average rainfall, and maximum temperature (Table 2). Using Stata statistical software (version 12) (StataCorp LLC, College Station, Texas), a robust regression analysis was conducted to predict the association between mosquito species population patterns, adjusting for year, month, rainfall, and high temperature. Relationships were considered to be significant if the P value was < 0.05. Given the history of DENV and WNV in South Texas, and the potential for autochthonous transmission of CHIKV in the region, the surveillance study focused on the vectors that circulate those three diseases. Regression analysis revealed a negative correlation between C. quinquefasciatus and month of the year and a positive correlation between Ae. aegypti and month of the year. Although the TDSHS in Austin considers the arbovirus season in South Texas to be finished in November, the surveillance teams found large numbers of mosquitoes in the months of December and January. Overall mosquito abundance decreased toward the end of the year, but Ae. aegypti mosquitoes continued to survive well into the months traditionally considered post-mosquito season (17). Also, while there seemed to be no significant relationship between average rainfall and mosquito species, there was a correlation between temperature highs and mosquito species.
Based on these analyses, the research team submitted recommendations to the City of Brownsville Public Health Department to guide and refine future surveillance and vector control strategies. Improving the surveillance strategy would allow Brownsville to more proactively mitigate arboviral risk in the community. Ongoing systematic collection of mosquito surveillance data helps establish associations between temporal and weather-related variables and mosquito abundance and increases understanding of the arboviral season. Based on the overall mosquito density and virus activity, the results of this study suggest that Brownsville is currently at high risk for DENV, WNV, and potentially CHIKV (9, 18). In addition, based on the temporal and weather-related data, Brownsville appears to be at risk of arboviral transmission year-round. While other studies have found a correlation between mosquito abundance and rainfall, this study did not. However, this study did find a positive correlation between mosquito species abundance and both temperature highs and month of the year. These results, based on the longitudinal surveillance, could be used to identify and target higher-risk neighborhoods and thus facilitate more efficient allocation of the city's limited resources by directing public health outreach efforts where they are needed most.
As arboviral threats become more numerous in South Texas, it is vital to prioritize mosquito control programs and support surveillance capacity in areas of highest risk. This includes bolstering entomologic surveillance, enhancing local laboratory infrastructure to increase mosquito and human testing capacity, and allotting resources for community education focused on prevention. Vector surveillance helps the City of Brownsville Public Health Department 1) detect the presence of various mosquito species and arboviruses that affect human health; 2) understand vector and disease dynamics over time for better risk assessment; and 3) evaluate the impact of vector control measures and public outreach. All of these data combined can provide better understanding of the local arbovirus epidemic potential (11, 19).
The limitations of this study are largely related to a lack of local funding and resources to support adequate arboviral surveillance in the border regions. The City of Brownsville only employs one or two part-time personnel to conduct weekly surveillance activities. Furthermore, the number of mosquito traps available for use has steadily declined since 2009, as traps have broken but have not been replaced due to lack of funding. On the other hand, the use of traps presented another potential limitation. When looking at species and species abundance, the trap itself can be a study confounder if, due to the design or other characteristics, including location, it attracts only certain species of mosquitoes. For example, almost all sites sampled were in urban areas, which could have contributed to a bias in the species caught.
Through better understanding of seasonality and areas of mosquito density, public health outreach and community education in high-risk areas can be prioritized. The City of Brownsville Public Health Department's vector surveillance study represents the first step in unifying arbovirus surveillance systems in South Texas and may serve as a model for similar improvements in other border areas susceptible to arbovirus epidemics.
Acknowledgments. The authors would like to thank the City of Brownsville Department of Public Health, the Kleberg Medical Research Scholars Foundation, and the University of Texas Health Science Center at San Antonio (UTHSCSA) Center for Medical Humanities and Ethics for lending their time, resources, and expertise.
Conflicts of interest. None.
Disclaimer. Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the RPSP/ PAJPH or the Pan American Health Organization (PAHO).
(1.) U.S. Centers for Disease Control and Prevention. CDC Health Alert Network (HAN) Health Advisory: CDC guidance for travel and testing of pregnant women and women of reproductive age for Zika virus infection related to the investigation for local mosquito-borne Zika virus transmission in Brownsville, Cameron County, Texas [Internet]. Atlanta: CDC; 2016. (CDCHAN-00399). Available from: https://emergency.cdc.gov/ han/han00399.asp Accessed on 11 January 2017.
(2.) Brunkard JM, Lopez JL, Ramirez J, Cifuentes E, Rothenberg SJ, Hunsperger EA, et al. Dengue fever seroprevalence and risk factors, Texas-Mexico border, 2004. Emerg Infect Dis. 2007;13(10): 1477-83.
(3.) Clark GG. Dengue and dengue hemorrhagic fever in northern Mexico and south Texas: do they really respect the border? Am J Trop Med Hyg. 2008;78(3):361-2.
(4.) Smith BR. Dengue, Region 11, 2013 [PowerPoint]. Harlingen, Texas: Texas Department of State Health Services; 2014. Available from: http://www.dshs.texas.gov/IDCU/ Dengue-Region-11-2013-(3).doc Accessed on 10 January 2017.
(5.) Thomas DL, Santiago GA, Abeyta R, Hinojosa S, Torres-Velasquez B, Adam JK, et al. Reemergence of dengue in southern Texas, 2013. Emerg Infect Dis. 2016;22(6):1002-7.
(6.) Knowlton K, Solomon G, Rotkin-Ellman M. Fever pitch: mosquito-borne dengue fever threat spreading in the Americas. New York: Natural Resources Defense Council; 2009.
(7.) Eisen L, Moore CG. Aedes (Stegomyia) aegypti in the continental United States: a vector at the cool margin of its geographic range. J Med Entomol. 2013;50(3): 467-78.
(8.) Bouri N, Sell TK, Franco C, Adalja AA, Henderson DA, Hynes NA. Return of epidemic dengue in the United States: implications for the public health practitioner. Public Health Rep. 2012;127(3):259-66.
(9.) Champion SR, Vitek CJ. Aedes aegypti and Aedes albopictus habitat preferences in South Texas, USA. Environ Health Insights. 2014; 8(Suppl 2):35-42.
(10.) National Weather Service Forecast Office. NOAA online weather data. Norman, Oklahoma: National Oceanic and Atmospheric Administration; 2006.
(11.) Luz PM, Vanni T, Medlock J, Paltiel AD, Galvani AP. Dengue vector control strategies in an urban setting: an economic modelling assessment. Lancet. 2011;377(9778): 1673-80.
(12.) Arunachalam N, Tana S, Espino F, Kittayapong P, Abeyewickreme W, Wai KT, et al. Eco-bio-social determinants of dengue vector breeding: a multicountry study in urban and periurban Asia. Bull World Health Organ. 2010;88(3): 173-84.
(13.) Ramos MM, Mohammed H, Zielinski-Gutierrez E, Hayden MH, Lopez JL, Fournier M, et al. Epidemic dengue and dengue hemorrhagic fever at the Texas-Mexico border: results of a household-based seroepidemiologic survey, December 2005. Am J Trop Med Hyg. 2008;78(3):364-9.
(14.) World Health Organization. Dengue fact sheet. Geneva: WHO; 2012.
(15.) Bolling B. Mosquito testing program for arbovirus surveillance in Texas [PowerPoint]. Austin: Texas Department of State Health Services; 2015. Available from: https://www.dshs.texas.gov/lab/ directorsconference.shtm
(16.) Lee J, Stahl M, Sawlis S, Suzuki S, Lee JH. A potential risk assessment of a dengue outbreak in north central Texas, USA (part 1 of 2): abundance and temporal variation of dengue vectors. J Environ Health. 2009;71(10):24-9.
(17.) Weaver SC, Reisen WK. Present and future arboviral threats. Antiviral Res. 2010;85(2):328-45.
(18.) World Health Organization. Vector surveillance and control. In: WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. 2nd ed. Geneva: WHO; 1997. p. 48. Available from: http://www.who. int/csr/resources/publications/ dengue/Denguepublication/en/
(19.) Hadler JL, Patel D, Nasci RS, Petersen LR, Hughes JM, Bradley K, et al. Assessment of arbovirus surveillance 13 years after introduction of West Nile virus, United States. Emerg Infect Dis. 2015;21(7): 1159-66.
Manuscript submitted 31 October 2015. Revised version accepted for publication on 12 July 2016.
Krithika Srinivasan,  Beatriz Tapia,  Arturo Rodriguez,  Robert Wood,  and Jennifer J. Salinas 
Suggested citation Srinivasan K, Tapia B, Rodriguez A, Wood R, Salinas JJ. Species abundance and temporal variation of arbovirus vectors in Brownsville, Texas. Rev Panam Salud Publica. 2017;41:e28.
 University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America. Send correspondence to: Krithika Srinivasan, email@example.com
 City of Brownsville Department of Public Health, Brownsville, Texas, United States of America.
 Texas Tech University Health Sciences Center at El Paso, El Paso, Texas, United States of America.
TABLE 1. Mosquito species collected in an arbovirus vector surveillance program using gravid traps, Brownsville, Texas, 2009-2013 Aedes aegypti Aedes albopictus Aedes bimaculatus Aedes epacitus Aedes sollicitans Aedes taeniorhynchus Anopheles pseudopunctipennis Anopheles punctipennis Anopheles spp. Anopheles crucians Anopheles quadrimaculatus Culex coronator Culex (Melanoconion) spp. Culex nigripalpus Culex quinquefasciatus Culex salinarius Culiseta inornata Deinocerites mathesoni Mansonia titillans Toxorhynchites rutilus septentrionalis Uranotaenia spp. Source: Prepared by the authors based on the study results. TABLE 2. Weather statistics and arbovirus mosquito vector species incidence by year based on pooled data from gravid traps (n = 35), Brownsville, Texas, 2009-2013 Variable 2009 2010 Average rainfall per month (inches) (SD (a)) 2.9 (4.4) 3.9 (4.4) Average temperature highs ([degrees]F) (SD) 86.6 (11.2) 89.3 (5.2) Proportion (%) of insect trap collections by species (b) (SD) Culex q. 95.1 (5.0) 76.4 (25.8) Aedes spp. 4.7 (4.8) 23.0 (25.6) Ae. aegypti 4.6 (4.7) 21.4 (25.7) Variable 2011 2012 Average rainfall per month (inches) (SD (a)) 1.7 (3.0) 1.3 (1.1) Average temperature highs ([degrees]F) (SD) 90.9 (4.8) 91.9 (3.0) Proportion (%) of insect trap collections by species (b) (SD) Culex q. 76.6 (19.4) 80.4 (14.5) Aedes spp. 23.0 (19.8) 22.0 (11.3) Ae. aegypti 22.9 (19.8) 22.0 (11.3) Variable 2013 Average rainfall per month (inches) (SD (a)) 0.95 (0.902) Average temperature highs ([degrees]F) (SD) 91.5 (3.6) Proportion (%) of insect trap collections by species (b) (SD) Culex q. 80.8 (20.7) Aedes spp. 17.6 (21.2) Ae. aegypti 15.2 (20.9) Source: Prepared by the authors based on the vector surveillance results. Temperature data are from the National Oceanic and Atmospheric Administration (NOAA) station in Brownsville, Texas. (a) Standard deviation. (b) The three most common species caught in the surveillance traps were 1) Culex quinquefasciatus, which transmits West Nile virus (WNV) and Saint Louis encephalitis; 2) Ae aegypti, which transmits dengue virus (DENV) and yellow fever virus; and 3) Ae. Albopictus, which transmits chikungunya virus, Eastern equine encephalitis virus, WNV, and DENV. However, because so few Ae. albopictus mosquitoes were found, they were combined with Ae. aegypti to form a new, third category ("Aedes spp.") for the analysis of species incidence over time.