Li-Lian Chao, et al.
Received: April 21, 2009; Revised: May 8, 2009;
Accepted: May 26, 2009
Chien-Ming Shih, Department of Parasitology and
Tropical Medicine, National Defense Medical Center,
No. 161, Sec. 6, Minquan East Road, Taipei 114,
Taiwan, Republic of China. Tel: +886-2-87923152; Fax:
+886-2-87921821; E-mail: firstname.lastname@example.org
J Med Sci 2009;29(5):249-255
Copyright © 2009 JMS
Genetic Analysis of the Flagellin Gene of Lyme Disease Spirochetes
(Borrelia burgdorferi) Isolated from Rodents in Taiwan
Li-Lian Chao and Chien-Ming Shih*
Department of Parasitology and Tropical Medicine, National Defense Medical Center,
Taipei, Taiwan, Republic of China
Background: The fl agellin gene of Borrelia burgdorferi is highly conserved, and genomic analysis among Borrelia iso-
lates using sequence similarity of a fl agellin gene has been proven useful for the species identifi cation and genomic typing
of Borrelia spirochetes isolated from various biological and geographical sources. Thus, we analyzed the fl agellin gene
of Lyme disease spirochetes for the fi rst time in Taiwan. Methods: The genetic identities of Taiwan isolates (TWKM1-7)
were determined by comparing their sequence similarities of the PCR-amplifi ed fl agellin genes with three major genospe-
cies of Lyme disease spirochetes. Their phylogenetic relationships were also analyzed by neighbour-joining and maxi-
mum parsimony methods. Results: The sequence similarity of Taiwan isolates revealed a highly homogeneous genotype,
ranging from 97.8% to 100%, within the genospecies of B. burgdorferi sensu stricto and was clearly distinguished from
other genospecies of Lyme disease spirochetes with a high sequence variability (>4.9%) of the fl agellin gene. In addition,
phylogenetic analysis based on the fl agellin gene also revealed a highly genetic divergence between Taiwan isolates and
other Borrelia strains that were isolated from different biological and geographical sources. Conclusions: Our results
provide the fi rst investigation on the genetic identity of the fl agellin gene of these Taiwan isolates and confi rm that these
Taiwan isolates are genetically related to the genospecies of B. burgdorferi sensu stricto.
Key words: lyme disease, Borrelia burgdorferi, Flagellin gene, Taiwan
Lyme disease (LD) is an emerging tick-borne spiro-
chetal infection1 that can cause multisystem human ill-
ness with varying degrees of clinical symptoms among
infected persons, ranging from relatively benign skin le-
sions to severe arthritic, neurologic, and cardiac manifes-
tations2,3. On the basis of immunoreactivity with Borrelia
burgdorferi-specific monoclonal antibodies, plasmid
profi les, and the clinical manifestations of patients, the
causative agents of Lyme disease can be classifi ed into
three major genospecies, i.e., B. burgdorferi sensu stric-
to, B. garinii, and B. afzelii (group VS461)4,5. Although
human cases of Lyme disease had been reported in Tai-
wan6-11 and Borrelia spirochetes have also been isolated
from rodents in the Taiwan area12, the genetic diversity
of spirochetes as well as the tick vectors responsible for
transmission in Taiwan need to be further identifi ed.
The heterogeneity of molecular and immunological
characteristics among isolates of B. burgdorferi from dif-
ferent geographical and biological origins has been dem-
onstrated previously13-17. Genomic analysis among Bor-
relia isolates by sequence similarity of a specifi c target
gene has been proven useful for the species identifi cation
and genomic typing of Borrelia isolates from various bi-
ological and geographical sources18-20. The fl agellin gene
of B. burgdorferi is highly conserved and located on a
linear chromosome of Lyme disease spirochetes21-24. Its
diversity is valuable for distinguishing genetic heteroge-
neity among different Borrelia isolates25-29. Thus, the ob-
jective of this study was to determine the genetic identity
of these Borrelia spirochetes isolated from rodent hosts
in Taiwan by analyzing the sequence similarity of their
PCR-amplifi ed fl agellin gene.
A total of twenty-six strains of Borrelia isolates be-
Genetic analysis of Borrelia burgdorferi in Taiwan
longing to the three major genospecies of B. burgdorferi
sensu lato and other Borrelia species were analyzed
in this study (Table 1). Of which, seventeen strains of
Borrelia spirochetes including seven Taiwan isolates
(TWKM1-7) were cultured and maintained in our labo-
ratory, as described previously12. Borrelia strains of B.
garinii (PBi), B. afzelii (PKo), B. andersonii (21123), B.
tanukii (OR1eR), B. turdi (Kt501), B. valaisiana (CKA4a
and CMN1b), and B. sinica (CMN3 and CMN1a) were
included as the recorded information described in the
Preparation of Spirochete DNA
Total genomic DNA from all Borrelia strains was ex-
tracted as described previously30. Briefl y, samples (3 ml)
of cultured spirochetes were grown to a density of about
2× 108 cells per ml of medium and were centrifuged for
10 min at 12,000× g to pellet the spirochetes. The pel-
lets were washed twice with PBS (pH 7.2) containing
5 mM MgCl2, resuspended in 150μ l of distilled water,
and boiled for 10 min. After centrifugation at 10,000×
g for 10 sec, the supernatant was collected and the DNA
concentrations were determined spectrophotometrically
by using a DNA calculator (GeneQuant II; Pharmacia
Biotech, Uppsala, Sweden).
PCR Amplifi cation of Spirochete DNA
DNA samples extracted from the Taiwan isolates and
other spirochetes representative of the three major geno-
species of B. burgdorferi sensu lato were used as tem-
plates to perform the PCR amplifi cation of the fl agellin
gene DNA. A fl agellin-specifi c primer set of 1F (forward)
5’-GCATTAACGCTGCTAATC-3’ and M-2F (reverse)
5’-TGCAGGCTGCATTCCAAG-3’ were synthesized by
a custom oligonucleotide synthesis service (Gibco BRL,
Taipei, Taiwan) and were used to amplify the flagellin
gene of Lyme disease spirochetes (B. burgdorferi), as
described previously31. All PCR reagents and TaqGold
DNA polymerase were obtained from the GeneAmp kit
and were used as recommended by the supplier (Applied
Biosystem, Taipei, Taiwan).
Briefl y, a total of 20-pmol of the appropriate primer
set and various amounts of template DNA were used in
each 50-μ l reaction mixture. PCR amplification was
performed with a Perkin-Elmer Cetus thermocycler
(GeneAmp system 9700) and with amplifi cation for 30
cycles of denaturation at 92oC for 30 s, annealing at 41oC
for 30 s, and extension at 72oC for 90 s. PCR-amplifi ed
DNA products were electrophoresed on 2% agarose gels
in Tris-Borate-EDTA (TBE) buffer and the electropho-
resed gel was visualized under UV light after staining
with ethidium bromide. To determine the molecular size,
a 1Kb plus DNA ladder (catalogue no. 10787-018, Gibco
BRL, Taipei, Taiwan) was used as the standard marker
Sequence Alignments and Phylogenetic Analysis
The nucleotide sequences of the fl agellin gene of the
Borrelia isolates used in this study were sequenced by a
dye-deoxy terminator reaction method using the bigdye
terminator-Taq cycle sequencing kit under an ABI Prism
377-96 DNA sequencer (Applied Biosystems Inc., Foster
city, CA, USA). The determined sequences were initially
aligned with the CLUSTAL W software32 and further
analyzed by the neighbour-joining and maximum par-
simony methods to estimate the phylogeny of the entire
alignment using MEGA 4.0 software package33. A simi-
larity matrix was also constructed using the DNASTAR
program (Lasergene 7.0). All phylogenetic trees were
constructed and performed with 1000 bootstrap replica-
Table 1. Genospecies and strains of Borrelia isolates
analyzed in this study
Origin of Borrelia strain
Borrelia burgdorferi sensu stricto
U. S. A.
U. S. A.
U. S. A.
U. S. A.
U. S. A.
U. S. A.
B. andersonii 21123
B. tanukii OR1eR
B. turdi Kt501
U. S. A.
*GenBank accession numbers (AF416433-416449) were submitted by this
Li-Lian Chao, et al.
tions to evaluate the reliability of the constructions, as
Nucleotide Sequence Accession Numbers
The nucleotide sequences of the PCR-amplifi ed fl agel-
lin genes determined in this study have been registered
and assigned the following GenBank accession num-
bers: strains B31 (AF416433), TWKM1 (AF416434),
TWKM2 (AF416435), TWKM3 (AF416436),
TWKM4 (AF416437), TWKM5 (AF416438), TWKM6
(AF416439), TWKM7 (AF416440), JD1 (AF416441),
K48 (AF416442), VS461 (AF416443), CT20004
(AF416444), CT27985 (AF416445), ECM-NY86
(AF416446), N40 (AF416447), TB (AF416448), and
VS219 (AF416449). For phylogenetic analysis, the
nucleotide sequences of the fl agellin genes of other Bor-
relia spirochetes were included for comparison and their
GenBank accession numbers were recorded as the infor-
mation described in the GenBank data (Table 1).
To clarify the genomic identity of these Taiwan iso-
lates, PCR amplification of the flagellin gene was per-
formed to generate the flagellin DNA of all the strains
of Taiwan isolates (TWKM1-7) and the other ten strains
of Borrelia isolates belonging to three major genospe-
cies of Lyme disease spirochetes. A DNA fragment of
approximately 400 bp was generated and observed on a
2% agarose gel (Fig. 1). These results demonstrate that
the fl agellin genes were highly conserved in all strains of
Borrelia isolates regardless of the origin of isolation and
the genospecies of Lyme disease spirochetes.
The sequence similarity of the fl agellin gene was ana-
lyzed to identify the homogeneity of these Taiwan iso-
lates in relation to the genospecies of B. burgdorferi spi-
rochetes. As shown in Table 2, the nucleotide sequences
of the fl agellin genes were highly homogeneous, ranging
from 97.8% to 100% among Taiwan isolates and the oth-
er eight Borrelia isolates (strains B31, JD1, N40, ECM-
NY86, CT27985, CT20004, TB, and VS219) within the
genospecies B. burgdorferi sensu stricto. However, the
homogeneity of fl agellin nucleotide sequences among the
strains of K48 (B. garinii) and VS461 (B. afzelii) showed
a homogeneity of only 93.8-95.5% and 92.2-94%, re-
spectively, in comparison to the genospecies B. burg-
dorferi sensu stricto (Table 2). Thus, all of these Taiwan
isolates are genetically affi liated with the genospecies B.
burgdorferi sensu stricto.
Phylogenetic analysis based on the sequence align-
ment of the fl agellin gene were also performed to con-
struct the genetic divergence among twenty-six strains
of Borrelia isolates investigated in this study. Bootstrap
analysis was used to analyze the repeatability of the clus-
tering of specimens represented in phylogenetic trees. All
Borrelia isolates from Taiwan constitute a monophyletic
group separated from other strains of Borrelia spirochetes
(i.e., B. garinii and B. afzelii) within the same clade of
B. burgdorferi sensu lato and can be distinguished from
Fig. 1 PCR analysis with a primer sets specifi c for the fl a-
gellin genes of B. burgdorferi sensu lato (Bbsl).
Lane B, isolate B31; lanes 1 to 7 represent the Tai-
wan isolates of TWKM1-7, respectively; lane J, iso-
late JD 1 of B. burgdorferi sensu stricto (Bbss); lane
K, isolate K48 of B. garinii; lane V, isolate VS461
of B. afzelii; lanes 8 to 13 represent the B. burgdor-
feri sensu stricto strains CT20004, CT27985, ECM-
NY86, N40, TB, and VS219, respectively; lanes M,
1Kb plus DNA ladder (Gibco BRL). The amplifi ca-
tion products were electrophoresed on 2% agarose
gels (Agarose-LE, USB, Cleveland, Ohio, U.S.A.)
and DNA fragments of approximately 400 bp were
visualised under UV light with ethidium bromide
Genetic analysis of Borrelia burgdorferi in Taiwan
other genospecies of Borrelia spirochetes derived from
different biological and geographical origins in both
neighbour-joining and maximum parsimony methods
(Fig. 2). Within the same clade, all of these Taiwan iso-
lates (TWKM1-7) are highly related within the genospe-
cies of B. burgdorferi sensu stricto.
Our report describes the first genomic characteriza-
tion and classifi cation of the fl agellin gene among Lyme
disease spirochetes (B. burgdorferi) isolated in Taiwan.
In previous investigations, the protein profiles of these
Taiwan isolates were consistent with the major protein
bands of other documented strains of Lyme disease spi-
rochetes and their antigenicity was also verifi ed by their
reactivities with MAbs specifi c for B. burgdorferi sensu
lato12. Although the heterogeneity among major protein
bands and the immunoreactivity with B. burgdorferi-
specifi c MAbs have been used for the genomic typing or
species identifi cation of B. burgdorferi spirochetes, the
validity of these methods for identifying genospecies was
not fully satisfi ed17,35. Thus, genomic analysis based on
the sequence similarity of fl agellin gene may provide a
reliable and useful method for genospecies identifi cation
of Borrelia spirochetes isolated from various animal res-
ervoirs and vector ticks of Taiwan.
Genetic analysis based on genospecies-specifi c PCR
primers has been recognized as a rapid and distinguish-
able assay for the species identifi cation of Borrelia spi-
rochetes isolated from various biological and geographi-
cal origins17-20,35. Indeed, genetic heterogeneity can be
further classifi ed among Borrelia isolates that have been
previously identifi ed as the same genospecies or atypi-
cal strains of spirochetes36. In our previous studies, the
genetic relationship of these Taiwan isolates was clarifi ed
as the same genospecies by their differential reactivi-
ties with genospecies-specifi c PCR primers based on the
OspA37 and OspC38 genes of B. burgdorferi sensu lato.
Results from the present study further clarify the genetic
identity of these Taiwan isolates by analyzing the fl agel-
lin gene. Moreover, all of these Taiwan isolates are ge-
netically classifi ed into the genotype within B. burgdor-
feri sensu stricto.
Table 2. Sequence similarity between fl agellin gene sequences from Taiwan isolates and isolates of other genospecies of
% Sequence similarity to
B31Twkm1 Twkm2 Twkm3 Twkm4 Twkm5 Twkm6 Twkm7 JD-1N40
CT27985 CT20004TB VS219K48 VS461
B31100.0100.0 98.2 100.099.2 100.0100.0100.0 100.0 100.099.5100.0100.0 100.0 100.095.594.0
Twkm-1 100.098.2100.0 99.2 100.0 100.0 100.0100.0100.099.5100.0 100.0 100.0100.095.594.0
Twkm-2100.098.298.598.2 98.2 98.298.2 98.297.898.298.298.2 98.293.892.2
Twkm-3100.0 99.2 100.0100.0100.0100.0 100.0 99.5100.0100.0100.0100.095.594.0
Twkm-4100.099.299.299.299.299.298.899.2 99.2 99.2 99.294.893.2
Twkm-5 100.0 100.0100.0 100.0100.0 99.5 100.0100.0 100.0100.0 95.594.0
Twkm-6100.0100.0100.0100.0 99.5100.0100.0 100.0100.0 95.594.0
Twkm-7 100.0100.0 100.099.5100.0100.0100.0 100.095.594.0
JD-1 100.0100.0 99.5100.0 100.0 100.0100.095.594.0
N40100.0 99.5100.0100.0 100.0100.095.594.0
＊Strains: B31, JD1, CT20004, CT27985, ECM-NY86, N40, TB, and VS219, B burgdorferi sensu stricto; K48, B garinii; VS461, B. afzelii.
Li-Lian Chao, et al.
The phylogenetic relationships among Borrelia iso-
lates can be constructed by analyzing the sequence
similarity of a specifi c target gene. Indeed, the sequence
analysis of the fl agellin gene among Borrelia isolates has
been proven useful in the analysis of the genetic related-
ness of Borrelia isolates derived from various geographi-
cal and biological sources20,29,39. In addition, nucleotide
sequence variation of a target gene may actually repre-
sent the genetic divergence between or within the geno-
species of Borrelia isolates40-42. In this study, phyloge-
netic analysis based on the fl agellin gene demonstrates
a high sequence homogeneity among Borrelia isolates
of Taiwan and within the same genospecies of B. burg-
dorferi sensu stricto (Fig. 2). However, a high sequence
heterogeneity was also observed between different geno-
species of Borrelia spirochetes. Further investigation on
the sequence analysis of the flagellin gene of Borrelia
spirochetes isolated from different localities of Taiwan
would help to clarify the genetic divergence of Borrelia
spirochetes in the Taiwan area.
In conclusion, our study provides the fi rst investiga-
tion on genetic identifi cation of the fl agellin gene among
Lyme disease spirochetes (B. burgdorferi) isolated in
Taiwan. On the basis of the sequence similarity of the fl a-
gellin gene, all of these Taiwan isolates (TWKM1-7) are
genetically related to the genospecies of B. burgdorferi
sensu stricto. Further application of this molecular tool
to identify the genetic variability of the flagellin gene
among Borrelia spirochetes isolated from patients, reser-
voir animals, and vector ticks may help to illustrate the
genetic divergence of Borrelia spirochetes in relation to
the epidemiological features of human Lyme borreliosis
This work was supported in part by a grant from the
Department of Defense (DOD98-34), Taipei, Taiwan,
Republic of China.
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