Molecular Methodology for Tick Speciation and Tickborne Pathogen Surveillance

Abstract

Purpose: The goal of this study was to utilize quantitative polymerase chain reaction (qPCR) to identify field collected ticks and determine the prevalence of thirteen tickborne pathogens. Methods & Materials: Ticks were collected by drag/flag sampling from seven west Michigan counties. Ticks were found at 22 out of 35 total field sites. and were preliminarily identified by microscopy. Following transfer to the University of New England, total nucleic acids were extracted and quantified. Species-specific primers were developed targeting diverse regions of the mitochondrial cytochrome oxidase B (CytB) gene for Ixodes scapularis, I. cookei, I. pacificus, Amblyomma americanum, A. maculatum, Ripicephalus sanguineus, Dermacentor andersoni, and D. variabilis. Previously described primer sets were synthesized to detect the presence of Bourbon virus, Heartland virus, Powasson virus, Babesia microti, Borrelia miyamotoi, B. burgdoferi, B. mayonii, Anaplasma phagocytophilum, Ehrlichia chaffiensis, Bartonella spp., Rickettsia spp., Franciscella tularensis, and Coxiella burnetii. Nucleic acid templates for all tick species and tickborne pathogens were obtained from culture collections to serve as positive controls. All detections were made by SYBR green qPCR. Results: All tick species determined by microscopy were confirmed by qPCR. Seven of the thirteen tickborne pathogens have been detected to date. The most abundant pathogen detected was B. burgdoferi, and the most abundant tick species collected was I. scapularis. Conclusion: Here we present a single methodology to process field-collected ticks that allow for the determination of vector species and pathogen carriage. This methodology holds the potential to eliminate the need for manual, microscopic identification of vectors used for habitat/disease surveillance.