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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.

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