Identification of Borrelia burgdorferi ospC genotypes in host tissue and feeding ticks by terminal restriction fragment length polymorphisms

Abstract

We developed a high-throughput method based on terminal restriction fragment length polymorphisms (T-RFLP) to identify ospC genotypes from field-collected samples of Borrelia burgdorferi. We first validated the method by analyzing B. burgdorferi ospC previously identified by sequencing. We then analyzed and compared ospC genotypes detected from ear biopsy tissue from natural populations of the white-footed mouse, a major B. burgdorferi reservoir host species in the eastern United States, and larval ticks feeding on those individual mice. The T-RFLP method enabled us to distinguish all 17 ospC genotypes tested, as well as mixed samples containing more than one genotype. Analysis costs compare favorably to those of alternative ospC identification methods. The T-RFLP method will facilitate large-scale field studies to advance our understanding of genotype-specific transmission patterns.

Fig 1

Schematic diagram of ospC PCR product digestion of ospC genotype A. Filled triangles, fluor-labeled primers; open triangles, unlabeled primers; asterisks, restriction enzyme cut sites; numbers, lengths (bp) of labeled fragments.

Fig 2

Fragment analysis chromatograms. (Top) Mouse host tissue with genotypes A and G, with peaks at 163 (green), 401 and 407 (blue), and 557 (black) bp, respectively. (Bottom) Larval tick from the same mouse, with genotypes A and D, with peaks at 163, 407 and 455, and 557 and 560 bp. Fragments smaller than 100 bp were disregarded as primer peaks.

Fig 3

Cost comparison of ospC identification using sequencing, reverse line blotting, and T-RFLP. The per-sample costs for reverse line blotting are equal to those of sequencing at 1,700 samples and match those of T-RFLP at 5,500 samples.