Lyme disease (LD) is caused by infection with Borrelia burgdorferi (Bb) and represents a growing problem in the United States due to increasing prevalence and expanding geographic range. It is estimated that 300,000 cases of LD occurred in the United States in 2017. The disease course is highly variable and can include organ-specific and disseminated involvement with acute and chronic manifestations. A knowledge gap exists regarding the immune events during the dissemination of Bb in blood during acute LD. Our objective is to bridge this knowledge gap through the development of novel tools that measure neutrophil-Bb interactions in blood. In preliminary work, we demonstrate the feasibility of microfluidic assays for the study of host-pathogen interactions in blood. Specifically, we found (A) a neutrophil spontaneous motility phenotype after spiking as few as one hundred Bb in blood, (B) a similar phenotype in blood samples from acute LD patients, and (C) a key role for complement in the activation on neutrophils in blood during early interactions with Bb. Continuing these efforts, we will optimize the neutrophil-assay-design to (1) study the interactions between neutrophils and live, motile Bb at single-cell resolution (2) identify signature neutrophil responses to Bb, and (3) probe the relay function of neutrophils during complement activation in blood by small numbers of Bb. We will begin applying the new tools to measuring neutrophil functions in patients during acute LD before specific antibodies are produced, and during treatment, to evaluate the Bb clearance from blood. Overall, our study will design new tools for the diagnostic and monitoring of LD and will expand our knowledge of host responses to LD.