ABSTRACT Lyme disease is a tick-borne disease caused by spirochetes of the Borrelia burgdorferi sensu lato complex. Murine models of B. burgdorferi infection allows for the assessment of bacterial dissemination and presence through culture of infected tissue and dark-field microscopic examination after a period of outgrowth. Although valuable, dark-field microscopy can be subjective, complex, and time-consuming. We propose that the limitations of current culture-screening methodologies could be overcome using a fluorescent B. burgdorferi stain for murine challenge studies. To test this, we generated A3-68 Δbbe02 pBBE22G-flgBpGFP, a strain of B. burgdorferi that expresses the fluorescent protein GFP encoded on the plasmid pBBE22G. We then validated that the A3-68 Δbbe02 pBBE22G-flgBpGFP strain ( Bb- GFP) expressed detectable GFP in vitro and that it grew and had a similar infection profile in mice compared to its parental strain, A3-68 Δbbe02 . Next, we compared the accuracy and sensitivity of three different methods to detect B. burgdorferi from infected tissues: flow cytometry, dark-field microscopy, and PCR. In a Bb- GFP murine challenge model, screening of tissue cultures with flow cytometry exhibited sample positivity similar to that of traditional methods, with increased objectivity, speed, and efficiency. Overall, the Bb -GFP fluorescent strain and associated methodology described here provide additional resources for the study of B. burgdorferi pathogenesis. IMPORTANCE Studying Borrelia burgdorferi pathogenesis can be challenging due to reliance on labor-intensive techniques such as dark-field microscopy to determine the infection status of animals. In this study, we demonstrated that flow cytometry could be used as an alternative approach, allowing for fast, reliable, and comparable results to those of dark-field microscopy in the context of mouse infection models.
Huckaby et al. (Mon,) studied this question.