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Journal of Clinical Microbiology, November 2000, p. 4026-4033, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular and Pathogenic Characterization of Borrelia
burgdorferi Sensu Lato Isolates from Spain
Raquel
Escudero,1
Marta
Barral,2
Azucena
Pérez,3
M. Mar
Vitutia,4
Ana L.
García-Pérez,2
Santos
Jiménez,3
Ricela
E.
Sellek,1 and
Pedro
Anda1,*
Servicio de Bacteriología1
and Servicio de Parasitología,4
Centro Nacional de Microbiología, Instituto de Salud
Carlos III, 28220 Majadahonda, Madrid, Servicio de
Investigación y Mejora Agraria (AZTI-SIMA), Departamento de
Agricultura, Gobierno Vasco, 48160 Derio,
Vizcaya,2 and Consejería de
Salud, Consumo y Bienestar Social del Gobierno de la Rioja, 26071 Logroño,3 Spain
Received 25 May 2000/Accepted 29 August 2000
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ABSTRACT |
Fifteen Borrelia burgdorferi sensu lato isolates from
questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including
the two human isolates, three B. burgdorferi sensu stricto,
two B. valaisiana, and one B. lusitaniae),
showing a diverse spectrum of B. burgdorferi sensu lato
species. B. garinii isolates were highly variable in terms
of pulsed-field gel electrophoresis pattern and OspA serotype, with
four of the seven serotypes described. One of the human isolates was
OspA serotype 5, the same found in four of seven tick isolates. The
second human isolate was OspA serotype 3, which was not present in
ticks from the same area. Seven B. garinii isolates were
able to disseminate through the skin of C3H/HeN mice and to cause
severe inflammation of joints. One of the two B. valaisiana
isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary
bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice
or to infect internal organs. In summary, there is substantial
diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme
disease is endemic.
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INTRODUCTION |
Lyme borreliosis (LB) is considered
the most prevalent tick-borne disease worldwide. In Europe, the
causative agent, Borrelia burgdorferi sensu lato, is diverse
and has been divided into several species or genomic groups, three of
which (B. burgdorferi sensu stricto [29]
[B. burgdorferi in this paper], B. garinii
[7], and B. afzelii [13])
are pathogenic for humans. The pathogenicity of B. lusitaniae (35), also present in Europe, remains to be elucitated, since it has been isolated only from Ixodes ricinus. B. valaisiana (61), isolated for the first time in
Switzerland (45), has been detected by PCR in skin lesions
of erythema migrans (EM) patients (52), and there is some
evidence of pathogenic potential in humans (53). There have
also been descriptions of genotypic and phenotypic similarities of
human European isolates to strain 25015 of B. bissettii
(47, 58), but this strain has been isolated only from ticks
and small mammals (50). In addition, Wang et al.
(62) have suggested that apart from the established
genospecies, there is another Borrelia genomic group with
culture-confirmed pathogenic potential for causing human LB.
Investigations into the geographical distribution of B. burgdorferi sensu lato in Europe have revealed that B. garinii is the most frequently cultured species, followed by
B. afzelii, B. burgdorferi, and B. valaisiana in that order (25, 54). B. valaisiana and B. lusitaniae have been isolated
from or detected in I. ricinus in a few countries
(25). A genospecies specificity has been proposed in
Eurasia, with rodents as the main host for B. afzelii
(19, 24, 26, 27, 38, 39), and birds as the main host for
B. garinii (33, 41), where a migration
restlessness-associated transient spirochetemia occurs (23).
However, there are some descriptions of the existence of such cycles
for B. garinii and B. valaisiana in
different Eurasian countries (19, 26, 33, 38). Other authors
argue against this, describing an even distribution of
Borrelia species in local ticks and rodents (51),
proposing a one-vector-one-reservoir system. In addition, B. garinii has been detected in small rodents in other studies
(28, 32), and all three genospecies were detected in larval
ticks feeding on birds (42).
In Spain, the first isolation of B. burgdorferi (strain
Esp1) from I. ricinus was reported in 1992 (17). Previously, Oteo Revuelta et al. (44) had
described spirochetes in the midgut of I. ricinus in a
different area from the one where the strain Esp1 was isolated.
Although LB has been reported in Spain since 1977 (60) and
several series of cases have been studied (2, 20, 55), it
was not until 1998 that the first isolation of B. garinii
from an EM lesion was described (43), confirming the role of
this strain as a human pathogen in Spain.
Since information about the prevalence of Borrelia
spirochetes in tick populations and about the different genospecies is essential for our understanding of the epidemiology, diagnosis, and
prevention of LB, we have conducted the first study involving the
molecular and pathogenic characterization of B. burgdorferi sensu lato isolates from ticks from different areas of
Spain known to harbor populations of I. ricinus
(8, 16), as well as from skin biopsy specimens from patients
with LB.
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MATERIALS AND METHODS |
Isolation of the spirochetes.
Questing I. ricinus ticks were collected by flagging at three regions in the
northern half of Spain (Basque Country, La Rioja, and
Castilla-León), in areas known to harbor dense populations of
I. ricinus (8, 16). The ticks were
disinfected by serial passages of 2 min in 2-propanol and 70% ethanol,
serially washed in phosphate-buffered saline and Barbour-Stoenner-Kelly
medium II (BSKII), and placed in fresh BSKII, where the specimens were broken up with two needles. The suspension was either filtered through
a syringe filter (µStar 0.45-µm-pore-size filter; Corning Inc.,
Corning, N.Y.) and added to a 5-ml culture tube containing 4.5 ml of
BSK supplemented with 6% rabbit serum (BSK-RS) (14) or
directly added without previous filtration to a BSK-RS tube supplemented with 0.4 µg of ciprofloxacin per ml and 40 µg of rifampin per ml (BSK-CR) (11). The second type of medium
used, to which unfiltered tick suspension was added, was composed of BSK-RS supplemented with 8 µg of kanamycin per ml and 230 µg of 5-fluorouracil per ml (BSK-K5) (30). Blind passages were
done always at 24 to 48 h of inoculation to avoid possible
toxicity of tick debris and to prevent any adverse effects of the
antibiotics on the growth of the spirochetes (6). Cultures
were examined by dark-field microscopy weekly for the first month and
twice a month for the second and third months after inoculation.
Spirochetes from positive cultures were frozen at 
70°C in BSK
supplemented with 10% dimethyl sulfoxide (Sigma-Aldrich
Química S.A., Alcobendas, Madrid, Spain). When possible, only
isolates from the first blind passage in antibiotic-free medium were
used throughout all the study. Skin biopsy specimens from patients with
EM were shipped to the laboratory in complete BSK medium. They were
processed as described previously (43).
In addition to the spirochetes isolated in this study, a total of 30 B. burgdorferi sensu lato strains were used for comparison as shown in Table 1.
Sequencing of the 16S rRNA gene and phylogenetic analysis.
Partial sequencing of the 16S rRNA gene was done by PCR with
primers constructed as described previously (3). The primers used were based on the published sequences of the bacterial 16S rRNA
(4, 18, 36, 56). For this study we used primer 16-1 (5'-CGAAGAGTTTGATCCTGGCTTAG-3') as the forward primer and
primer 16-3 (5'-GCGGCTGCTGGCACGTAATTAGC-3') as the
reverse primer. The amplified fragment was 519 bp long. Products
were purified using the Qiaquick PCR purification columns (Qiagen Inc.,
Chatsworth, Calif.) as specified by the manufacturer and sequenced
using the ABI PRISM Dye Terminator cycle-sequencing ready reaction kit
(Perkin-Elmer Co.) on an ABI 377 DNA sequencer.
The DNASTAR package (DNASTAR, Inc., Madison, Wis.) and the Clustal
method were used for sequence alignment and construction
of the
phylogenetic tree. 16S rRNA sequences from other
B. burgdorferi strains were used in the analysis to construct the
phylogenetic
tree. These included
B. burgdorferi 272 (GenBank accession number
X85189), 297 (
X85204), and Esp1
(
U28501);
B. garinii DK27 (
X85193), R-IP9 (
M89937), and
Rio1 (
U28500);
B. afzelii DK1 (
X85190) and DK2
(
X85188);
B. valaisiana M49
(
U78155) and VS116
T
(
X98232);
B. lusitaniae POTIB1 (
X98226) and
POTIB2
T (
X98228);
B. japonica H014
T
(
L40597) and IKA2 (
L40598);
B. andersonii 19857 (
L46688)
and
21038
T (
L46701); and
B. bissettii
DN127
T (
L40596). The 16S rRNA sequence from
Treponema
pallidum (
M88726)
was used as
well.
PFGE.
Pulsed-field gel electrophoresis (PFGE) was done as
described previously (10, 46). Two restriction endonucleases
were used: MluI and SmaI (MBI Fermentas, Amherst,
N.Y.). A contour-clamped homogeneous electric field pulsed-field
apparatus (CHEF-DRII; Bio-Rad Laboratories, Richmond, Calif.) was used
for all separations. For the separation of undigested genomic DNA, we
used a pulse time ramped from 1 to 6 s for 24 h at 200 V; for
the separation of digested DNA, pulse times were ramped from 3 to
40 s for 20 h. Lambda concatamers with a monomer size of 48.5 kbp (Boehringer, Mannheim, Germany) and a high-molecular-weight marker
(Gibco-BRL Life Technologies, Inc., Gaithersburg, Md.) were used as
standards. In describing MluI- and SmaI PFGE
profiles, we followed the definition and nomenclature previously
devised by Belfaiza et al. (10) and Picken et al
(46), with the inclusion of the pulsotypes MLv1 and SMv1 for
the pattern observed in the B. valaisiana isolates tested.
SDS-PAGE.
For protein analysis, whole-cell sonicates of
cultured spirochetes were prepared from Borrelia isolates
from ticks and EM patient biopsy specimens, as well as from B. burgdorferi (strain B31T and strain Esp1), B. garinii (strain PBiT), and B. afzelii
(strain VS461T). Proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
using Laemmli's discontinuous buffer system and 10%
polyacrylamide gels (34). The gels were stained with
Coomassie brilliant blue R-250 (Merk AG, Darmstadt, Germany). Protein
molecular weight standards (GIBCO BRL, Life Technologies, Inc.,
Gaithersburg, Md.) were used to determine the relative molecular mass
of major proteins by comparison. Monoclonal antibody 84C (15) was used to assess the expression of OspB.
Western blotting.
Sera from the mice inoculated with the
different isolates were tested for reactivity to the respective
homologous strain by Western blotting, following the previously
described protocol (2) with the minor modification of using
NuPAGE Bis-Tris System (Novex, San Diego, Calif.).
Partial sequencing of ospA genes.
Nested PCR was
carried out as described elsewhere (22). Each PCR
amplification product was purified using the Qiaquick PCR purification
columns (Qiagen Inc.) as specified by the manufacturer and sequenced as
above. The DNASTAR package and the Clustal method were used for
sequence alignment and construction of the phylogenetic tree. For
comparison of the sequences, a series of other strains were included in
this study: Phei (GenBank accession number X80251), TN (X80252), PWudII
(X80253), T25 (X80254), PBr (X80256), WABSou (X85441), TIs I (X85440),
DK29 (X63412), Gö2 (X60300), DK6 (L38657), PBi (S48323), and PLa
(X95355) (genospecies for these strains are indicated in Table 1).
Animal studies.
The C3H/He Lyme disease mouse model
(9) was used to assess the pathogenicity of the strains from
this study. A total of 20 mice were injected intradermally in the lower
back with 104 spirochetes of each isolate. The percentage
of mice that developed arthritis after injection was determined for
each isolate by monitoring signs of inflammation of the tibiotarsal
joints (TTJ) daily during the first week after injection, every other
day during the second week, and twice a week until the end of the
fourth week. The level of spirochete dissemination through the skin was
determined on day 15 by culturing in BSK-RS 3-mm-diameter ear punch
biopsy specimens (EPB) from two mice in each group that had shown signs
of inflammation (in the groups where no mice showed signs of
inflammation, the two mice were selected on the basis of the level of
antibodies to the homologous strain). On day 30, the selected mice were
euthanized in CO2 chambers and necropsy material from the
liver, kidneys, heart, brain, spleen, and bladder was collected and
cultured in BSK-RS. Citrated blood samples were also cultured for each
mouse to ensure that the isolates were tissue and not blood associated.
A score for pathogenicity was constructed as explained in Table
2.
Level of IL-6 in serum.
Quantification of interleukin-6
(IL-6) in the sera of the same mice selected for culture was done using
the InterTest-6X mouse IL-6 enzyme-linked immunosorbent assay ELISA kit
(Genzyme Diagnostics, Cambridge, Mass.) as specified by the manufacturer.
Nucleotide sequence accession numbers.
Partial sequences of
the ospA gene generated in this study were deposited in
GenBank under the following accession numbers: AF227323 (Rio1),
AF227319 (Rio2), AF227320 (Rio3), AF227321 (Rio4), AF227322 (Rio5),
AF227316 (PV4), AF227317 (PV5), AF227318 (PV6), and AF227315 (CL1).
Partial sequences of the 16S RNA generated in this study were deposited
in GenBank under the following accession numbers: AF245110 (Rio1),
AF245111 (Rio2), AF245097 (Rio3), AF245102 (Rio4), AF245108 (Rio5), AF245109 (Rio6), AF245103 (PV1), AF245105 (PV2), AF245106 (PV3),
AF245098 (PV4), AF245107 (PV5), AF245100 (PV6), AF245099 (PV7),
AF245101 (PV8), and AF245104 (CL1).
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RESULTS |
Four B. burgdorferi genospecies are present in
Spain.
A total of 13 isolates were obtained from pooled
I. ricinus. Eight isolates (PV1 to PV8) were derived
from ticks collected in the Basque Country, one isolate (CL1) was
derived from Castilla-León, and four isolates (Rio3 to Rio6) were
derived from La Rioja. We also obtained one more isolate from a skin
biopsy specimen of an EM patient in La Rioja (Rio2). The human strain
Rio1, previously isolated in our laboratory (43), was also
used in this study. For isolate PV8, only PCR-related tests were
possible, due to its slow growth.
Sequencing of a fragment at the 3' end of the 16S rRNA (519 bp long)
and subsequent phylogenetic analysis grouped our isolates
as follows
(Fig.
1): Rio1, Rio2, Rio3, Rio4, Rio5,
PV4, PV5, PV6,
and CL1 grouped with other
B. garinii
strains; PV1, PV2, and PV3
grouped with the
B. burgdorferi
cluster; PV7 and Rio6 formed a
branch group with the recently named
B. valaisiana species; and
PV8 grouped with the
B. lusitaniae strains.
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FIG. 1.
Phylogenetic tree of B. burgdorferi strains
based on the sequence of the 16S rRNA gene as described in the text.
The scale under the tree measures the distance between sequences.
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There is genotypic and phenotypic variability in the isolated
strains.
Figure 2A and Table
3 show the results of the restriction
fragment length polymorphism patterns of MluI-digested total
genomic DNA by PFGE. According to nomenclature previously described
(10, 46), 3 of the 14 isolated strains were B. burgdorferi (PV1 is MLb13, and PV2 and PV3 are MLb2). All of them
had the 135-kbp characteristic band. There were nine B. garinii strains (PV4, PV5, PV6, Rio3, Rio4, Rio5, and CL1 from
ticks and Rio1 and Rio2 from skin biopsy specimens). These had the two
B. garinii characteristic bands of 220 and 80 kbp, and all
corresponded to pattern MLg2. PV7 and Rio6, which grouped with B. valaisiana in the phylogenetic analysis, shared an atypical
pattern, with three fragments of 380, 320, and 90 kbp. This pattern was
named MLv1 in this study. There was a total correlation of these
results with the ones obtained by analyzing the 16S rRNA gene. Both
analyses yielded the same result with respect to the genospecies of
each isolate.
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FIG. 2.
PFGE separation of MluI-digested (A) and
SmaI-digested (B) genomic DNA. Lanes: L, DNA lambda
concatemers (48.5 to 485 kb); 1, strain TI-1; 2, Esp1; 3, PV2; 4, PV1;
5, PBi; 6, Rio4; 7, PV6; 8, PV4; 9, Rio1; 10, Rio2; 11, Rio3; 12, Rio5;
13, PV7; 14, Rio6; 15, PV3; 16, PV5; 17, CL1. The arrowhead indicates
the 135-kbp band characteristic of B. burgdorferi sensu
stricto, and the arrows indicate the 220- and 80-kbp bands
characteristic of B. garinii.
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A higher variability was found when
SmaI was used, since
some isolates that had the same
MluI pulsotype had different
SmaI
patterns. PV2 and PV3 (MLb2) showed different
restriction bands
(named types SMb2 and SMb1, respectively). PV1, which
was MLb13,
had the same pulsotype as PV3 (SMb1). For the nine
B. garinii isolates, there were four different patterns (named SMg1
to SMg4).
The two strains that grouped with
B. valaisiana in
the phylogenetic
analysis had the same
SmaI pattern (named
SMv1). Figure
2B and
Table
3 show the results of the LRFP patterns of
the
SmaI-digested
total
genome.
When the total genome (chromosome and plasmids) of the isolates was
separated by PFGE, there was a variable number of plasmids,
ranging
between two and seven, per strain (Fig.
3, Table
3).
All isolates contained a
large plasmid in the 45- to 50-kbp range,
which was identified as the
linear
ospAB-containing plasmid (
12).
The size of
this plasmid varied, but the variation did not correlate
with the
different genospecies. The diffuse band of DNA immediately
below the
chromosome in same strains could correspond to a multimeric
form of a
small circular plasmid. The plasmid content of each
strain, expressed
as the number of bands seen in four size ranges
(60 to 40, 39 to 30, 29 to 20, and <20 kb), is shown in Table
3.
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FIG. 3.
PFGE separation of the total undigested genome of the
B. burgdorferi sensu lato isolates and reference strains.
Lanes: M, DNA molecular size markers of 8.3, 8.6, 10.1, 12.2, 15.0, 17.1, 19.4, 22.6, 24.8, 29.9, 33.5, 38.4, and 48.5 kb; 1, Rio4; 2, Rio3; 3, Rio5; 4, Rio2; 5, CL1; 6, Rio6; 7, PV7; 8, PV6; 9, PV4; 10, PV5; 11, Esp1; 12, PV2; 13, PV1; 14, PV3.
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The phenotypic variability of the isolates was demonstrated by SDS-PAGE
(Fig.
4). The protein profiles were
compared with
the profile of the reference strains, demonstrating a
correlation
with the results obtained in the genotypic analysis. All
the isolates
had a protein profile consistent with that for each
B. burgdorferi sensu lato species. All had protein bands of
various sizes ranging
from 13 to 97 kDa. They were homogeneous with
regard to the size
and level of expression of their
higher-molecular-mass proteins
(>41 kDa) but heterogeneous in their
lower-molecular-mass proteins
(<41 kDa). The sizes of OspA and OspB of
the
B. burgdorferi isolates
correlated with those previously
described by Baranton et al.
(
7). Seven of the
B. garinii isolates and the two
B. valaisiana isolates
expressed a protein with a molecular mass higher than
that of the OspA
protein, which was confirmed to be OspB (by reactivity
with monoclonal
antibody 84C [data not shown]). The level of expression
of a protein
in the appropriate size range for OspC (22 to 25
kDa) (
64)
varied highly among the genospecies (Fig.
4).
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FIG. 4.
SDS-PAGE and Coomassie blue staining of B. burgdorferi sensu lato isolates. In each panel, the protein
profiles of B31T (B. burgdorferi sensu stricto),
PBi (B. garinii), and VS461T (B. afzelii) are also given. Lanes M contain molecular mass markers of
the sizes shown. (A) Isolates from Basque Country. Lanes: 1, B31; 2, Esp1; 3, PV2; 4, PV1; 5, PV3; 6, PBi; 7, PV6; 8, PV4; 9, PV5; 10, VS461; 11, PV7. (B) Isolates from La Rioja. Lanes: 1, B31; 2, VS461; 3, Rio6; 4, PBi; 5, Rio4; 6, Rio2; 7, Rio3; 8, Rio5.
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Partial sequencing of the
ospA gene was performed, and a
phylogenetic tree was constructed using representative strains of
each
serotype described for
B. garinii (
63,
64) (Fig.
5).
Among the
B. garinii
isolates, PV4, PV5, PV6, Rio1, and Rio3 are
serotype 5, Rio4 and Rio5
are serotype 6, Rio2 is serotype 3,
and CL1 is serotype 8.
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FIG. 5.
Phylogenetic tree of B. burgdorferi strains
based on the sequence of the ospA gene as described in the
text. The scale under the tree measures the distance between sequences.
OspA serotypes of each strain are given in parentheses.
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B. burgdorferi isolates have low pathogenicity in C3H
mice.
Of the three B. burgdorferi isolates tested, PV2
was nonpathogenic (Table 3) for mice, PV1 did not induce inflammation
of the TTJ but was recovered from urinary bladder, and PV3 induced inflammation of the TTJ in only 5 of 20 mice and was considered to be
of low pathogenicity (Table 3). The sera of the mice inoculated with
PV1 and PV3 showed a reactivity in Western blots (with the respective
homologous strain) to the 41-kDa protein, OspA, and OspB (Fig.
6). The isolate that was recovered from
urinary bladder also induced secretion of IL-6 at a low level.
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FIG. 6.
Reactivity of sera from the B. burgdorferi
PV1, PV3, PV7, and Rio6 isolates by Western blotting to the respective
homologous strain. M, molecular mass markers of the sizes shown.
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B. garinii isolates are pathogenic for mice.
Eight
of nine B. garinii isolates disseminated through the
skin and induced inflammation of the TTJ (in 25% of mice for two strains, PV5 and Rio1). The rate of recovery from organs varied from
isolate to isolate. One isolate of human origin (Rio2) was recovered
from EPB and induced inflammation of the TTJ in 100% of mice and
secretion of IL-6 but was not recovered from internal organs,
suggesting low disseminating capabilities in this model; considering
this discrepancy, it was classified as having low pathogenicity. PV5
did not migrate through the skin and induced TTJ inflammation in only
25% of mice, was recovered only from the urinary bladder, and did not
induce IL-6 secretion; it was also classified as having low
pathogenicity. A third isolate, CL1, induced TTJ inflammation in 100%
of mice and secretion of IL-6 and was recovered from EPB but was
recovered only from the urinary bladder; it was therefore classified as
pathogenic. For the rest of the strains, a positive EPB, inflammation
of the TTJ in 100% of mice, secretion of IL-6, and recovery from at
least two organs were seen; they were also classified as pathogenic. All the B. garinii strains showed a strong antibody
reactivity to the homologous isolate by Western blotting (data not shown).
B. valaisiana is pathogenic for mice.
Of the two
B. valaisiana isolates analyzed, one (PV7) did not show any
sign of pathogenicity and the other (Rio6) was recovered from EPB,
induced TTJ inflammation in 25% of mice, and was recovered from the
urinary bladder and kidneys; it was classified as pathogenic. Both
isolates showed a high reactivity with the 41-kDa protein and OspA by
Western blotting to the homologous strain, and Rio6 was also reactive
with a band in the range of the 22-kDa protein (Fig. 6).
In summary, the pathogenicity for mice was higher among the
B. garinii isolates, one isolate of
B. valaisiana was
considered
pathogenic, and all the
B. burgdorferi isolates
showed milder
signs of pathogenicity. The isolates that were recovered
from
EPB were
B. garinii and
B. valaisiana
strains. Also, recovery
from the urinary bladder was considered of low
significance since,
even with no arthritogenicity or recovery from EPB,
some isolates
were found in this organ, suggesting that it was
preferentially
infected, with low pathogenic significance. The
secretion of IL-6
at low level (1+) did not always correlate with
inflammation of
TTJ in our system, but secretion at 2+ to 3+ level was
always
associated with positive
EPB.
| |
DISCUSSION |
Fifteen B. burgdorferi sensu lato isolates were
recovered from Spanish I. ricinus ticks and biopsy
specimens from EM patients. Of these, three were B. burgdorferi sensu stricto, nine were B. garinii, two
were B. valaisiana, and one was B. lusitaniae. These findings indicate greater genospecies
diversity of B. burgdorferi sensu lato in Spain than in
other parts of Europe. The only genospecies not present was B. afzelii, even though this species is the second most frequently
isolated throughout Europe (25, 54). Based on these results,
B. afzelii may be absent at the southwestern margin of
the continent. Accordingly, B. afzelii has not been detected
in patients in Spain (1), and there has been an absence of
descriptions of B. afzelii-related cutaneous manifestations in clinical series (2, 5, 20, 21). In contrast, B. lusitaniae is present in southern Europe and North Africa
(40, 65) but it is not frequent in eastern Europe
(49). Overlapping geographic areas in the Iberian peninsula
with highly diverse B. burgdorferi populations as well as
relapsing-fever borrelia (4) could create the necessary
conditions for genetic exchanges and for the origin of new genospecies.
The high intraspecies variability detected on the basis of all the
parameters studied is reflected in the behavior of the isolates in C3H
mice (Table 3). Seven of the nine B. garinii isolates (CL1,
Rio1, Rio3, Rio4, Rio5, PV4, and PV6) disseminated through the skin,
induced severe TTJ inflammation in 20 of 20 mice, and caused
disseminated infection in C3H mice. Organisms were recovered from a
battery of internal organs (Table 3). The two remaining B. garinii strains (Rio2 and PV5) showed low pathogenicity, even
though one of them was an isolate of human origin, which was the only
one that disseminated through the skin of C3H mice. None of the three
B. burgdorferi isolates were virulent to C3H mice (only
strain PV1 was recovered from urinary bladder, and strain PV3 induced
TTJ inflammation in 25% of mice). None of them were recovered from
EPB. Interestingly, one of the two B. valaisiana isolates
(Rio6) was able to disseminate through the skin and to induce severe
TTJ inflammation in 25% of mice and was recovered from the kidneys and
urinary bladder, suggesting that a tick-mouse cycle could maintain this
isolate in nature.
Several authors have suggested that B. afzelii is
preferentially or exclusively maintained in cycles involving small
rodents and ticks (19, 24, 26, 27, 38). B. burgdorferi has been largely associated with small rodents
(57). Associations for B. garinii appear to be
more heterogeneous: a cycle involving sea birds has been well
characterized for this species (41), and a mechanism of
transient spirochetemia associated with migrating birds has been
described (23). Several other studies have pointed out the
existence of such cycles for B. garinii and B. valaisiana in different Eurasian countries (19, 23, 26, 33,
38). However, other descriptions have found B. garinii associated with small rodents (28, 51). Whether
a bird-tick or rodent-tick cycle or perhaps both maintain local
variants of B. garinii and B. valaisiana in
nature remains to be elucidated, but, given the high frequency of
B. garinii isolates in the areas studied and the data from
the animal model, we have shown that at least some variants of B. garinii can infect mice and disseminate through the skin from day
7 after infection until at least day 90 (data not shown). Since
B. garinii is a very heterogeneous species in terms of
pulsotype (Fig. 2, Table 3), plasmid content (Fig. 3, Table 3) and OspA
serotype (Fig. 5, Table 3), some of these differences could account for
a distinct susceptibility of different hosts. Data about the
transmission of the human isolate Rio1 from syringe-infected C3H mice
to xenodiagnostic larval I. ricinus and the subsequent
transmission of the organisms to mice via a tick bite from the derived
nimphal ticks (M. M. Vitutia, unpublished data) support this
hypothesis. We can assume that other B. garinii isolates
that exhibited a full spectrum of pathogenicity could at least be
equally and efficiently maintained in a tick-mouse cycle.
We did not find an association between OspA serotype and pathogenicity
to mice. In fact, the only B. garinii strain that was not
recovered from EPB was OspA serotype 5 (PV5), in common with four
additional isolates that were cultured with this method (PV4, PV6,
Rio1, and Rio3). Consequently, different degrees of pathogenicity to
mice are found among serotype 5 B. garinii isolates. In
summary, in this study, isolates belonging to OspA serotypes 5, 6, and 8 were pathogenic to mice and a serotype 3 isolate had low
pathogenicity, suggesting that other factors seem to influence the
behavior of B. garinii in mice.
These differences in pathogenicity to C3H mice found in this work for
each isolate could be used, given the constraints of extrapolation to
humans, to hypothesize about the risk for humans of contracting
Lyme disease in a certain area. Given that the isolates represent a
highly variable population, they could form the basis for a variable
clinical spectrum in humans.
| |
ACKNOWLEDGMENTS |
Raquel Escudero participated in this study while supported by a
contrast from the DGICYT (Dirección General de
Investigación en Ciencia y Tecnología, Spanish Ministry
of Education and Culture) program of "Incorporación de Doctores
y Tecnólogos." Ricela E. Sellek was supported by a Beca de
Iniciación of the Instituto de Salud Carlos III (ref. 97/4181).
This work was supported by Instituto de Salud Carlos III grants
98/0026-01 and 98/0026-02.
We are grateful to Angel del Pozo for the photographic work. We
acknowledge the excellent technical work done by Isabel
Rodríguez and Cati Chaparro. We also thank Gerardo Dominguez
Peñafiel (zona de Salud de Soncillo, Burgos), Rufino
Álamo Sanz (Consejería de Salud, Juntas de
Castilla-León), and José Antonio Oteo (Servicio de Medicina
Interna, Hospital de La Rioja) for providing ticks and patient samples
for isolation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Servicio de
Bacteriología, Centro Nacional de
Microbiología-Instituto de Salud Carlos III,
28220-Majadahonda, Madrid, Spain. Phone: (34) 91 509 7901. Fax:
(34) 91 509 7966. E-mail: panda{at}isciii.es.
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Abstract
Introduction
