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Clinical and Diagnostic Laboratory Immunology, March 2001, p. 376-384, Vol. 8, No. 2
Infectious
Diseases
Received 20 June 2000/Returned for modification 20 September
2000/Accepted 11 December 2000
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.
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.
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
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
Immunology1 and DNA
Laboratory,2 Institut Central des
Hôpitaux Valaisans, CH-1950 Sion, Switzerland
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C until use.
TABLE 1.
B. burgdorferi sensu lato isolates
evaluated in this studya
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 |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
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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).
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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).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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* 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.
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Discussion
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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.
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