![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, March 2001, p. 376-384, Vol. 8, No. 2
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.2.376-384.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Specificities and Sensitivities of Four Monoclonal
Antibodies for Typing of Borrelia burgdorferi Sensu
Lato Isolates
A. G.
Bretz,1
K.
Ryffel,1
P.
Hutter,2
E.
Dayer,1 and
O.
Péter1,*
Infectious
Diseases
Immunology1 and DNA
Laboratory,2 Institut Central des
Hôpitaux Valaisans, CH-1950 Sion, Switzerland
Received 20 June 2000/Returned for modification 20 September
2000/Accepted 11 December 2000
 |
ABSTRACT |
Borrelia burgdorferi, the agent of Lyme
borreliosis, is genetically more heterogeneous than previously
thought. In Europe five genospecies have been described from
the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii,
B. lusitaniae, and B. valaisiana. In the United
States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East
Coast were differentiated. In Asia, B. japonica has been
identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of
four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies
by immunoblot and confirmed genospecies by restriction fragment length
polymorphism (RFLP) of rrf (5S)-rrl (23S)
intergenic spacer amplicon. Monoclonal antibody H3TS had 100%
sensitivity for 55 B. burgdorferi ss isolates but
showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100%
specificity and sensitivity for 45 B. afzelii isolates.
Monoclonal antibody D6 was 100% specific for
B. garinii but missed 1 of 64 isolates (98.5%
sensitivity). Monoclonal antibody A116k was 100% specific for
B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with
results of reactivity and confirmed efficacy of the phenotypic typing
of these antibodies. Some isolates showed atypical RFLP. Therefore,
both phenotypic and genotypic analyses are needed to characterize new
Borrelia isolates.
| |
INTRODUCTION |
Borrelia burgdorferi, the
agent of Lyme borreliosis, has been found to be genetically more
heterogeneous than previously thought. In Europe, five genospecies have
been described from the original B. burgdorferi, now
called B. burgdorferi sensu lato: B. burgdorferi sensu stricto, B. garinii, B. afzelii,
B. lusitaniae, and B. valaisiana (3, 9,
19, 32). Barbour et al. (4) and Wilske et al.
(35) first provided early evidence of the heterogeneity among European isolates of B. burgdorferi, whereas U.S.
isolates appeared to be a homogeneous group except for a few variants
observed in California (6, 25) and on the East Coast from
Ixodes dentatus (B. andersonii). In Asia,
some new species have been differentiated as B. japonica and B. turdae. B. garinii,
B. afzelii, and B. valaisiana also appear to
be endemic species (11; T. Masuzawa, Y. Imai, and Y. Yanagihara,
Abstr. VIII. Int. Conf. Lyme Borreliosis Other Tick-Borne Dis.,
1999, abstr. O5, p. 5).
The European borreliae apparently have distinct reservoir hosts.
Apodemus mice and Clethrionomys glareolus voles
appear to be the main reservoirs for B. afzelii
(14, 16). Similarly, birds of the genus Turdus
are reservoirs for B. garinii and B. valaisiana (15), and the red squirrel Sciurus
vulgaris might be a reservoir for B. burgdorferi
sensu stricto and B. afzelii (13).
Although not absolute, associations of particular clinical manifestations in humans with distinct species of B. burgdorferi sensu lato have been documented. Acrodermatitis
chronica atrophicans is clearly associated with infection due to
B. afzelii (9, 10). Patients with Lyme
arthritis are more often infected with B. burgdorferi
sensu stricto (18) or show higher serological reactions
with this particular Borrelia species, and patients with
neuroborreliosis are more frequently infected with B. garinii (8) or present serological reactions in
accordance with this association (1, 2, 24, 29). We have
previously reported serological evidence for a pathogenic potential of
B. valaisiana in humans (29). Sera from
three patients with neuroborreliosis and from one patient with Lyme
arthritis showed higher reactivity with this Borrelia species.
Genetic analysis based on 16S rDNA, restriction fragment length
polymorphism (RFLP), arbitrarily primed PCR, and other methods for
phylogenetic study of bacterial population, such as multilocus enzyme
electrophoresis, all confirmed the subdivision of B. burgdorferi sensu lato into different species worldwide.
The serotyping method developed by Wilske et al. (34) and
classification based on protein profiles provided similar data. Monoclonal antibodies specific to some of these species have been described (3, 9, 22), and a new monoclonal antibody to B. valaisiana has been produced in our laboratory. In
the present study, we evaluated the specificity and sensitivity of four
species-specific monoclonal antibodies based on the analysis of 210 isolates of B. burgdorferi sensu lato.
| |
MATERIALS AND METHODS |
Culture of Borrelia isolates.
All isolates
(Table
1)
were cultured in BSK II medium at 34°C, and spirochetes were
harvested during the late log phase by centrifugation at
10,000 × g for 10 min. The pellet was washed twice in
phosphate-buffered saline with 5 mM MgCl2 and finally resuspended in distilled water. Protein concentration was adjusted to 1 mg/ml. The preparation was frozen at 
20°C until use.
T. Balmelli, G. Baranton, A. G. Barbour, S. Bergström, A. van den Bogaard, W. Burgdorfer, S. J. Cutler, A. J. van Dam,
L. Gern, A. Gylfe, I. Heinzer, P. F. Humair, K.-J. Hwang, T. Masuzawa, S. Nuncio, D. Postic, V. Preac-Mursic, S. Rijpkema, V. Sambri, J. Schmidli, T. Schwan, J. Wilhelm, M. M. Wittenbrink, and
B. Wilske kindly provided us with various isolates.
Phenotypic typing of B. burgdorferi sensu
lato.
Electrophoresis and immunoblots were performed as previously
described (23). Briefly, a suspension of washed borreliae
(protein concentration, 1 mg/ml) was dissolved (1:1) in sample buffer
with 0.6% sodium dodecyl sulfate (final concentration) and 50 mM
dithiothreitol as a reducing agent. The samples were boiled for 5 min
before undergoing electrophoresis (constant voltage, 170 V) on a
polyacrylamide gel at 12.5% for the separating gel. Standards (Bio-Rad
low-range protein molecular weight standards) were used as a reference
for the calculation of relative molecular masses. After
electrophoresis, proteins were transferred by Western blot to
polyvinylidene difluoride (Immobilon; Millipore, Bedford, Mass.) membranes.
After transfer, the membrane was stained with Coomassie blue. The
membrane was then cut at the level of OspA and OspB as well as below
the 14.4-kDa marker, and these two pieces were destained in a bath of
pure methanol for a few seconds. They were saturated with 5% gelatin
in a Tris-NaCl buffer (pH 7.5) for 1 h at 37°C and washed three
times for 5 min each in a Tris-Tween 20 (0.05%) buffer containing
0.1% gelatin. The pieces containing OspA and OspB were incubated for 2 h at room temperature with monoclonal antibodies H3TS (Symbicon,
Stockholm, Sweden) or A116k (K. Ryffel, unpublished data) and I17.3
(kindly provided by G. Baranton) (9) diluted 1:500,
1:1,000 and 1:500,000, respectively, in the same buffer with 1%
gelatin. The piece below 14.4 kDa was incubated as described above with
monoclonal antibody D6 (22) diluted 1:100. After washing,
monoclonal antibodies fixed specifically on the antigens were
demonstrated by a second goat anti-mouse immunoglobulin for H3TS,
A116k, and I17.3 monoclonal antibodies or goat anti-mouse
immunoglobulin M (µ-chain specific) for D6 monoclonal antibody
conjugated to alkaline phosphatase, followed by three washes and the
addition of 5-bromo-4-chloro-3-indolyl p-toluidine phosphate
and p-nitroblue tetrazolium chloride substrate (Kirkegaard
and Perry Laboratories, Gaithersburg, Md.).
At least one isolate each of B. burgdorferi sensu
stricto, B. garinii, B. afzelii, and B. valaisiana were run in each blot as positive controls for
reactivity with the monoclonal antibodies.
Genotypic typing.
The method described by Postic
(25) was used for typing, using the restriction pattern of
amplicons in the rrf (5S)-rrl (23S) intergenic
spacer region.
In short, 50 µl of reaction mixture containing 5 µl of bacterial
thermolysate (95°C for 10 min) with 2 U of Extra-Pol II DNA polymerase (Eurobio, Les Ulis, France) and 200 µmole of
deoxynucleoside triphosphate mix were subjected to 40 cycles (94°C
for 1 min, 55°C for 1 min, 72°C for 1 min) in a Perkin-Elmer
GeneAmp PCR System 9600.
Several negative controls were included in each run as well as
reference strains. Amplified products were electrophoresed on 2%
agarose gels stained with ethidium bromide at 0.5 µg/ml.
A total of 210 amplicons were digested overnight at 37°C with
restriction endonucleases MseI (2.5 U/10 µl of PCR
product), and if needed (43 amplicons) with DraI (10 U/10
µl of PCR product) (Gibco-BRL, Life Technologies, Paisley, United
Kingdom). The digested products were electrophoresed on 16%
acrylamide-bisacrylamide (19:1) (Bio-Rad, Hercules, Calif.) for 3 h at 100 V.
To resolve particular RFLP, amplicons were purified using a Qiaquick
PCR purification kit (Qiagen, Hilden, Germany). DNA sequencing was
performed on a Li-Cor 4000 automated sequencer, using IRD800-labeled primers (Lincoln, Neb.) and Thermo-sequenase (Nycomed, Amersham, United Kingdom).
| |
RESULTS |
Protein profiles based on OspA and OspB allowed us to easily
recognize three of the five European groups. Two of these showed both
OspA and OspB with distinct electrophoretic mobility (Fig. 1). B. burgdorferi sensu
stricto had an OspA of 31 kDa and an OspB of 34 kDa, and B. afzelii had an OspA of 32 kDa and an OspB of 35 kDa. The other
groups presented a pattern with an OspA without an apparent OspB, also
with distinct electrophoretic mobility, B. garinii of
32.5 kDa and B. valaisiana of 33 to 33.5 kDa.
B. lusitaniae showed an OspA profile very similar to
that of B. valaisiana, which did not provide sufficient
characteristics to differentiate them. We used these distinct protein
patterns to predict the reactivity of the isolates with
species-specific monoclonal antibodies.
View larger version (93K):
[in this window]
[in a new window]
|
FIG. 1.
(Top) Protein profiles of B. burgdorferi
sensu lato isolates after polyacrylamide gel electrophoresis and
Western blot on polyvinylidene difluoride membrane stained with
Coomassie blue. Representative isolates of each Borrelia
genospecies are ordered according to their RFLP pattern. Sizes are
shown in kilodaltons. Lanes 1 to 3, B. burgdorferi
sensu stricto (VS215, VS619, and NE49). Lanes 4 to 6, B. bissettii (CA128, DN127, and 25015). Lane 7, B. andersonii (21123). Lanes 8 to 11, B. garinii
(VS102, VSDA, AS19s, and NT29). Lane 12, B. afzelii
(VS461). Lane 13, B. valaisiana (VS116). Lane 14, B. japonica (FiAE2). Lanes 15 and 16, B. lusitaniae (PotiB3 and IR345). (Bottom) Reactivity with monoclonal
antibodies. (+), weak reactivity.
|
|
H3TS was confirmed to react specifically with OspA of all B. burgdorferi sensu stricto isolates expressing an OspA of 31 kDa and an OspB of 34 kDa. All 55 isolates originally classified as B. burgdorferi sensu stricto were reactive with H3TS.
In addition, four B. bissettii evaluated in this study,
two isolates from Ixodes neotomae (CA128 and CA55), one from
Ixodes scapularis (25015), and one from Ixodes
pacificus (DN 127) were also reactive (Table 2). This last isolate
reacted very weakly. Isolate NE49, whose OspA and OspB profiles are
typical of B. burgdorferi sensu stricto, showed at most
very weak reactivity with H3TS. The 156 other isolates were not
reactive with this monoclonal antibody.
Among the 64 isolates classified as B. garinii with
respect to their protein profile (OspA of 32.5 kDa), only strain 935T isolated from a Korean Ixodes persulcatus did not react with
monoclonal antibody D6. We observed that a few isolates (NT29,
IP89, and A19S) presented a protein reactive with monoclonal
antibody D6 of lower molecular mass (<12 kDa) than the other
B. garinii isolates (Table 2). Monoclonal antibody D6
did not react with any of 151 isolates belonging to other species.
Similarly, only the 45 isolates of B. afzelii were all
specifically recognized by monoclonal antibody I17.3. These isolates
typically had an OspA of 32 kDa and an OspB of 35 kDa (Table 2). No
other of 170 isolates were reactive with this monoclonal antibody.
The specificity and sensitivity of monoclonal antibody A116k were
evaluated with 111 Borrelia isolates, including all the B. valaisiana isolates available (Table 2). This
monoclonal antibody appears to be specific but was not reactive with 4 of the 24 B. valaisiana isolates.
The four monoclonal antibodies tested did not show any reaction with
B. anserina, B. coriaceae, B. hermsii, B. parkeri, B. turicata, B. japonica, B. lusitaniae,
or B. andersonii.
Monoclonal antibodies D6, I17.3, and A116k showed 100% specificity,
whereas H3TS had a specificity of 92.5% (12 of 13 evaluated species)
or 98% with respect to all isolates examined (211 of 215). Sensitivity
of 100% was observed with monoclonal antibodies H3TS (55 of 55) and
I17.3 (45 of 45), 98.5% (63 of 64) with monoclonal antibody D6, and
83.5% (20 of 24) with monoclonal antibody A116k (Table
2).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Sensitivity and specificity of four monoclonal antibodies
to 210 B. burgdorferi sensu lato isolates and five
relapsing-fever Borrelia species
|
|
One isolate (H11) was received as a mixture of two Borrelia
species (B. burgdorferi sensu stricto and B. garinii). In the first passages, H11 was confirmed to be
B. burgdorferi sensu stricto, and after two to four
additional passages in our BSK II medium, a mixed population of
B. garinii and B. burgdorferi sensu
stricto was observed. In further passages, only the B. garinii population of spirochetes persisted. Similar results were
obtained from several aliquots of an original culture that we have
received. We could clearly observe the appearance of the mixed
populations with the Osp profiles on the Coomassie blue staining as
well as with the reactivity with monoclonal antibodies H3TS and D6.
Genotypic typing.
Confirmation of the phenotypic determination
was made at the genetic level. Amplicons generated by PCR in the
rrf (5S)-rrl (23S) intergenic spacer region had
sizes of about 250 bp (226 to 266 bp). The isolate IKA2 generated a
nonspecific fragment of about 700 bp.
Several restriction patterns were observed for B. burgdorferi sensu stricto, B. garinii, and
B. lusitaniae, including some hitherto undescribed
patterns (Fig. 2 and Table
3).
View larger version (69K):
[in this window]
[in a new window]
|
FIG. 2.
Msel restriction patterns of
rrf-rrl intergenic spacer amplicons of B. burgdorferi sensu lato. Representative isolates of each
Borrelia genospecies are ordered according to their RFLP
pattern. Lanes 1 to 3, B. burgdorferi sensu stricto
(VS219, VS619, and NE49). Lanes 4 to 6, B. bissettii
(CA128, DN127, and 25015). Lane 7, B. andersonii
(21123). Lanes 8 to 11: B. garinii (VS618, VSDA, AS19s,
and IP89). Lane 12, B. afzelii (VS18). Lane 13, B. valaisiana (AG1). Lane 14, B. japonica (FiAE2). Lanes 15 and 16, B. lusitaniae
(PotiB3 and IR345).
|
|
The majority of B. burgdorferi sensu stricto isolates
(37 of 55, 67%) showed the typical MseI pattern A, whereas
17 isolates (31%), including 13 isolates from our region (Valais,
Switzerland), revealed a pattern referred to as A1 (Table 3). The
isolates belonging to pattern A1 possessed a particular fragment of 40 bp instead of the 38-bp fragment. One isolate, NE49, presented a unique
pattern, with a fragment of 26 bp instead of 28 bp. After DraI digestion, 11 isolates with the A or A1 RFLP exhibited
identical patterns, but NE49 (A2) showed three fragments of 144, 80, and 29 bp instead of four fragments of 144, 53, 29, and 28 bp.
Among the 64 B. garinii isolates analyzed, 54 (84%)
had RFLP pattern B, identical to reference strain 20047 after digestion with MseI. Two isolates showed pattern C, and seven other
isolates had a particular pattern called B1, with a fragment of 98 bp
instead of the expected 95-bp fragment. In one isolate from the
Netherlands, A19S, a fragment of 93 to 94 bp was observed (not
sequenced). This pattern was called B2. DraI digestion of 12 amplicons with RFLP patterns B, B1, and B2 resulted in the same RFLP,
with fragments of 204 and 49 bp, respectively.
All 45 B. afzelii isolates analyzed showed pattern D
after digestion of amplicons with MseI, although the 20-bp
fragment was rarely observed in our gels. The 24 B. valaisiana amplicons (pattern F) consistentely showed the typical
fragment of 175 bp after MseI digestion, along with the
other fragments, though the 7-bp fragment was never detected.
B. andersonii isolates (pattern L) were also easily
identified by their 120-bp fragment, characteristic of this
Borrelia species, after MseI digestion. The
B. lusitaniae isolates presented two patterns, named G
and H. Since the expected 16-bp fragment was not visible on our gel,
pattern H was almost indistinguishable from pattern E described for
B. japonica. The four B. bissettii fell
within three different patterns named I, J, and K, with pattern J being
close to pattern A of B. burgdorferi sensu stricto.
All the isolates classified by phenotypic characteristics were
confirmed by genetic analysis. By RFLP analysis, we were also able to
show the presence of two Borrelia species (B. burgdorferi sensu stricto and B. garinii) in the
H11 isolate, as detected by phenotypic analysis as well.
| |
DISCUSSION |
Comparison of both phenotypic and genotypic analyses revealed an
excellent agreement for isolates belonging to B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana, each specifically
recognized by species-specific monoclonal antibodies. Monoclonal
antibody H3TS had an estimated sensitivity of 100% to B. burgdorferi sensu stricto, but was also reactive with some B. bissettii isolates. Monoclonal antibody I17.3
recognized all and only the B. afzelii isolates,
revealing both a sensitivity and a specificity of 100%. Monoclonal
antibody D6 was reactive to all B. garinii isolates
except one isolate from Korea. Specificity was 100%, and estimated
sensitivity was 98.5% (63 of 64).
The specificity of monoclonal antibody A116k was 100%, and the
sensitivity was about 83.5%. The OspA to which this monoclonal antibody is reactive appears to be quite heterogeneous in B. valaisiana isolates, showing different electrophoretic
mobilities, as reported previously (32). Based on both the
electrophoretic mobility of the OspA and OspB (22) and the
reactivity of monoclonal antibodies H3TS, I17.3, D6, and A116k, the
phenotypic characterization of new European Borrelia
isolates is efficient. At the regional level, we were able to define
the Borrelia species merely from the electrophoretic profile
of OspA and OspB. For example, of 50 local isolates, 48 were
classified correctly, and two isolates belonging to B. valaisiana would not have been differentiated from B. lusitaniae isolates. We realize that our observation still needs
confirmation by specific methods with monoclonal antibodies and further
genetic analysis.
The genetic analysis by RFLP with MseI, as described by
Postic et al. (25, 26), is an excellent and powerful
typing method for B. burgdorferi sensu lato isolates.
Isolate NE49 was readily identified as a new subgroup in B. burgdorferi sensu stricto. This observation was confirmed by
Postic et al. (27). All the other RFLP groups were
previously described by Postic et al. (25). However, about
10% of all isolates needed a second step of digestion with the enzyme
DraI. Some RFLP patterns were not easily resolved, above all
when a difference of ±2 bp was observed on one fragment (i.e., groups
A, A1, A2, and J or groups E and H). In order to interpret this, the
sequence of such amplicons will have to be determined. One additional
problem arose with short restriction fragments (<20 bp). These
fragments were usually not visible in our gels and thus were not
informative. Only full sequencing of the amplicons allowed us to
determine the original size of such small fragments.
Among all the isolates investigated, we noticed that different
B. burgdorferi sensu lato isolates have identical
names, such as M7, one isolated from Ixodes ricinus in the
Netherlands (B. valaisiana), and one isolated from
I. persulcatus in China (B. afzelii), or
IP3, isolated from human cerebrospinal fluid (CSF) in France
(B. burgdorferi sensu stricto) and one isolated from I. persulcatus in the Commonwealth of Independent
States (B. afzelii). One B. garinii
referred to in this study as isolate M50 (isolated from I. ricinus in the Netherlands) was typed as B. valaisiana in a previous report (32). Similarly,
isolate A76S was clearly identified as B. afzelii in
this study but was previously described as B. garinii
(31). We do not know whether these isolates were not
initially pure and if further passages in culture medium in the two
laboratories have selected two different isolates, or if these isolates
were incorrectly labeled. In our hands, these isolates were never found
to be a mixture of two Borrelia species.
Other genetic methods often used, such as DNA-DNA hybridization
(3), pulsed-field electrophoresis (5),
multilocus enzyme electrophoresis (7), arbitrarily primed
PCR (33), specific typing of 16S rDNA
(20), and species-specific hybridization (28), allow us to type B. burgdorferi
sensu lato isolates. All these methods have confirmed the
presence of these different species among B. burgdorferi sensu lato, with some particular geographical distribution (30). There is no doubt that the description
of these different Borrelia species allowed us to better
understand the complex natural history of Lyme borreliosis in Europe.
For example, different reservoir hosts were described for some
Borrelia species (13-15). Similarly, several
reports have suggested an association of particular clinical symptoms
in human with some Borrelia species (1, 2, 10, 24,
29). The typing of B. burgdorferi sensu lato
isolates is essential to clarify this specific point and consequently
to understand the physiopathology of each Borrelia species
(12, 17, 21).
Our results have shown that some B. burgdorferi sensu
lato isolates cannot be easily typed by genetic methods and need
cumbersome techniques. In this respect, monoclonal antibodies may
greatly help to type closely related isolates at one particular point of the genome. Therefore, phenotypic and genotypic methods
appear to be complementary. Phenotypic methods, particularly monoclonal antibodies, are helpful epidemiological tools that may be essential for
laboratories which lack facility in genetic methods.
| |
ACKNOWLEDGMENTS |
We are grateful to all the colleagues who provided us with
Borrelia isolates as well as with monoclonal antibodies.
This work was supported by the Institut Central des Hôpitaux
Valaisans and the Fonds National Suisse de la Recherche Scientifique (grant 32-52739.97).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Infectious
diseases and Immunology, Institut Central des Hôpitaux Valaisans,
CH-1950 Sion, Switzerland. Phone: 41 27 603 47 90. Fax: 41 27 603 48 93. E-mail: olivier.peter{at}ichv.vsnet.ch.
| |
REFERENCES |
| 1.
|
Anthonissen, F. M.,
M. De Kessel,
P. P. Hoet, and G. H. Bigaignon.
1994.
Evidence for the involvement of different genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium.
Res. Microbiol.
145:327-331[Medline].
|
| 2.
|
Assous, M. V.,
D. Postic,
G. Paul,
P. Nevot, and G. Baranton.
1993.
Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Borrelia strains used as antigens.
Eur. J. Clin. Microbiol. Infect. Dis.
12:261-268[CrossRef][Medline].
|
| 3.
|
Baranton, G.,
D. Postic,
I. Saint Girons,
P. Boerlin,
J. C. Piffaretti,
M. Assous, and P. A. Grimont.
1992.
Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.
Int. J. Syst. Bacteriol.
42:378-383[Abstract/Free Full Text].
|
| 4.
|
Barbour, A. G.,
R. A. Heiland, and T. R. Howe.
1985.
Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates.
J. Infect. Dis.
152:478-484[Medline].
|
| 5.
|
Belfaiza, J.,
D. Postic,
E. Bellenger,
G. Baranton, and I. S. Girons.
1993.
Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed- field gel electrophoresis.
J. Clin. Microbiol.
31:2873-2877[Abstract/Free Full Text]. (Erratum, 32:2040, 1994.)
|
| 6.
|
Bissett, M. L., and W. Hill.
1987.
Characterization of Borrelia burgdorferi strains isolated from Ixodes pacificus ticks in California.
J. Clin. Microbiol.
25:2296-2301[Abstract/Free Full Text].
|
| 7.
|
Boerlin, P.,
O. Péter,
A. G. Bretz,
D. Postic,
G. Baranton, and J. C. Piffaretti.
1992.
Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis.
Infect. Immun.
60:1677-1683[Abstract/Free Full Text].
|
| 8.
|
Busch, U.,
C. Hizo-Teufel,
R. Boehmer,
V. Fingerle,
H. Nitschko,
B. Wilske, and V. Preac-Mursic.
1996.
Three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. afzelii, and B. garinii) identified from cerebrospinal fluid isolates by pulsed-field gel electrophoresis and PCR.
J. Clin. Microbiol.
34:1072-1078[Abstract].
|
| 9.
|
Canica, M. M.,
F. Nato,
L. du Merie,
J. C. Mazie,
G. Baranton, and D. Postic.
1993.
Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.
Scand. J. Infect. Dis.
25:441-448[Medline].
|
| 10.
|
Dunand, V.,
A. G. Bretz,
A. Suard,
G. Praz,
E. Dayer, and O. Péter.
1998.
Acrodermatitis chronica atrophicans and serologic confirmation of infection due to Borrelia afzelii and/or Borrelia garinii by immunoblot.
Clin. Microbiol. Infect.
4:159-163[Medline].
|
| 11.
|
Fukunaga, M.,
A. Hamase,
K. Okada, and M. Nakao.
1996.
Borrelia tanukii sp. nov. and Borrelia turdae sp. nov. found from ixodid ticks in Japan: rapid species identification by 16S rRNA gene-targeted PCR analysis.
Microbiol. Immunol.
40:877-881[Medline].
|
| 12.
|
Garcia-Monco, J. C.,
B. Fernandez-Villar,
R. C. Rogers,
A. Szczepanski,
C. M. Wheeler, and J. L. Benach.
1992.
Borrelia burgdorferi and other related spirochetes bind to galactocerebroside.
Neurology
42:1341-1348[Abstract/Free Full Text].
|
| 13.
|
Humair, P. F., and L. Gern.
1998.
Relationship between Borrelia burgdorferi sensu lato species, red squirrels (Sciurus vulgaris) and Ixodes ricinus in enzootic areas in Switzerland.
Acta Trop.
69:213-227[CrossRef][Medline].
|
| 14.
|
Humair, P. F.,
O. Péter,
R. Wallich, and L. Gern.
1995.
Strain variation of Lyme disease spirochetes isolated from Ixodes ricinus ticks and rodents collected in two endemic areas in Switzerland.
J. Med. Entomol.
32:433-438[Medline].
|
| 15.
|
Humair, P. F.,
D. Postic,
R. Wallich, and L. Gern.
1998.
An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes.
Zentralbl. Bakteriol.
287:521-538[Medline].
|
| 16.
|
Humair, P. F.,
O. Rais, and L. Gern.
1999.
Transmission of Borrelia afzelii from Apodemus mice and Clethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwintering maintenance.
Parasitology
118:33-42.
|
| 17.
|
Isaacs, R.
1994.
Borrelia burgdorferi bind to epithelial cell proteoglycan.
J. Clin. Investig.
93:809-819.
|
| 18.
|
Jaulhac, B.,
I. Chary-Valckenaere,
J. Sibilia,
R. M. Javier,
Y. Piemont,
J. L. Kuntz,
H. Monteil, and J. Pourel.
1996.
Detection of Borrelia burgdorferi by DNA amplification in synovial tissue samples from patients with Lyme arthritis.
Arthritis Rheum.
39:736-745[Medline].
|
| 19.
|
Le Fleche, A.,
D. Postic,
K. Girardet,
O. Péter, and G. Baranton.
1997.
Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis.
Int. J. Syst. Bacteriol.
47:921-925[Abstract/Free Full Text].
|
| 20.
|
Marconi, R. T., and C. F. Garon.
1992.
Identification of a third genomic group of Borrelia burgdorferi through signature nucleotide analysis and 16S rRNA sequence determination.
J. Gen. Microbiol.
138:533-536[Medline].
|
| 21.
|
Parveen, N.,
D. Robbins, and J. M. Leong.
1999.
Strain variation in glycosaminoglycan recognition influences cell type-specific binding by Lyme disease spirochetes.
Infect. Immun.
67:1743-1749[Abstract/Free Full Text].
|
| 22.
|
Péter, O., and A. G. Bretz.
1992.
Polymorphism of outer surface proteins of Borrelia burgdorferi as a tool for classification.
Zentralbl. Bakteriol.
277:28-33[Medline].
|
| 23.
|
Péter, O.,
A. G. Bretz, and D. Bee.
1995.
Occurrence of different genospecies of Borrelia burgdorferi sensu lato in ixodid ticks of Valais, Switzerland.
Eur. J. Epidemiol.
11:463-467[CrossRef][Medline].
|
| 24.
|
Péter, O.,
A. G. Bretz,
D. Postic, and E. Dayer.
1997.
Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland.
Clin. Microbiol. Infect.
3:423-431[Medline].
|
| 25.
|
Postic, D.,
M. V. Assous,
P. A. Grimont, and G. Baranton.
1994.
Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons.
Int. J. Syst. Bacteriol.
44:743-752[Abstract/Free Full Text].
|
| 26.
|
Postic, D.,
N. M. Ras,
R. S. Lane,
M. Hendson, and G. Baranton.
1998.
Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127).
J. Clin. Microbiol.
36:3497-3504[Abstract/Free Full Text].
|
| 27.
|
Postic, D.,
N. M. Ras,
R. S. Lane,
P. Humair,
M. M. Wittenbrink, and G. Baranton.
1999.
Common ancestry of Borrelia burgdorferi sensu lato strains from North America and Europe.
J. Clin. Microbiol.
37:3010-3012[Abstract/Free Full Text].
|
| 28.
|
Rijpkema, S. G.,
M. J. Molkenboer,
L. M. Schouls,
F. Jongejan, and J. F. Schellekens.
1995.
Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes.
J. Clin. Microbiol.
33:3091-3095[Abstract].
|
| 29.
|
Ryffel, K.,
O. Péter,
B. Rutti,
A. Suard, and E. Dayer.
1999.
Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana in humans.
J. Clin. Microbiol.
37:4086-4092[Abstract/Free Full Text].
|
| 30.
|
Saint Girons, I.,
L. Gern,
J. S. Gray,
E. C. Guy,
E. Korenberg,
P. A. Nuttall,
S. G. Rijpkema,
A. Schonberg,
G. Stanek, and D. Postic.
1998.
Identification of Borrelia burgdorferi sensu lato species in Europe.
Zentralbl. Bakteriol.
287:190-195[Medline].
|
| 31.
|
van Dam, A. P.,
H. Kuiper,
K. Vos,
A. Widjojokusumo,
B. M. de Jongh,
L. Spanjaard,
A. C. Ramselaar,
M. D. Kramer, and J. Dankert.
1993.
Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis.
Clin. Infect. Dis.
17:708-717[Medline].
|
| 32.
|
Wang, G.,
A. P. van Dam,
A. Le Fleche,
D. Postic,
O. Péter,
G. Baranton,
R. de Boer,
L. Spanjaard, and J. Dankert.
1997.
Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19).
Int. J. Syst. Bacteriol.
47:926-932[Abstract/Free Full Text].
|
| 33.
|
Welsh, J.,
C. Pretzman,
D. Postic,
I. Saint Girons,
G. Baranton, and M. McClelland.
1992.
Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups.
Int. J. Syst. Bacteriol.
42:370-377[Abstract/Free Full Text].
|
| 34.
|
Wilske, B.,
V. Preac-Mursic,
U. B. Gobel,
B. Graf,
S. Jauris,
E. Soutschek,
E. Schwab, and G. Zumstein.
1993.
An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.
J. Clin. Microbiol.
31:340-350[Abstract/Free Full Text].
|
| 35.
|
Wilske, B.,
V. Preac-Mursic,
G. Schierz,
R. Kuhbeck,
A. G. Barbour, and M. Kramer.
1988.
Antigenic variability of Borrelia burgdorferi.
Ann. N.Y. Acad. Sci.
539:126-143[Abstract].
|
Clinical and Diagnostic Laboratory Immunology, March 2001, p. 376-384, Vol. 8, No. 2
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.2.376-384.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
