Tick-borne diseases (TBDs) affect companion and food production animals around the world. As emerging TBDs are identified, there is increasing need for reliable and appropriate test methods that can be used in animal specimens and ticks for clinical treatment and surveillance. The prevalences of newly identified agents are largely unknown, especially in animals. Furthermore, appropriate animal specimen types are not well understood for some emerging agents. Current testing methodologies are largely focused on Lyme disease alone and do not offer comprehensive detection of animal health concerns. In molecular testing, which is ideal for host species-independent detection of multiple pathogen types at once, scaling up the number of targets in typical reactions is limited due to cost and/or loss of sensitivity when combining assays.