Detection of established virulence genes and plasmids to differentiate Borrelia burgdorferi strains

Borrelia burgdorferi sensu stricto is the major causative agent of Lyme disease in the United States, while B. garinii and B. afzelii are more prevalent in Europe. The highly complex genome of B. burgdorferi is comprised of a linear chromosome and a large number of variably sized linear and circular plasmids. Many plasmids of this spirochete are unstable during its culture in vitro. Given that many of the B. burgdorferi virulence factors identified to date are plasmid encoded, spirochetal plasmid content determination is essential for genetic analysis of Lyme pathogenesis. Although PCR-based assays facilitate plasmid profiling of sequenced B. burgdorferi strains, a rapid genetic content determination strategy for nonsequenced strains has not yet been described. In this study, we combined pulsed-field gel electrophoresis (PFGE) and Southern hybridization for detection of genes encoding known virulence factors, ribosomal RNA gene spacer restriction fragment length polymorphism types (RSTs), ospC group determination, and sequencing of the variable dbpA and ospC genes. We show that two strains isolated from the same tick and both originally named N40 are in fact very distinct. Furthermore, we failed to detect bbk32, which encodes a fibronectin-binding adhesin, in one "N40" strain. Thus, two distinct strains that show different plasmid profiles, as determined by PFGE and PCR, were isolated from the same tick and vary in their ospC and dbpA sequences. However, both belong to group RST3B.