Development of multiplex real-time PCR assays for the surveillance of tick-borne pathogens in Delaware

Abstract

Tick-borne pathogens are responsible for most vector-borne human diseases in the United States. In the United States, close to 650,000 cases of vector-borne diseases were reported from 2004–2016. Of the 650,000 cases, tick-borne diseases accounted for >75% of these cases. From 2016-2019, there were 206,568 reported cases of tick-borne diseases. This demonstrates increasing numbers of tick-borne pathogen cases reported in the United States. Given the increasing recognition of tick-borne diseases, as well as the increase in the range and distribution of ticks, it is imperative to understand which pathogens, and in what prevalence, are carried by tick species in areas populated by humans. Few studies exist surveying the presence and distribution of tick-borne pathogens in the state of Delaware. The goal of this study was to create multiplex real-time PCR assays to identify Borrelia burgdorferi sensu stricto, Babesia microti, Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii from their respective reservoir tick species. ☐ Three multiplex, real-time PCR assays were developed and tested on 1527 ticks comprising Ixodes scapularis, Dermacentor variabilis, and Amblyomma americanum, three species of ticks relevant to Delaware. The results showed that of a sample of 500 Ixodes scapularis ticks from Delaware, 30.20% were positive for Borrelia burgdorferi, 2.60% were positive for Babesia microti, and 1% were positive for Anaplasma phagocytophilum. Testing of 500 D. variabilis ticks revealed that 0.00% were positive for E. chaffeensis and 0.20% were positive for A. phagocytophilum. Finally, of the 527 A. americanum ticks tested, 4.74% were positive for E. chaffeensis and 1.14% were positive for E. ewingii. These findings support the notion that real-time PCR assays can be used to successfully identify and monitor tick-borne pathogen activity in Delaware.