Borrelia burgdorferi Organism specific information

Introduction

Lyme borreliosis (LB) is the most frequent vector-borne disease in humans in the temperate zone of the Northern Hemisphere. Clinical symptoms include arthritis, skin manifestations, such as acrodermatis chronica atrophicans (ACA) or erythema migrans (EM), and neurological disorders, such as facial palsy. The zoonotic disease is caused by several species belonging to the LB group of spirochaetes within the genus Borrelia. LB group spirochaetes have complex life cycles that involve ixodid (hard) ticks as vectors and a large number of vertebrate species as reservoir hosts, in particular small mammals and several bird species. Relapsing fever (RF) spirochaetes constitute another major group within the genus Borrelia. Except for B. recurrentis, which is transmitted by body lice, RF spirochaetes are transmitted by argasid (soft) ticks. Relapsing fevers are occurring mainly in subtropical and tropical regions.

A novel MLST scheme has recently been developed for B. burgdorferi by Gabriele Margos and Klaus Kurtenbach (University of Bath, United Kingdom), in collaboration with researchers from the U.S. (Yale University, NYMC, Centers for Disease Control, University of Utah), U.K. (University of Oxford, Imperial College London, University of Bath) and France (Institut Pasteur). Unlike a previous MLSA scheme of LB group spirochaetes, this MLST scheme is based exclusively on chromosomal housekeeping genes. Due to primer design, this MLST scheme is currently being extended to MLSA comprising other species within the genus Borrelia. The MLSA scheme has the power to characterize the spirochaetes at all the different phylogenetic levels required for evolutionary, epidemiological and fine-scale population/landscape genetic studies. Importantly, this scheme can be applied to infected ticks without the need to culture the spirochaetes, which can be fastidious and selective.
This feature renders the MLSA scheme a highly useful tool in large-scale epidemiological studies of these vector-borne bacteria.

The development and validation of the scheme is funded mainly by The Wellcome Trust, London, U.K. (to K. Kurtenbach) and the NIH, U.S.A. (to D. Fish, Yale University, K. Kurtenbach, and I. Schwartz, NYMC). David M. Aanensen is funded through a Wellcome Trust grant to Brian Spratt, Imperial College London, U.K.

Margos, G., A.G. Gatewood, D.M. Aanensen, K. Hanincová, D. Terekhova,  S.A. Vollmer, M. Cornet, J. Piesman, M. Donaghy, A. Bormane, M.A. Hurn, E.J. Feil, D. Fish, S. Casjens, G.P. Wormser, I. Schwartz, and K. Kurtenbach. 2008. MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA., published online 24/06/08

Housekeeping genes and PCR primers

PCR conditions for MLST housekeeping genes.

HotstarTaq Mastermix (Qiagen, Germany), 25 pmol of each primer, forward and reverse, and 2.5 ml of template DNA (purified DNA of isolates or tick lysates) were used for the first set of amplification cycles (25 ml final reaction volume). For PCR on tick-derived material, the MgCl2 concentration was adjusted to 2.5 mM. Bioline Immomix Red, 50 pmol of each primer and 5 ml of product derived from the primary set of cycles were used for the second set of amplification cycles (50 ml final reaction volume). 

The PCR conditions for the housekeeping genes, except for recG, were as follows: for the first set of cycles, touchdown PCR was used with annealing temperatures starting from 55 oC and decreasing 1 oC each cycle. Specific conditions were 95 oC for 15 min, 94 oC for 30 sec, annealing temperature from 55 oC to 48 oC for 30 sec, and an extension step of 72 oC for 30 sec. An additional 20 cycles were run at 94 oC for 30 sec, annealing temperature of 48 oC, and extension at 72 oC for 30 sec. After a final extension step for 5 min at 72 oC, the samples were kept at 15 oC until further analysis. The conditions for the second set of 35 cycles were 95 oC for 7 min, 94 oC for 30 sec, 50 oC for 30 sec, 72 oC for 30 sec. After a final extension step for 5 min at 72 oC, the samples were kept at 15 oC.

For recG, the PCR conditons for the first set of cycles were 95 oC for 15 min, 94 oC for 30 sec, 55 oC for 30 sec, 72 oC for 30 sec, 30 cycles, and extension at 72 oC for 5 min. The conditions for the second set of cycles were identical, except for an initial denaturing step at 95 oC for seven minutes.