Development of zebrafish (Danio rerio) in vivo model for Borrelia burgdorferi infection

Borrelia burgdorferi is the spirochetal bacterium that causes Lyme disease. Despite the fact that antimicrobial sensitivity of B. burgdorferi has been widely studied, there is still a need to develop an affordable, practical, high-throughput in vivo model which can be used to find effective antibiotic therapies, especially for the recently discovered persister and biofilm forms. Recent studies showed that Zebrafish (Danio rerio) could offer a novel, high-throughput, affordable model in antibiotic therapies for various infections agents can be studied. Therefore, in this study, we developed a straightforward, standardized infection procedure and tested it for zebrafish survival rate, morphological or behavioral changes post-infection as well as providing evidence that B. burgdorferi persists in zebrafish using polymerase chain reaction (PCR) and immunohistochemical (IHC) techniques. Morphological and physiological examination showed a significant size difference between control and infected zebrafish at 2 wpi, and infected zebrafish exhibited slower heart rates through 72 hpi. Furthermore, our results showed B. burgdorferi DNA can be detected and active replication of the B. burgdorferi 16S rRNA gene can be confirmed through 10 days post-infection via PCR and Reverse Transcription PCR respectively. Fluorescent microscopy and immunohistochemical staining revealed spirochetes present in the eyes, gills, heart, liver, tail, and hindbrain tissues though 72 hpi as well as the stomach and digestive tract at 2 weeks post-infection, respectively. These findings demonstrate that zebrafish could serve as a promising animal model to study the mechanism of B. burgdorferi infection as well as in vivo antibiotic sensitivity.