Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species

We describe the development, optimization, and validation of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of Babesia, Bartonella, and Borrelia spp. DNA from several sample matrices, including clinical blood samples from animals and humans, vectors, in-vitro infected human and animal cell lines, and tissues obtained from animal models (infected with Bartonella and/or B. burgdorferi). The multiplex ddPCR assay was able to detect 31 Bartonella, 13 Borrelia, and 24 Babesia species, including Theileria equi, T. cervi, and Cytauxzoon felis. No amplification of Treponema or Leptospira spp. was observed. Sensitivity of 0.2–5 genome equivalent DNA copies per microliter was achieved for different members of the Bartonella and Borrelia genus, depending on the species or matrix type (water or spiked blood DNA) tested. The ddPCR assay facilitated the simultaneous detection of co-infections with two and three vector-borne pathogens comprising four different genera (Babesia, Bartonella, Borrelia, and Theileria) from clinical and other sample sources.