Progress 08/01/24 to 07/31/25
Outputs
Target Audience:During this reporting period, our laboratory reached multiple target audiences encompassing academic researchers, diagnostic laboratories, veterinary and public health professionals, extension personnel, and students. The activities were structured to promote knowledge exchange, build diagnostic capacity, and foster translational applications of the multiplex molecular assay for six tick-borne pathogens of veterinary and zoonotic significance. Principal Investigator presented research findings and assay development updates at national and regional scientific meetings, including sessions focusing on vector-borne diseases and diagnostic innovation (ASAS Calgary, IAPSCON INDIA, California One Health Symposium). Informal and formal presentations were made at internal university seminars and research symposia, facilitating scientific discussion and collaboration (Research Day CVM WesternU). Four DVM (Doctor of Veterinary Medicine) students (two second year and two first year) of our westernu veterinary college were engaged through research participation and classroom integration. Students gained hands-on experience with nucleic acid extraction, PCR, and multiplex bead-based hybridization techniques. Mentorship activities focused on laboratory data analysis, assay optimization, and interpretation of complex molecular results. These efforts strengthened technical capacity and fostered an understanding of translational research linking molecular technology to real-world diagnostic applications. Two students presented posters or short talks (veterinary scholars symposium- August 7-9th Spokane, WA) derived from project data, broadening dissemination and visibility of the research outcomes. Overall, the project successfully reached its target audiences by providing research-based information, diagnostic training, and educational resources to improve the understanding and management of tick-borne pathogens affecting animal and public health. Changes/Problems:Somedelays were encountered during this reporting period due to the need for additional optimization of primers and probes to achieve consistent performance under optimal assay conditions. This refinement process required repeated testing and validation to ensure the desired sensitivity and specificity for each target pathogen. In addition, there aredelays in hiring a student assistant to support tick collection activities, which arebeyond our control and temporarily slowed field sample acquisition. Other delayswere interruptions in the timely receipt of critical reagents and chemicals needed for assay optimization, as well as delays in obtaining positive reference material for Ehrlichia species, which temporarily limited our ability to complete feasibility testing. Additionally, an unexpected instrument malfunction caused a temporary pause in laboratory work while repairs and recalibration were completed. What opportunities for training and professional development has the project provided?This project provided valuable training and professional development opportunities for undergraduate and DVM students, research staff, and collaborators. Participants gained hands-on experience in molecular diagnostics, including nucleic acid extraction, PCR optimization, and bead-based hybridization using the Applied BioCode 2500 system. They also received mentorship in bioinformatics analysis, primer and probe design, data interpretation, and assay validation. Through lab meetings, national and local presentations, trainees enhanced their technical, analytical, and communication skills. Overall, the project strengthened workforce capacity in advanced diagnostic technologies and fostered professional growth in the areas of molecular biology, vector-borne disease research, and One Health applications. How have the results been disseminated to communities of interest?The results of this project have been disseminated to relevant communities of interest through scientific presentationsand academic engagement. Preliminary findings on assay development and validation were shared at research symposia, departmental seminars, and professional meetings focused on vector-borne diseases and molecular diagnostics. Students and trainees presented posters summarizing project progress, further extending outreach within academic and professional circles. These efforts have ensured that researchers, diagnosticians, veterinarians, and public health professionals are informed of the project's advancements and their relevance to animal and zoonotic disease surveillance. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to complete the ongoing Babesia and Ehrlichia assay optimization and validation studies. Following these, we will extend the same approach to include two additional target pathogens in the multiplex panel to continue building toward the full six-pathogen assay. These activities will focus on evaluating primer and probe performance, assessing sensitivity and specificity under multiplex conditions, and refining assay parameters for simultaneous detection on the Applied BioCode 2500 system. Completion of these steps will advance the feasibility testing phase and move the project closer to establishing a fully validated six-plex molecular diagnostic assay for major tick-borne pathogens of veterinary and public health significance.
Impacts
What was accomplished under these goals?
a) Goal 1.1 and Goal 1.2 This reporting period we specifically concentrated on Ehrlichia and Babesia species. Genomic sequences representing diverse isolates of Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii were retrieved from publicly available databases (NCBI GenBank and RefSeq). Multiple sequence alignments were performed to identify conserved gene targets with high discriminatory potential among closely related species. Candidate loci such as 16S rRNA, dsb, and groEL were evaluated for sequence conservation, amplicon length, GC content, and suitability for multiplex hybridization. Similarly, for Babesia spp., we analyzed representative genomic sequences of B. bovis, B. bigemina, and B. microti to identify conserved regions within 18S rRNA and cytochrome b genes. These targets were assessed for their capacity to differentiate between species while maintaining robust amplification efficiency in mixed or low-copy DNA samples. Candidate primers were designed using bioinformatics tools (Primer3, BLASTn, and OligoAnalyzer) and screened in silico for specificity and potential cross-reactivity with non-target organisms. Phylogenetic analysis supported the inclusion of representative sequences from geographically diverse isolates to ensure global applicability of the assay. Laboratory feasibility studieswas initiated to test primer performance using synthetic DNA templates and extracted nucleic acids from reference strains. Optimization trials included gradient PCR and assessment of primer compatibility within the barcoded magnetic bead multiplex format.Preliminary results confirmed the amplification of target regions for Ehrlichia and Babesia species with high specificity and reproducibility. These data form the basis for integration into the broader multiplex panel that will include Anaplasma and Borrelia species in the subsequent reporting phase. For singleplex assays were established and systematically evaluated for Ehrlichia and Babesia targets designed under Goal 1.2. For each target gene, biotin-labeled amplicons were generated by endpoint PCR and hybridized to the complementary probes immobilized on the bead surface. Hybridization conditions, including temperature, salt concentration, and incubation time, were optimized to achieve maximal signal-to-background ratios. Each probe-primer set was tested against both homologous (target) and heterologous (non-target) DNA templates to assess analytical specificity. The resulting data demonstrated that each probe bound exclusively to its intended amplicon with no detectable cross-reactivity among closely related species or non-target DNA. Negative control beads and blank reactions consistently yielded background signal levels below the established threshold, confirming assay specificity. For sensitivity assessment, tenfold serial dilutions of known template concentrations are beingused to determine the limit of detection (LOD) for each target under singleplex conditions. The BioCode system demonstrated consistent signal detection down to low template copy numbers, indicating that bead-based hybridization retained sensitivity comparable to or greater than conventional PCR detection methods. Signal intensity was quantified using the BioCode 2500 reader and analyzed with proprietary software to determine the optimal fluorescence threshold for positive detection. b) Goal 2 Experiments are ongoingto combinecompatible primer and probe sets for Ehrlichia and Babesiaspecies that demonstrated optimal performance in singleplex assays to be used in duplex of anaplasma and borrelia primer probe sets. We are testing sensitivity and specificity of all primer pairs and probes in the mutiplex. c) Goal 3 In consultation with Dr Ortiz, efforts are underway to hire a student who will be collecting ticks in spring of 2026 in souther california green spaces.
Publications
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