Human babesiosis is an important emerging vector-borne disease in North America. Most documented cases are caused by the protozoan parasite, Babesia microti, in the Eastern and Midwestern United States. On the Pacific Coast, rare cases of human babesiosis have been ascribed to Babesia duncani, first identified in 1991, as well as closely related wildlife Babesia species. While the tick vector and mammalian reservoir hosts for B. microti are well characterized, the vector and reservoir hosts for B. duncani are unknown despite previous investigations of common human-biting ticks. Identification of potential hosts and vector species has been hampered by the lack of a specific and sensitive molecular diagnostic tool that could identify piroplasm species and did not co-amplify other eukaryotic DNA. To further the study of this zoonotic disease, we attempted to develop a nested PCR assay that would be both specific and sensitive enough to screen ticks for the pathogen. We sequenced the target gene in two wildlife isolates and one human isolate to create nested primers which would pick up all B. duncanisubtypes and not amplify host DNA. We then tested the assay against mammalian and tick DNA for specificity, and in dilution to test sensitivity.
Our novel PCR assay is highly specific to Babesia spp. The target sequence includes a highly variable intron, which allows for differentiation between B. duncani and Babesia odocoilei, another wildlife Babesia species common in the suspected reservoir species, based on band size on a gel. This assay has a sensitivity of 2.72x10-12 pg/µl of purified template DNA and does not cross-react with mammalian and tick DNA, even in the absence of pathogen DNA. We have successfully used this assay to screen ticks and identify samples based on their similarity to the WA-1 isolate from the index-case patient and two wildlife isolates. Based on these findings, this new assay is a valuable tool for the study of the ecology and epidemiology of this zoonotic pathogen, and will permit sample screening and identification of potential vector and reservoir species, ultimately allowing for spatial analysis and disease risk management of this pathogen.