Microbial Pathogenesis Unit, Catholic
University of Louvain Medical School, International Institute of
Cellular and Molecular Pathology, B-1200 Brussels, Belgium
Received 30 April 1997/Returned for modification 12 June
1997/Accepted 19 September 1997
Species-specific sequences were shown to be carried by plasmids of
the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids
of B. burgdorferi sensu stricto, Borrelia
garinii, and Borrelia afzelii, respectively, were
then built and used to isolate species-specific sequences. After
sequencing of the cloned inserts, three sets of primers were designed.
They were shown to determine species-specific PCR amplification
products. The sensitivities of the PCR assay with these primers were
100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The
usefulness of these primers for the identification of species in
biological samples (tick, serum, and cerebrospinal fluid samples) was
ascertained.
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TEXT |
Borrelia burgdorferi
sensu lato spirochetes, which cause Lyme disease, are transmitted to a
wide range of vertebrates by the bite of infected ticks of the genus
Ixodes. Genetic and immunological studies (4, 7, 11,
19) have led to the delineation of three distinct species among
pathogenic B. burgdorferi sensu lato isolates: B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. Recently, Borrelia japonica and
Borrelia andersonii were recognized as two novel species of
B. burgdorferi sensu lato (16, 20, 25). Up to
now, these two species were not found in Lyme disease patients. The
identification of the different species of B. burgdorferi
sensu lato might be clinically relevant, since they seem to cause
different complications (2, 3) by distinct pathogenic
mechanisms (13, 14).
B. burgdorferi sensu lato strains have a small linear
chromosome of approximately 950 kb. In addition, they harbor several circular and linear plasmids. The contribution of plasmids, considered minichromosomes, to the genetic makeup and variability of
Borrelia strains has long been recognized (5, 8, 31,
32). In view of the fact that different clinical outcomes depend
on the species of the infecting spirochete, the isolation of
species-specific sequences might help provide an understanding of the
distinct pathogenic mechanisms. We recently isolated a species-specific sequence of B. burgdorferi sensu stricto repeated on several
plasmids of this species (21).
Among the methods able to distinguish the B. burgdorferi
sensu lato species, PCRs have the advantage of not requiring previous cultivation of the organisms. In the present study, we extended the
isolation of phylogenetically significant plasmid target sequences to
the three pathogenic B. burgdorferi sensu lato genospecies. These sequences were exploited for the development of PCR tests. The
chosen primers were tested for their ability to amplify DNA from
cultured bacteria, biological fluids from patients, and infected ticks
in order to facilitate the study of risk and clinical outcome of Lyme
disease in areas inhabited by ticks.
Isolation of species-specific B. burgdorferi sensu lato
plasmid sequences.
The Borrelia strains used in this
study, listed in Table 1, were provided
by I. Saint Girons (Institut Pasteur, Paris, France). Their isolation
has been described previously (4, 6). We previously showed
in each species plasmids with distinct pulsed-field gel electrophoretic
profiles, allowing us to identify for each species a plasmid that was
present in all tested strains of a given species and that had no
prevalent counterpart with an equivalent molecular weight in strains
belonging to the other two species (21). These plasmids were
chosen as possible carriers of species-specific sequences: a 16-kb
plasmid of B. burgdorferi sensu stricto IP1, a 33-kb plasmid
of B. garinii N34, and a 25-kb plasmid of B. afzelii UO1. They were excised from the gel, radiolabeled by
random priming, and hybridized onto a dot blot with DNAs of different
strains of each species (28). As indicated in Fig.
1, the 16-kb plasmid of the B. burgdorferi sensu stricto strain hybridized strongly with DNA of
this species and poorly with the DNAs of B. garinii and
B. afzelii. The 33-kb plasmid of the B. garinii
strain hybridized strongly with the DNAs of the other strains of this
species and moderately with the DNAs of B. burgdorferi sensu
stricto B31 and B. afzelii VS461 and UMO1. It hybridized
poorly with the DNAs of other strains of B. burgdorferi
sensu stricto and B. afzelii species. The 25-kb plasmid of
the B. afzelii strain hybridized strongly with the DNAs of
the other strains of B. afzelii but also hybridized with the
DNA of B. garinii 20047. It hybridized poorly with DNAs of
other strains of B. burgdorferi sensu stricto and B. garinii. It seemed that these three plasmids contained largely
species-specific sequences.
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FIG. 1.
Hybridization of the 16-kb plasmid of B. burgdorferi sensu stricto, the 33-kb plasmid of B. garinii, and the 25-kb plasmid of B. afzelii with DNAs
of the three species of B. burgdorferi sensu lato involved
in Lyme disease. DNAs (100 ng) of the B31 (dot 1), IP1 (dot 2), IP2
(dot 3), IP3 (dot 4), and IRS (dot 5) strains of B. burgdorferi sensu stricto, the 20047 (dot 6), N34 (dot 7), G25
(dot 8), and P/Bi (dot 9) strains of B. garinii, and the
VS461 (dot 10), UO1 (dot 11), UMO1 (dot 12), and Iper3 (dot 13) strains
of B. afzelii were denatured and spotted onto a nylon
membrane. The membrane was incubated in the presence of the 16-kb
radiolabeled plasmid of B. burgdorferi sensu stricto IP1
(A), the 33-kb radiolabeled plasmid of B. garinii N34 (B),
or the 25-kb radiolabeled plasmid of B. afzelii UO1 (C). The
membrane was autoradiographed.
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For isolation of those specific sequences, the chosen plasmids, excised
from the agarose gel, cleaved with Sau3A1, and separately ligated into the BamHI site of the pBluescript
SK+ vector. These recombinant plasmids were introduced in
Escherichia coli DH5
F'. Recombinant colonies were
hybridized in parallel with total cleaved radiolabeled DNA of B. burgdorferi sensu stricto IP1, B. garinii N34, and
B. afzelii UO1 (28). Three species-specific recombinants were further studied. Plasmid pMC52, carrying cloned sequences of the 16-kb plasmid of a B. burgdorferi
sensu stricto strain, hybridized in a Southern blot analysis to several
plasmids of strains belonging to the same species (21).
Plasmids pMC159 and pMC18, carrying sequences of the 33-kb plasmid of
B. garinii N34 and of the 25-kb plasmid of B. afzelii UO1, respectively, hybridized exclusively to the
equivalent plasmid of strains belonging to their respective species
(data not shown).
Both strands of the spirochetal inserts were sequenced (29).
The insert of plasmid pMC52, specific for B. burgdorferi
sensu stricto, contained 1,271 bp. The inserts of plasmid pMC159,
specific for B. garinii, and plasmid pMC18, specific for
B. afzelii, contained 254 and 347 bp, respectively. These
sequences, analyzed by the DNA Strider (17) and oligo4-s
(National Biosciences, Inc., Plymouth, Minn.) programs, were analyzed
in a database search with the BLAST program for homology with
nucleotide and amino acid sequences (1). They did not show
homology with any other sequences (BLAST [release, June 1997]).
Selection of primers and specificity of the PCR amplification
products.
To select PCR primers suitable for the differentiation
of the three species of B. burgdorferi sensu lato involved
in Lyme disease, we have chosen a set of primers in each specific
sequence derived from each recombinant plasmid (pMC52, pMC159, and
pMC18). The nucleotide sequences of the primers and their target
species are presented in Table 2.
All primers were tested for their ability to amplify DNA from 10 B. burgdorferi sensu lato isolates and 3 relapsing fever
agents.
For PCR, the reaction mixture (50 µl) contained 1.5 mM
MgCl2, 175 µM (each) deoxyribonucleotide triphosphates
(dATP, dCTP, dTTP, and dGTP), 0.1% Triton X-100, 50 mM KCl, 10 mM
Tris-HCl (pH 8.0), 2.5 U of Thermus brockianus DNA
polymerase (Dynazyme; Techgen), 50 pmol of each primer, and 10 ng of
spirochetal DNA (or volumes of biological samples, as described below).
Samples were overlaid with 50 µl of mineral oil (Sigma) and amplified for 35 cycles (with the c/c' and MC16 primers), 30 cycles (with MC33
primers), or 40 cycles (with MC25 primers) in a thermocycler (New
Brunswick Scientific, Benelux b.v.) under the following conditions: 1 min at 96°C; 1 min at 54°C (with the c/c' and MC16 primers), 60°C (with MC33 primers), or 48°C (with MC25 primers); and 1 min at
72°C. The last cycle was terminated by elongation for 10 min at
72°C. The PCR amplification products were resolved by electrophoresis in a 1.5% agarose gel containing 0.5 µg of ethidium bromide per ml
and were visualized under UV light. Possible inhibition of DNA
polymerase, yielding a false-negative result for any given sample, was
checked by adding 10 ng of DNA of B. burgdorferi sensu lato
to the reaction mixture.
PCR with the MC16 primers amplified an expected 395-bp fragment from
the DNA of B. burgdorferi sensu stricto B31, IP1, and IRS
but not from the DNA of B. garinii and B. afzelii
isolates (Fig. 2A). Fragments of 236 bp
from B. garinii 20047, N34, and G25 were amplified in the
PCR assay with the MC33 primers but not from the strains of B. burgdorferi sensu stricto or B. afzelii (Fig. 2B).
Finally, the MC25 primers amplified an expected 125-bp fragment from
the DNA of B. afzelii VS461, UO1, and Iper3 but not from the
DNA of B. burgdorferi sensu stricto or B. garinii (Fig. 2C). The MC16, MC33, and MC25 primers did not amplify the DNA of
B. japonica Cow611C, B. hermsii, B. parkeri, or B. turicatae. No signal was observed when
template DNA was omitted from the amplification reaction mixture (faint
signals are nucleotides and oligonucleotides not used in the reaction).
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FIG. 2.
Specificity of the chosen primers for amplification of
B. burgdorferi sensu stricto, B. garinii, or
B. afzelii DNA. DNAs from B. burgdorferi sensu
stricto B31 (lanes 3), IP1 (lanes 4), and IRS (lanes 5), B. garinii 20047 (lanes 6), N34 (lanes 7), and G25 (lanes 8),
B. afzelii VS461 (lanes 9), UO1 (lanes 10), and Iper3 (lanes
11), B. japonica Cow611C (lanes 12), B. hermsii
(lanes 13), B. parkeri (lanes 14), and B. turicatae (lanes 15) were amplified with the MC16 (A), MC33 (B),
or MC25 (C) primers. The amplified products were separated on a 1.5%
agarose gel and revealed under UV light after the addition of ethidium
bromide (10 µg/ml). Lanes 2, control without DNA; lanes 1, DNA
molecular mass markers (587, 540, 504, 458, 434, 267, 234, 213, 192, 184, and 124 bp).
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The species-specific primers described here recognize different
plasmids. Since only strains with high passage numbers were analyzed,
these plasmids must be stably maintained and the primers should be able
to distinguish most if not all B. burgdorferi sensu lato
strains in nature belonging to the three pathogenic species. Other
species-specific primers were described on chromosomal (fla, orfX, rRNA) (15, 18, 24, 27) or
plasmid (ospA) genes (10, 22). These primers were
able to distinguish the species by their ability to recognize specific
sequences within a given gene shared by all spirochetes. A current
comparison of two different sets of primers for species identification
underlines the genetic heterogeneity of spirochetes in nature
(20a).
Sensitivity of the PCR amplification assay.
The detection
threshold of the PCR assay was determined by performing amplification
reactions with serially diluted samples of DNA from B. burgdorferi sensu stricto, B. garinii, or B. afzelii. Amplification reactions were performed with aliquots
containing amounts of DNA equivalent to given numbers of spirochetes
(from 107 to 1) and with the MC16 (Fig. 3A), MC33 (Fig.
3B), and MC25 (Fig. 3C) primers. It was
found that a template DNA input corresponding to 100 spirochetes was
sufficient for the detection of the amplified fragment of B. burgdorferi sensu stricto and that 1,000 spirochetes was
sufficient for the detection of the amplified fragments of B. garinii and B. afzelii.
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FIG. 3.
Sensitivity of the B. burgdorferi sensu
stricto, B. garinii, and B. afzelii
species-specific primers. DNA corresponding to 107 (lanes
2), 106 (lanes 3), 105 (lane 4),
104 (lanes 5), 103 (lanes 6), 102
(lanes 7), 10 (lanes 8), 1 (lanes 9), or 0 (lanes 10) spirochetes of
B. burgdorferi sensu stricto IP1 (A), B. garinii
N34 (B), or B. afzelii UO1 (C) was amplified with the MC16
(A), MC33 (B), or MC25 (C) primers. The amplified products were
electrophoretically separated in a 1.5% agarose gel and revealed under
UV light after the addition of ethidium bromide (10 µg/ml). Lanes 1, DNA molecular mass markers (587, 540, 504, 458, 434, 267, 234, 213, 192, 184, and 124 bp).
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The usefulness of these primers for the identification of species in
biological samples was ascertained. Biological samples came from
patients with Lyme disease diagnosed during the summer of 1996 at the
Belgian Reference Center for Borreliosis (G. Bigaignon, Infectious
Serology Laboratory, Saint-Luc Hospital, Brussels, Belgium) by positive
enzyme-linked immunosorbent assay (Diagast, Lille, France) and PCR
(Biocode, Sclessin, Belgium) test results. Sera and cerebrospinal
fluids (150 µl) were centrifuged (13,000 × g, 20 min), and the pellets were washed three times with phosphate-buffered saline and resuspended in 60 µl of H2O. After heat
denaturation (10 min at 100°C), 20 µl was added to the PCR mixture.
Ticks were collected in July 1996 from vegetation on the ground in the
forest near Matagne-la-Petite, Namur Province, Belgium. Ticks were kept
at 4°C in alcohol until use. Ticks were dried and incubated in 100 µl of TE (10 mM Tris-HCl [pH 7.8], 1 mM EDTA) containing 200 µg
of proteinase K (Boehringer Mannheim, Mannheim, Germany) per ml. After
overnight incubation at room temperature, the ticks were crushed with a
pipette tip, boiled for 10 min, and then placed on ice for 10 min. The samples were centrifuged at 13,000 × g for 10 min, and supernatants were collected and stored at 
20°C. A total of
5 µl of the supernatants was added to the PCR mixture.
Three serum samples, two cerebrospinal fluid samples, and four ticks
containing B. burgdorferi sensu lato DNA, as shown by amplification with the c/c' primers of Rosa et al. (27),
were analyzed further to identify the Borrelia species in
these samples (Table 3). Only one sample
from a human contained DNA from a unique species, whereas the four
others contained DNA originating from at least two different species of
B. burgdorferi sensu lato. PCR with the MC16 or the MC33
primer amplified DNA from three samples from patients, whereas PCR with
the MC25 primer, amplified DNA from four samples. Of the four ticks,
one contained the DNA of only one species and the three others
contained the DNAs of two or three B. burgdorferi sensu lato
species. The three primer sets were thus able to amplify borrelial DNA
in biological samples.
The sensitivities of the PCR amplification assays with the primers
designed in this work seemed adequate and at least equivalent to those
with the general c/c' primers (27). Indeed, the infecting species could be identified with the primers being studied in all the
samples, which were selected by the ability of the c/c' primers to
amplify DNA. The detection of B. burgdorferi sensu stricto
seemed more sensitive than the detection of the other two species (Fig.
3). This is probably due to the presence of the target sequence of the
corresponding primers (MC16) in several plasmids of this species
(21). Interestingly, homologous regions of DNA have been
found in different plasmids of Borrelia strains (9, 30,
31, 33). These shared sequences are not specific for any one of
the described B. burgdorferi sensu lato genospecies, however.
The PCR data indicated the usefulness of these primers with tick and
clinical samples (Table 3). This analysis revealed several cases of
infection with multiple Borrelia species, in agreement with
observations with ticks (12, 23, 26) and biological fluids
from patients with Lyme disease (10).
Nucleotide sequence accession numbers.
The accession
numbers for the nucleotide sequences (EMBL Nucleotide Sequence
Database) reported here are U12332 for B. burgdorferi sensu
stricto, U83998 for B. garinii, and U84145 for B. afzelii.
M. C. Misonne received a predoctoral grant from Institut
d'Encouragement de la Recherche Scientifique dans
l'Industrie et l'Agriculture. P. Hoet is research director of FNRS
(National Fund for Scientific Research, Brussels, Belgium).
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Zückert, W. R., and J. Meyer.
1996.
Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.
J. Bacteriol.
178:2287-2298[Abstract/Free Full Text].
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