Novel Molecular Strategies for the Detection and Characterization of Tick-Borne Pathogens in Domestic Dogs

Tick-borne diseases (TBD) are common across the United States and can result in critical and chronic disease states in a variety of veterinary patients, specifically domesticated dogs. Borreliosis, anaplasmosis, rickettsiosis, ehrlichiosis, and babesiosis have been cited as the most common TBDs. Despite recent reports revealing past exposure of TBD, there are no molecular epidemiological reports for dogs in Texas. Therefore, data to support the level of actively infected dogs in the population is inadequate. Limited molecular data for TBDs is due, in part, to the lack of consolidated molecular tools available to researchers. Real-time PCR (qPCR) assays are a commonly utilized tool for molecular detection of TBDs, and achieve species specificity by assigning each pathogen a unique fluorogenic label. However, current limitations of qPCR instruments include restricting the number of fluorogenic labels that can be differentiated by the instrument per a given reaction. As such, this dissertation explored the development of a qPCR methodology, termed layerplexing, that would allow for the simultaneous detection and characterization of 11 pathogens responsible for causing common TBDs in domestic dogs. Additionally, an endogenous internal positive control was designed and integrated into the assay for quality assurance of attained molecular results. Analysis revealed that the layerplex assay format was comparable in terms of target sensitivity and specificity to other qPCR assays utilized in the field. The layerplex assay was then applied to conducting a molecular prevalence investigation of TBDs affecting dogs across Texas ecoregions. By conducting molecular prevalence studies for TBDs, updated rates of active exposure and specific regions that may contain sentinels of disease could be identified. Results obtained indicated molecular prevalence of borrelial, rickettsial, and babesial pathogens varied across the Texas study area and indicated specific regions where susceptible hosts may be at higher risk for infection with TBD. Furthermore, the layerplex assay lead to the first reported molecular detection of Anaplasma platys in Texas and coinfection with Ehrlichia canis and A. platys in Texas dogs. Overall, findings from this dissertation provided substantial evidence that the layerplex technique can be utilized for grouping multiple targets under a single fluorogenic label without impeding diagnostic efficacy. The layerplex technique also demonstrated utility in facilitating large scale molecular analyses of animals in Texas. Surveillance data obtained from this study may aid public health agencies in updating maps depicting high-risk areas of disease and provide baseline data for future research aiming to characterize TBDs in additional animal models