borelioza_usa-europa.pdf

DOI: 10.3201/eid1407.070880

Suggested citation for this article : Qiu W-G, Bruno JF, McCaig WD, Xu Y, Livey I, Schriefer

MM, et al. Wide distribution of a high-virulence Borrelia burgdorferi clone in Europe and North

America. Emerg Infect Dis. 2008 Jul; [Epub ahead of print]

Wide Distribution of a High-Virulence

Borrelia burgdorferi Clone

in Europe and North America

Wei-Gang Qiu,* John F. Bruno,† William D. McCaig,* Yun Xu,† Ian Livey,‡

Martin M. Schriefer,§ and Benjamin J. Luft†

*Hunter College of the City University of New York, New York, New York, USA; †Stony Brook University, Stony

Brook, New York, USA; ‡Baxter Vaccine AG, Orth/Donau, Austria; and §Centers for Disease Control and Prevention,

Fort Collins, Colorado, USA

The A and B clones of Borrelia burgdorferi sensu stricto, distinguished by outer surface protein C ( ospC )

gene sequences, are commonly associated with disseminated Lyme disease. To resolve phylogenetic

relationships among isolates, we sequenced 68 isolates from Europe and North America at 1

chromosomal locus (16S–23S ribosomal RNA spacer) and 3 plasmid loci ( ospC , dbpA , and BBD14). The

ospC -A clone appeared to be highly prevalent on both continents, and isolates of this clone were uniform

in DNA sequences, which suggests a recent trans-oceanic migration. The genetic homogeneity of ospC -A

isolates was confirmed by sequences at 6 additional chromosomal housekeeping loci ( gap, alr, glpA,

xylB, ackA, and tgt ). In contrast, the ospC -B group consists of genotypes distinct to each continent,

indicating geographic isolation. We conclude that the ospC -A clone has dispersed rapidly and widely in

the recent past. The spread of the ospC- A clone may have contributed, and likely continues to contribute,

to the rise of Lyme disease incidence.

Multilocus sequence typing (MLST) is the use of DNA sequences at multiple

housekeeping loci to characterize genetic variations of natural populations of a bacterial

pathogen ( 1 , 2 ). MLST studies showed that local populations of a bacterial species typically

consist of discrete clusters of multilocus sequence types called “clonal complexes,” rather than a

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multitude of randomly assorted genotypes ( 2 ). Remaining to be tested are how such factors as

natural selection, low recombination rate, and genetic drift due to geographic structuring

contribute to the formation and maintenance of these clonal complexes in natural bacterial

populations ( 3 , 4 ). Recently, a multilocus sequence analysis approach was proposed to

reconstruct phylogenetic histories of bacterial clonal complexes by using concatenated sequences

of housekeeping genes when within-loci and between-loci recombinations are infrequent ( 5 ).

Lyme disease is a multisystem infection, with inflammatory complications that

commonly affect the skin, joints, and central nervous system in humans ( 6 ). Its causative agent,

Borrelia burgdorferi , a spirochete that parasitizes vertebrates, is transmitted by hard-bodied ticks

throughout the temperate zones of the Northern Hemisphere ( 7 ). Although humans are accidental

hosts of B. burgdorferi, Lyme disease is the most common vector-borne disease in the United

States with >20,000 annual reported cases, 93% of which occurred in 10 northeastern, mid-

Atlantic, and north-central states ( 8 ). Small mammals such as white-footed mice ( Peromyscus

leucopus ) and eastern chipmunks ( Tamias striatus ) serve as the main reservoirs of B. burgdorferi

( 9 , 10 ). In Europe, B. burgdorferi is transmitted by Ixodes ricinus ticks ( 11 ) and is carried by a

large variety of hosts, including birds and small- to medium-sized mammals ( 12 ).

B. burgdorferi sensu stricto is the primary pathogen of Lyme disease in the United States

and is the only pathogenic genospecies that causes Lyme disease in both North America and

Europe. More than 12 distinct outer surface protein C ( ospC ) major sequence types coexist in

local B. burgdorferi sensu stricto populations in the northeastern United States ( 13 – 15 ).

Sequence variability at ospC is the highest among known genomic loci and is strongly linked to

variations at other genome-wide loci, with occasional recombinant genotypes caused by plasmid

exchanges ( 16 – 19 ).

B. burgdorferi sensu stricto intraspecific clonal complexes may differ in their host

specificity and degree of human pathogenecity. Different clonal complexes may prefer different

host species ( 9 ). A restriction fragment length polymorphism type of intergenic spacer (IGS)

sequence (corresponding to the ospC- A and -B groups) is associated with hematogenous

dissemination in patients with early stage Lyme disease ( 20 , 21 ). Four ospC clonal complexes (A,

B, I, and K groups) were found to be more likely than others to cause disseminated Lyme disease

( 22 ). Also, an association of ospC clonal types with invasive disease in humans has been found

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in other pathogenic genospecies such as B. afzelii and B. garinii ( 23 , 24 ). However, additional

ospC clonal types have been isolated in patients with invasive disease ( 14 ).

Previous molecular assays found a close relationship and overlapping genotypes between

the European and North American populations ( 25 – 27 ). These authors found greater genetic

diversity among American strains than European strains and proposed a North American origin

for this genospecies. Although these studies provided the first evidence for recent

intercontinental migrations, they left the phylogenetic relationships among clonal complexes

unresolved because of the use of either anonymous genome-wide markers (e.g., arbitrarily

primed PCR), genes with a high recombination rate (e.g., ospC ), or sequences at a single locus.

A phylogeographic approach with multiple molecular markers provides a more robust inference

on population history ( 28 ). Here we obtained a well-resolved phylogeny of B. burgdorferi sensu

stricto clonal complexes by using multilocus sequence typing at housekeeping loci as well as loci

under adaptive evolution. We found evidence of genetic endemism, recent migration events, and

recombinant genomic types. In fact, the highly pathogenic ospC- A clone seems to have spread

rapidly in recent years to infect a broad range of host species in 2 continents.

Materials and Methods

B. burgdorferi Isolates and DNA Isolation

The B. burgdorferi sensu stricto isolates were obtained from clinical and tick specimens

and cultures from animals in the United States and Europe and maintained as frozen stocks at –

70°C (Table 1). For in vitro propagation, a small amount of frozen culture was scraped from the

surface of each sample with a sterile inoculating loop and injected into complete Barbour-

Stoenner-Kelly II medium (Sigma-Aldrich Corp., St. Louis, MO, USA). Spirochetes were then

cultivated at 34°C. All cultures used in this study had undergone a maximum of 2 in vitro

passages after recovery from frozen stock. For isolation of genomic DNA, 10 mL of low-passage

log-phase bacteria was harvested by centrifugation at 10,000 rpm for 30 min at 4°C. The

bacterial pellet was washed twice with Tris-Cl buffer (10 mmol/L Tris [pH 7.5], 100 mmol/L

NaCl), and resuspended in 430 μL TES (10 mmol/L Tris [pH 7.5], 100 mmol/L NaCl, 10

mmol/L EDTA). Subsequently, 10 μL of freshly prepared lysozyme (50 mg/mL), 50 μL Sarkosyl

(10%), and 10 μL proteinase K (10 mg/mL) were then added, and the mixture was incubated at

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50°C overnight before RNase treatment. After incubation, DNA was extracted with

phenol/chloroform and chloroform, precipitated with ethanol, and finally resuspended in TE

buffer (1 mmol/L Tris [pH7.5], 1 mmol/L EDTA).

Genomic Markers, PCR Amplifications, and DNA Sequencing

PCR amplifications were attempted at 4 genomic loci for all isolates and at 6

chromosomal housekeeping loci for a genetically representative subset of isolates (Table 2). The

IGS locus was chosen for its phylogenetically informative polymorphisms ( 16 , 20 ). The IGS

locus and 6 housekeeping genes ( gap, alr, glpA, xylB, ackA, tgt ) were approximately evenly

distributed on the main chromosome based on the B31 genome ( 29 ). The 3 plasmidborne loci

were selected for their high sequence variability and for the absence of close paralogs based on a

genome comparison ( 17 , 19 ). IGS sequences were amplified by using a nested PCR procedure

( 30 ). Because of high sequence variability, dbpA sequences were amplified by using 2 alternative

forward primers. PCR amplification was performed in 50 μL containing 200 mmol/L of each

dNTP, 2.0 mmol/L MgSO 4 , 2.5 U of Platinum Taq DNA polymerase High Fidelity (Invitrogen,

Carlsbad, CA, USA), 0.5 μmol/L of each primer, and 100 ng of genomic DNA template.

Following denaturation at 94°C for 1 min, samples underwent 30 cycles of denaturation at 94°C

for 30 s, annealing at 55°C for 30s, initial extension at 68°C for 1.5 min, and a final extension

step at 68°C for 10 min. PCR products were purified by GFX chromatography (Amersham

Pharmacia Biotech, Inc., Piscataway, NJ, USA), resolved by agarose gel electrophoresis, and

visualized by ethidium bromide staining. Purified amplicons were sequenced by using standard

dideoxy terminator chemistry as outlined below with the forward and reverse PCR primers.

Absence of specific PCR products, indicating potential absence of particular genetic loci or

plasmids, was confirmed by follow-up amplifications of the flanking DNA segments.

Automated DNA sequencing of both strands of each fragment was performed by the

Stony Brook University Core DNA Sequencing Facility (Stony Brook, NY, USA) by using the

dye-terminator method with the same oligonucleotide primers used for PCR amplification or,

where required, appropriate internal primers. Sequences were inspected and assembled with the

aid of the Sequencher program (Gene Codes, Inc., Ann Arbor, MI, USA). DNA sequences were

analyzed by using the BLASTN program through GenBank at the National Center for

Biotechnology Information (www.ncbi.nlm.nih.gov). Nucleotide and protein sequence

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alignments were performed with MacVector version 6.5 (MacVector, Inc., Cary, NC, USA).

New sequences were deposited to GenBank under accession nos. EF537321–EF537573.

Phylogenetic Inference and Tests of Population Differentiation

The IGS sequences were used to resolve intraspecific phylogenetic relationships among

B. burgdorferi isolates ( 16 , 20 ). Two highly divergent tick isolates from Finland (SV1 and Ri5)

were used as outgroups for rooting the phylogenetic tree. IGS sequences were aligned by using

ClustalW ( 31 ). A Bayesian majority-rule consensus tree was estimated by using MrBayes

(version 2.1) ( 32 ) as described previously ( 19 ). Sequences at the 3 plasmid-borne protein–coding

loci were translated into protein sequences and aligned in a pairwise fashion with ClustalW ( 31 ).

Nucleotide alignments were obtained according to the protein alignments. Neighbor-joining trees

based on pairwise nucleotide sequence distances were inferred by using PHYLIP ( 33 ) and

plotted by using theAPE package of the R statistical package ( 34 ). Genetic differentiation among

geographic populations was tested by using the analysis of molecular variance (AMOVA)

method implemented in the software package Arlequin 3.1 ( 35 ). The 6 housekeeping genes were

used to infer the overall within- and between-genospecies phylogeny. Sequences of strains B31

and PBi ( B. garinii ) were downloaded from GenBank ( 29 , 36 ). Sequences of N40, JD1, DN127

( B. bissettii ), and PKo ( B. afzelii ) were from draft genomes (S. Casjens, pers. comm.). The 6

alignments were concatenated and tested for the presence of gene conversion by using

GENECONV with the “within-group fragments only” option ( 37 ). Two approaches, a Bayesian

method with codon site-specific evolutionary rates (using MrBayes) and the other maximum

likelihood method with 100 bootstrapped alignments (using DNAML in PHYLIP) ( 33 ), were

used for phylogenetic reconstruction based on concatenated sequences. Branch supports were

measured by the posterior probabilities in the Bayesian method and the bootstrap values in the

maximum likelihood method.

Results and Discussion

AMOVA Tests of Geographic Differentiation

We sequenced 68 isolates (including 30 from northeastern United States, 6 from the

midwestern United States, and 32 from Europe) at a single chromosomal locus (IGS) and 3

plasmid loci ( ospC , dbpA , and BBD14). Using AMOVA, we evaluated the genetic differentiation

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