Performing Department
(N/A)
Non Technical Summary
With the increasing tick population and expansion of their geographical ranges as well changes in farming conditions and climate conditions, Tick-borne diseases are becoming a major global health impediment both for humans as well as for efficient livestock production. On a global basis, the economic toll caused by tick-borne diseases is staggering and better global control of tick-borne diseases of livestock and their vectors would contribute substantially to improved meat and milk production. Increasing Tick-borne infections nationally and internationally have increased the demand of diagnostic assays for these infections using different multiplex detection platforms. We intend to develop a sensitive, high throughput multiplex diagnostic approach that can simultaneously detect 6 Tick-borne pathogen groups from a single sample using Barcoded Magnetic Bead technology. The pathogens include Anaplasma spp., Borrelia spp., Ehrlichia spp., Rickettsia spp, Babesia spp, and Theileria spp., which are the etiologic agents for most of the known Tick-borne diseases. The central focus of this grant proposal is to carry out the feasibility, development, and verification testing for simultaneous detection of 6 Tick-borne pathogens groups in the multiplex diagnostic assay. Such an assay will serve the Veterinary health and Surveillance for many decades to come. This application has direct impact on veterinary health worldwide. Our diagnostic assay will rapidly identify the correct etiologic agent and will facilitate the specific treatment along with early interventions to stop the spread among greater population.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
?a) Goal 1: Feasibility testing for detection of 6 groups of tick-borne infectious pathogensGoal 1.1: Bioinformatics analysis and primer testing-To allow the detection of all know species of a particular pathogens in an assay, it is important to classify and categorize the known pathogens that are in circulation.Goal 1.2: Testing the sensitivity and specificity of each probe and primer set in a singleplex assay using the BMB's on Applied BioCode 2500 system.The rationale behind this aim is to confirm that PCR amplicons generated by each primer set are specifically detected by their respective probes tethered to barcoded beads, without compromising the sensitivity and specificity for detection.b) Goal 2: Up-gradation from singleplex to miniplex PCR detection- The rationale behind the gradual development from singleplex to miniplex in the development process is to standardize the working conditions in which primer sets retain the target specificity without competing for undesired targets or interrupting the specificity and efficiency of other primers in the combined reaction.c) Goal 3: Screening the ticks of San Gabriel valley for tickborne infections- Urban greenspaces offer recreational opportunities for people and pets, but they also harbor wildlife that can act as tick hosts. Animals, wildlife, and human sharing these spaces face increased risks of tick bites and potential exposure to tickborne infections. Limited data exists on ticks in the eastern San Gabriel Valley and Southern California. Our research aims to fill these knowledge gaps by employing a novel tick collection approach and using a multiplex assay to screen for tickborne pathogens. The resulting data will shed light on the risk of tickborne infections and identify the periods of highest likelihood for such exposures.
Project Methods
More than10,000 genome sequences of the pathogen strain will be analyzed by using bioinformatic tools like Geneious, MegAlign, and Jalview to identify highly conserved genes. Using software such as Primer-BLAST and MacVector, we'll design 3-5 primer sets for each target pathogen. Commercial kits will be utilized for nucleic acid extractions from intact pathogens. DNA probes specific to the amplicon will be coupled to BMBs following standard procedures. Sensitivity testing will involve a ten-fold dilution series of extracted nucleic acid from each target pathogen. For specificity testing, the experiment will be repeated with nucleic acid from other pathogen groups, ensuring minimal cross-reactivity. This process will be repeated for 4-5 primer sets per pathogen to obtain sets with high sensitivity and specificity.Further, Pathogens will then be grouped into mini-panels (miniplex 1-3). Each miniplex's probe and primer pairs will undergo sensitivity and specificity testing to ensure their effectiveness in combined reactions. Once proven effective, miniplexes will be combined sequentially to form more complex panels.Further testing of all parmeters of feasibility testing will be carried out (linearity, sensitivity, specificity, cross reactivity, interference, Limit of detection and reproducibility).The working assay will be finaly used to detect presence of 6 tickborne pathogens in ticks found in southern California. In January 2022, twenty-five sites were established 1 km apart along a 26 km transect in the San Gabriel Valley, California, for wildlife documentation using motion-triggered cameras as part of the Urban Wildlife Information Network (UWIN) method. Twelve sites were chosen for tick sampling, targeting areas with human recreation. Transects within a 500-meter buffer zone around each site were used for sampling. A 1m x 1m flag/drag cloth was employed along each transect, dragged at a walking pace with regular checks for ticks. Tickborne disease diagnostics will be performed by extracting nucleic acid using Qiagen extraction, followed by BMB assays to detect six tickborne pathogen groups.?