Performing Department
Entomology
Non Technical Summary
Mosquito-borne infections such as dengue, chikungunya, and zika are a significant threat to people living in endemic areas for Aedes mosquitoes, travelers and deployed personnel. The diseases occurindividually and in outbreaks, but research on effective preventive measures is limited by the unpredictability of outbreaks and the relatively small numbers of individual cases with a specific diagnosis. The use of antibodies to Aedes specific salivary antigens as surrogate markers for bite exposure is a promising alternative to standard entomological measures. Additionally, IgG antibodies to an Aedes aegypti derived N-terminal extremity peptide of a 34 kDa protein (Nterm-34 kDa) have been used as biomarkers for Aedes bite exposure. However, more studies are needed to determine the seroprevalence and specificity of antibodies against Aedes salivary proteins in people living in endemic areas as well in military personnel during high-risk group deployments. We propose a cost-effective, scalable methodology to evaluate antibody levels against both, the mosquito vector and the pathogen through ELISA and using dried blood spots that do not need high tech refrigeration or storage capacity. The salivary biomarkers, in combination with serologic assays for arboviral infections, will provide estimates of mosquito exposure and disease transmission intensity that can be used to determine the effectiveness of response strategies to outbreaks (e.g. improving vector control and use of personal protective measures).
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
<i>Ae. albopictus</i> and <i>Ae. aegypti</i>, with a focus on surveillance, invasion ecology, genetics
New Control Tools, including socio-ecological approaches
Training and training tools
Project Methods
Aim 1: Evaluation of anti-salivary antibody levels in populations with different mosquito-human exposure riskStudy participants and recruitment process: A longitudinal population-based household survey will be employed to monitorarboviral transmission (Dengue and Zika). Blood samples will be collected and axillary temperature recorded for all persons older than 1 year within each participating household after informed consent is requested and granted. For molecular diagnosis, four (4) blots of blood on filter paper will be collected at the time of the visit by finger prick and refrigerated. Additionally, RDT for Dengue virus (DENV) and Zika virus (ZIKV) will be performed on each participant using the blood from the same finger prick. The head of the household will be invited to participate in a short survey. A questionnaire with general demographic questions including gender, age, geographic residency, history of travel, sleeping habits (outdoor and/or indoor), water service for the household and use of bed nets for the head and all household members.A cross-sectional study will also be performed including patients admitted for febrile illness at the Los Patios Hospital (Los Patios - Colombia). After informed consent is granted, the same type of sample (dried blood in filter paper) will be collected from each patient. A cohort with US military personnel deployed to Africa and South America will also be included in the study to evaluate the usefulness of salivary markers in travelers.Estimation of vector-human contact by ELISA: We plan to evaluateantibody levels against mosquito salivary gland proteins to correlate OD values and clinical presentation infection and to correlate mosquito abundance with antibody levels. The corrected OD values (sample OD average-negative control) will be interpreted as the specific subject antibody concentration. For the ELISAs, plates will be coated with either Ae. aegypti or Ae. albimanus salivary gland (SGE) or specific salivary recombinant proteins (Nterm34 kDa and D7). Specific anti-human IgG, IgM and IgE HRP-labeled antibodies will be used to detect the antibody types in the dried blood samples in filter paper. Detection of target antibodies will be visualized by adding Tetramethylbenzidine (TMB) to each well. Reading of the OD values will be done in an ELISA reader/spectrophotometer at 450nm.SGE immunoblotting: A subset of 100 samples will be randomly selected to represent infected and non-infected subjects in the study group to identify the most immunogenic proteins in whole SGE samples. Briefly, proteins will be separated by SDS-PAGE, then transferred onto a PVDF membrane using Trans-Blot® Turbo™ Transfer System. Membranes will be blocked overnight with 2% milk in 1X PBST (Phosphate buffer saline plus 0.01 Tween 20) and incubated with human sera diluted 1/100 in blocking buffer for 2 h at room temperature. After washing, each membrane will be incubated with HRP-conjugated Goat Anti-Human total IgG diluted 1/1000 in blocking buffer for 1 h at 37°C. Color development will be obtained with the HRP chromogenic substrate tetra-methyl-benzidine (TMB). Band corrected density will be measured using MyImageAnalysis Software.Arbovirus confirmation and serotyping: Dengue Duo rapid test will be performed at the field site hospital. To confirm results RNA will be isolated from blood samples (with an RNA preservative) and the viral genome will be detected by using the QuantiFast SYBR Green kit and a Biorad CFX cycler to amplify isolated RNA with DENV serotype-specific primers (DENV 1- 4).Pathogen antigen serology: Data acquisition will be performed by standard ELISA assaying for IgG antibodies to the DENV and ZIKV envelope (Ep) and non-structural protein 1 (NS1) proteins. The proteins will be sent for synthesis to GenScript. Appropriate parametric distributions and seropositivity cutoff values will be determined by statistical measures.Aim 2. Develop a mathematical model of mosquito bite exposure dynamics, incorporating anti-salivary protein antibodies to model emergence/re-emergence potential and infection dynamics.Environmental data collection: Models of species distribution will be developed for Ae. aegypti and Ae. albopictusin a presence-only distribution modeling algorithm, the Maximum Entropy (MaxEnt) v. 3.3.3 model. MaxEnt finds the maximum entropy in specie-presence geographic datasets in relation to a set of environmental variables, in this case, we will be using environmental predictor variables derived from 2015 Landsat 8 satellite imagery. A set of spectral indices from the Landsat 8 image covering the study area will be used based on Samson et al (2015). Four different indices will be derived: urban index (UI), soil and vegetation index (SVI), normalized difference impervious surface index (NDISI) and modified normalized difference water index (MNDWI). Several questions will be included in the survey to be used as part of the model in the category of human behavior.House inspection for mosquito larvae: A trained entomologist will be part of the field team. During the household visit, a permission to inspect the household premises will be requested to the head of the household. If granted, the entomologist will search inside and around the house for water container with potential for mosquito breeding site (i.e. water cans, unused plastics, discarded bottles). Each water container will be inspected for the presence of aquatic stages of mosquitos (i.e. larvae and pupae). If mosquitoes are present in small containers, a small pipette will be used to extract the specimen out of the water. In case of larger containers (i.e. water tanks, barrels) using an entomologic hand dipper. All specimens will be collected in snap cap vials and kept refrigerated until detection.Host-vector contact analysis: Host-vector contact index (VCI) will be calculated using the anti-SGE IgG OD value divided by the number of mosquitoes captured in the respective house. Another index is the infective bite exposure (IBE) calculated as the IgG OD value of the antibody concentration against the up-divided multiplied by the number of infected mosquitoes in the house.