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Journal of Clinical Microbiology, April 1998, p. 1015-1019, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Culture-Confirmed Reinfection of a Person with
Different Strains of Borrelia burgdorferi Sensu
Stricto
William T.
Golde,1,2,*
Barbara
Robinson-Dunn,3
Mary Grace
Stobierski,3
Daniel
Dykhuizen,1
Ing-Nang
Wang,1
Vernette
Carlson,4
Harlan
Stiefel,3
Susan
Shiflett,3 and
Grant
L.
Campbell2
State University of New York at Stony Brook,
Stony Brook, New York1;
Centers for
Disease Control and Prevention, Fort Collins,
Colorado2; and
Community Public Health
Agency, Michigan Department of Community Health,
Lansing,3 and
Daggett Medical Clinic,
Daggett,4 Michigan
Received 7 August 1997/Returned for modification 10 October
1997/Accepted 23 December 1997
 |
ABSTRACT |
In recent years, the utility of serum-based diagnostic testing for
Lyme disease has improved substantially; however, recovery by culture
of the bacterium from skin biopsies of suspected patients is still the
only definitive laboratory test. Reinfection of patients has been
assumed to occur but as yet has not been documented by serial isolates
from the same person. We present a case of culture-confirmed reinfection of a patient in Menominee County, Michigan. Borrelia burgdorferi was isolated from the skin punch biopsy specimens during each episode of erythema migrans (EM) and was subjected to
molecular strain typing, genetic analysis of two outer surface protein
genes, protein profile analysis, and serum antibody response testing.
Results show that these isolates are distinct strains of the bacterium
and that the two episodes of EM were caused by independent infections.
This report describes the documented, culture-confirmed reinfection of
a human by two different strains of B. burgdorferi.
| |
INTRODUCTION |
Lyme disease is a vector-borne
spirochetosis caused by Borrelia burgdorferi and transmitted
by ticks of the genus Ixodes (6, 21). B. burgdorferi has been isolated and shown to be the cause of endemic
disease in several areas of the United States. In Michigan, ecologic
studies have demonstrated that the tick vector of Lyme disease became
established in the 1980s in Menominee County in the Upper Peninsula
(23). Further studies have suggested that Menominee County
is the only area in the state where B. burgdorferi is
currently endemic (26). In conjunction with a statewide
active surveillance program for acute Lyme disease, culture-confirmed Lyme disease has been documented for eight Michigan residents, all of
whom had physician-observed erythema migrans (EM) lesions following
tick exposure in Menominee County (22).
Multiple distinct episodes of EM in the same patient have been well
described clinically (7, 10, 17, 19). To our knowledge,
however, no such cases have been documented by culture of the
etiological agent in each of two consecutive episodes of EM and
subsequent characterization of the two isolates, leaving open the
question of whether these cases were independent infections or
recrudescences of the initial infection.
The patient subject of the current report, a resident of Menominee
County, experienced two distinct episodes of EM separated by more than
2 years, in 1992 and 1994, and isolates of B. burgdorferi were obtained during each infection. This has provided the opportunity to determine whether the second episode was a result of persistence and
reactivation of the initial infection or a novel infection due to a new
exposure to infected ticks. Analysis of these two isolates demonstrated
numerous differences in protein profile, plasmid profile, and most
importantly, the alleles of outer surface protein A (OspA) and outer
surface protein C (OspC) genes of B. burgdorferi sensu
stricto. Serum samples taken during both episodes of infection
contained antibodies to B. burgdorferi.
The duration of immunity in humans to natural infection with B. burgdorferi has not been determined for patients that have been
treated and that are no longer infected. Such an analysis requires
separate, documented episodes of infection by isolation and
characterization of the spirochete. In laboratory experiments, natural
immunity in mice appears to wane at about 9 months after treatment and
clearance of infection. This immunity does not correlate with
anti-B. burgdorferi serum antibody titers (15).
This study begins to provide data on the immune status of Lyme disease
patients after treatment and resolution of infection and on their
susceptibility to new episodes of infection.
| |
MATERIALS AND METHODS |
Borrelia cultures.
Modified Barbour-Stoner-Kelly (BSK)
medium (2) (Sigma, St. Louis, Mo.) supplemented with 6%
rabbit serum (Sigma) was used for all borrelia cultures. It was
prepared as described previously, with the addition of rifampin (50 mg/liter), phosphomycin (100 mg/liter), and amphotericin B (2.5 mg/liter) (5, 13, 20). Biopsies were taken according to the
procedure described by Berger et al. (5). Upon receipt at
the laboratory, the punch biopsies were aseptically transferred to 3 ml
of fresh modified BSK medium. Cultures were standardly incubated at
30°C for 12 weeks and were examined on alternate days under
dark-field microscopy. Cultures which showed slender, highly motile,
helical spirochetes measuring approximately 25 µm in length were
tested by indirect immunofluorescence assay with monoclonal antibody
(MAb) H5332 (3) specific for the outer surface protein A
(OspA) lipoprotein of B. burgdorferi. The 1992 isolate was
designated U193, and the 1994 isolate was designated U14. Aliquots were
frozen in 50% glycerol in BSK-H medium and stored at 
70°C.
Serologic analyses.
In 1992, all serum samples were tested
for antibodies to B. burgdorferi by both immunoglobulin M
(IgM)- and IgG-specific enzyme immunoassays (EIA; Zeus Scientific,
Cranbury, N.J.) according to the manufacturer's instructions. In 1994, specimens were tested for IgM and IgG antibodies according to the
Centers for Disease Control and Prevention (CDC) and Association of
State and Territorial Public Health Laboratory Directors protocol
(1). This procedure recommends two-stage testing starting
with an EIA, which in our case involved testing a whole-cell sonicate
preparation of strain B31 at low passage. We also tested all serum
samples with the flagellin protein preparation obtained from CDC as the
antigen. All positive and equivocal samples were confirmed by Western
blot testing (MarDx Diagnostics, Carlsbad, Calif.) with a collection of
B. burgdorferi-specific mouse MAbs as a reference
(1).
Molecular analyses.
Isolates of B. burgdorferi
were cultured in BSK medium (Sigma) to a density of 108
spirochetes per ml. Lysates were prepared by pelleting 100 ml of the
bacterial culture and washing twice with phosphate-buffered saline
containing 5 mM MgCl2. Spirochetes were then lysed in a Dounce homogenizer and assayed for protein content. Lysates were diluted to 2 mg/ml and mixed with an equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer.
Samples were separated by SDS-10% PAGE under reducing conditions.
Immunoblot analysis was performed as reported previously (8), with the B31 strain of B. burgdorferi
included as a reference.
Plasmid analysis was performed as previously reported (9).
Samples of genomic DNA were prepared from spirochetes by pelleting 5 × 108 bacteria, washing the pellet with cold 10 mM
Tris-150 mM NaCl (Tris saline), and resuspending the bacteria in 0.5 ml Tris saline. An equal volume of 2% low-melt agarose (Bio-Rad,
Richmond, Calif.) in Tris saline was added, and plugs were cast in plug
molds in a volume of 0.1 ml. Agarose plugs were cooled at 4°C,
removed from the molds, and incubated in lysis buffer (10 mM Tris, 150 mM NaCl, 20 mM EDTA, 0.1% SDS) for 2 h with tumbling at room
temperature. Plugs were then washed in 0.5× Tris-borate-EDTA (TBE)
with two changes and stored in 0.5× TBE at 4°C.
Genomic DNA was separated by pulsed-field gel electrophoresis (PFGE) on
a CHEF Mapper (Bio-Rad) PFGE system in 0.5× TBE as described
previously (9). Gels were stained with ethidium bromide and
photographed under UV. Restriction fragment length polymorphism testing
was performed by the criteria of Belfaiza et al. (4) with
MluI.
Further differentiation of the two isolates was carried out by
PCR-single-strand confirmation polymorphism (PCR-SSCP) for
the OspA
gene by the method of Guttman et al. (
11). The PCR
amplification protocol is a combination of nested PCR and modified
"touchdown" PCR (
12). A volume of 0.5 µl of culture
was used
directly as the template for the nested PCR. Primers specific
for the central region of the OspA gene were amplified with a
cycling
profile of 1 min at 96°C for 1 cycle followed by 30 s
at 94°C,
30 s at 37°C, and 2 min at 72°C for 20 cycles in a PTC-100
thermocycler (MJ Research, Inc., Watertown, Mass.). For the touchdown
PCR, the protocol was 1 min at 96°C for one cycle followed by
30 s at 94°C, 30 s at 60°C, and 1 min at 72°C for 10 cycles.
The
next 10 cycles used an annealing temperature of 55°C, followed
by
50°C for 10 cycles. For the last five cycles, 45°C was used.
External primers spanned bp 4 through 695, and internal primers
were
targeted for bp 220 through 565. A final 345-bp fragment
internal to
the OspA gene was the predicted product after this
amplification
procedure.
For SSCP analysis, volumes of PCR product were normalized to 5 µl and
mixed with 0.4 µl of 1 M mercury hydroxide and 9.6 µl
of loading
buffer. After being heated at 96°C for 4 min, samples
were
immediately iced. Samples were run on a 20% polyacrylamide-TBE
nondenaturing gel (Novex, San Diego, Calif.) with a 1.5× TBE buffer
at
210 V for 16 h at a constant temperature of 4 to 8°C. Gels
were
stained with ethidium bromide and visualized with UV. This
assay
analyzes the electrophoretic mobility shift of single-stranded
DNA of
highly related genes and can detect a difference of as
little as a
single base.
| |
RESULTS |
Case history and isolation of B. burgdorferi.
The first
illness in the patient was previously reported (22).
Briefly, this 56-year-old male resident of Menominee County was seen by
his physician in August 1992 with a 5- to 7-day history of fever,
headache, myalgia, nausea, and stiff neck. Upon physical examination, a
14-cm-diameter lesion characteristic of EM was noted in the left
scapular region. He denied any knowledge of recent tick bites but 2 weeks previously had spent time outdoors in an area of southern
Menominee County in which B. burgdorferi-infected Ixodes scapularis ticks have been identified
(23). He had not traveled outside of Menominee County during
this time. A punch biopsy was obtained from the erythematous periphery
of the lesion (5) and placed into modified BSK medium. The
biopsy was positive for the presence of spirochetes after 11 days of
culture. Serum samples were obtained at the time of the skin biopsy, 1 month later, and then 3 months after diagnosis. Therapy with
doxycycline (100 mg twice a day for 3 weeks) was initiated after the
biopsy specimen was obtained. The patient reported the EM resolved
within 48 h of treatment, and other symptoms resolved within a
week.
The patient continued to be asymptomatic for Lyme disease until 11 October 1994 when he noted the appearance of another lesion
suggestive
of EM. He presented to his physician the following
day, and EM was
confirmed. The lesion measured approximately 5
cm in diameter and was
again located in the left infrascapular
area. He reported mild myalgia
and fatigue but was afebrile and
denied knowledge of a tick bite.
Generally, he reported feeling
much less ill than with his previous
episode of Lyme disease.
Once again a biopsy specimen was obtained from
the edge of the
EM lesion and submitted for culture of
B. burgdorferi to the Michigan
Department of Community Health
Laboratory. This culture of biopsy
material had detectable spirochetes
after 7 days of incubation.
In both cases, isolation by culture of
motile spirochetes occurred
within the expected range of time for
bacterial isolation of
B. burgdorferi. Serum samples were
obtained at the time of diagnosis
and again 39 days later. These
samples were tested for antibodies
to
B. burgdorferi by EIA
and immunoblotting studies. After the
skin punch biopsy specimen was
obtained, the patient was again
treated with doxycycline (100 mg twice
daily), this time for 4
weeks. Clinical recovery was rapid.
Protein analysis of patient isolates.
Spirochetes reacting by
indirect immunofluorescence assay to MAb H5332 specific for OspA, and
therefore nominally considered to be B. burgdorferi, were
isolated from skin biopsies taken from the EM lesions of the patient in
1992 and 1994. The protein profiles of these isolates were very similar
(Fig. 1); the majority of bands for the
two isolates were identical, with two notable exceptions. The isolate
from 1994 expressed bands corresponding to molecular masses of 23 and
66 kDa that were absent from the isolate from 1992. Both of the
isolates expressed a number of proteins in common with strain B31;
however, both isolates expressed an OspB protein that migrated slightly
faster than the OspB of B31.
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FIG. 1.
Protein profiles for lysates of the two patient isolates
and B. burgdorferi B31 separated by SDS-PAGE and stained
with Coomassie blue. Molecular weight reference standards are
indicated.
|
|
When lysates of these isolates were electrophoretically transferred to
nitrocellulose filters for Western blotting, distinct
patterns of
reactivity with a panel of antibodies were observed.
Both isolates
expressed proteins reactive with the panel of MAbs
(Table
1) with one exception. The isolate from
1992 lacked a
protein reactive with the MAb recognizing OspD, whereas
this protein
was detected in the isolate from 1994. When these same
nitrocellulose
filters were probed with serum samples from the patient,
only
the convalescent-phase serum from the 1994 episode of EM was found
to be significantly reactive with either of the isolates, and
it
reacted with both isolates. The convalescent-phase serum sample
from
the 1992 episode of EM had marginal reactivity with both
isolates,
whereas the acute-phase serum sample from the 1994 infection
was not
reactive with either.
Serum antibody testing.
Results of two-stage testing by EIA
followed by immunoblotting of serum samples taken in 1992 and 1994 are
shown in Table 2. The acute-phase serum
from 1992 was negative by EIA and immunoblotting. The second sample,
taken 1 month after the initial diagnosis and after treatment had
ended, was positive by EIA for IgM antibodies to B. burgdorferi but negative for IgG antibodies. Paradoxically, results of immunoblotting showed that there was reactivity with one
diagnostic band when the sample was assayed for IgM and reactivity with
five diagnostic bands in the IgG assay. The third serum sample, taken 3 months after the original diagnosis, contained no IgM antibodies
demonstrable by EIA but was positive for IgG antibodies to B. burgdorferi. There were too few bands present in this immunoblot for it to be considered positive.
The serum sample obtained at the time of diagnosis during the episode
of EM in 1994 had no antibody to
B. burgdorferi detectable
by EIA and was also negative for the Fla antigen by EIA. The second
specimen, drawn 39 days after diagnosis, contained IgG reactive
with
both the whole-cell
B. burgdorferi antigen and the Fla
antigen.
The results of immunoblotting correlated with testing results
obtained by EIA in that the first specimen was neither IgM nor
IgG
positive but the second specimen was positive for both IgM
and IgG
antibodies reactive with diagnostic bands.
Genetic analysis of the two isolates.
Restriction fragment
length polymorphism analysis was used to confirm the identification of
the two isolates and showed that both strains were B. burgdorferi sensu stricto (data not shown). It was found that the
genetic profiles of the isolates differed when the chromosomal DNA and
plasmids were separated by PFGE (Fig. 2).
The isolate from 1992 lacked an 8-kb plasmid present in the isolate
from 1994, as well as in the B31 strain. The lack of an 8-kb plasmid is
not likely to be an artifact of culture, as these analyses were done on
the first passage of the spirochete after isolation and no loss of
plasmids has been reported for such low-passage derivatives.
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FIG. 2.
Genomic DNA of the two patient isolates and B. burgdorferi B31 separated in agarose gels by PFGE. Reference size
standards are provided by a HindIII digest of
bacteriophage lambda (Lambda H3).
|
|
We have previously reported the use of PCR-SSCP to successfully
identify OspA gene alleles of
B. burgdorferi amplified from
a single tick (
11). A double-blind test of the two isolates
from this patient, with the addition of strain B31 as a standard,
was
performed. Duplicate cultures of the three strains were analyzed,
and
the results showed that the strain in one pair of cultures
was
identical to the known strain B31 used as the standard. The
other four
samples could be sorted into two pairs, each pair member
containing a
strain identical to that of the other member and
different from those
of any other pair in the analysis. Figure
3 shows a representation of these
results, with the isolates identified
and run with the allelic
standards previously reported. The banding
pattern clearly shows the
allelic differences between these isolates.
A similar system has been
developed for OspC, and the results
confirm the OspA analysis in that
all three isolates, B31 and
the two patient isolates, contain distinct
alleles of the OspC
gene (
27).
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FIG. 3.
SSCP allelic analysis of the OspA genes of the two
patient isolates, U14 (lane 5) and U193 (lane 7), and B. burgdorferi B31 (lane 2). Reference strains representing the four
alleles of the OspA gene identified by this analysis were arranged such
that each was run in the lane adjacent to that of the test strain that
also has the allele.
|
|
| |
DISCUSSION |
As has been reported earlier, prompt treatment of this patient did
not abrogate the immune response during the initial episode of disease
(22). Upon reexposure and a new episode of infection in this
patient, there was a serum antibody response to borrelia antigens
detected. During both episodes, IgM and IgG antibodies were present, as
detected by EIA or immunoblotting techniques, correlating with the
clinical diagnosis of Lyme disease and the isolation of B. burgdorferi from this patient. In neither case, however, was any
EIA positive at the time of clinical diagnosis and initiation of
treatment.
Protein analysis showed that although the two isolates were similar in
many respects, the 1994 isolate expressed bands corresponding to
molecular masses of 23 and 66 kDa that appeared to be absent from the
1992 isolate (Fig. 1). The OspD protein was also detected in the 1994 isolate, but no reactivity with the monoclonal anti-OspD was observed
with lysates from the 1992 strain (Table 1). These differences may be
significant but could also be explained by variation in the stage of
growth in vitro at the time of harvest or in the preparation of the
lysates. These analyses did not conclusively demonstrate that the
isolates were distinct and did not resolve the question of whether the
second episode of EM was a new infection or reactivation of sequelae
from the original infection.
Analysis of the genetic profiles of the two isolates however, yielded
more definitive results. For example, the 1992 isolate is lacking the
8-kb plasmid. As we previously have reported (9), this
plasmid may be lost without affecting transmission of spirochetes to
ticks or mice. Given that the 1992 isolate lacks this plasmid and the
1994 isolate still maintains it, we are very confident that these are
unique strains of B. burgdorferi. It has been shown that
B. burgdorferi can lose plasmids during chronic infection (16) and during multiple rounds of zoonotic transmission in laboratory animals (9), yet to date there has been no
description of B. burgdorferi being able to acquire genetic
elements in vivo or in vitro without active manipulation in a
laboratory system, such as by electroporation.
Finally, we analyzed the outer surface protein A (OspA) and outer
surface protein C (OspC) genes of these isolates by SSCP. Since the
OspC gene has a reported variability in nucleotide sequence (14) even in closely related strains, we predicted that
these isolates would have different alleles of the OspC gene, whereas the alleles of the less-variable OspA gene would likely be similar or
identical. Surprisingly, the results of these analyses show that both
of the isolates from this patient have different alleles of the OspA
gene and that these alleles are both distinct from those of the
reference strain, B31. The more-likely result was also observed: all
strains differ in the OspC gene allele. These genetic analyses
conclusively demonstrate that this patient was infected by independent
isolates of B. burgdorferi, and the clinical data indicate
that infection resulted from two independent exposures separated by 2 years.
Previous reports have described episodes of persistence of infection
after resolution of symptoms (24) and treatment failure in
cases of Lyme disease (25). Further, case studies describing reinfection resulting from independent exposures to ticks, with from
two years to as little as 17 weeks between episodes of infection, have
been published (17, 19). Though the clinical data and risk
behavior of the subjects were strong indications of independent infections, in no case were isolates made during both infections. A
complicating factor illuminated by the reinfection reports is that the
prompt, successful treatment of patients infected with B. burgdorferi may interfere with the generation of sufficient immunity to prevent reinfection. These reports show the necessity of
careful documentation of reinfection of an individual after apparently
successful antibiotic treatment of Lyme borreliosis.
The present case provides a unique opportunity to evaluate the
spirochete B. burgdorferi after natural infection and
treatment. To our knowledge, this is the first report of sequential,
culture-confirmed infections in a single individual. Minimally, this
report demonstrates that natural immunity to B. burgdorferi
as a result of infection had waned in this individual 2 years after
initial infection and that it is possible that new episodes of
infection in Lyme disease patients can be a result of an independent
exposure to a different strain of the bacterium.
These results are important to the diagnosis and treatment of this
infection. First, previous exposure to the spirochete complicates serum-based diagnostics in subsequent episodes of infection since anti-B. burgdorferi antibodies are still likely to be
present in the serum of these individuals. Also, it is important to
differentiate between reactivation of infection in patients that were
diagnosed and treated and a new infection. This further emphasizes the
importance of the clinical diagnosis of Lyme disease for patients with
a history of risk of exposure and clinical symptoms. As more such cases
can be documented and a sufficient number of subjects can be studied,
we may reach an understanding of the duration of natural immunity as
well as the incidence of reinfection as opposed to the incidence of
treatment failure for Lyme disease.
| |
ADDENDUM |
During the review and revision of the manuscript, Nowakowski et
al. published the description of a culture-confirmed reinfection of a
patient with B. burgdorferi (13a). As in the
present report, the episodes of infection were apparently caused by two
different strains of the bacterium.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Allergy, Department of Medicine, HSCT-16-040, State University of New
York at Stony Brook, Stony Brook, NY 11794-8161. Phone: (516) 444-2714. Fax: (516) 444-3475. E-mail:
wgolde{at}epo.hsc.sunysb.edu.
| |
REFERENCES |
| 1.
|
Association of State and Territorial Public Health Laboratory Directors.
1995.
Proceedings from the Second National Conference on Serologic Diagnosis of Lyme Disease.
Association for State and Territorial Public Health Laboratory Directors, Washington, D.C.
|
| 2.
|
Barbour, A. B.
1984.
Isolation and cultivation of Lyme disease spirochetes.
Yale J. Biol. Med.
57:521-525[Medline].
|
| 3.
|
Barbour, A. G.,
S. L. Tessier, and W. J. Todd.
1983.
Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigen determinant defined by a monoclonal antibody.
Infect. Immun.
41:795-804[Abstract/Free Full Text].
|
| 4.
|
Belfaiza, J.,
D. Postic,
E. Bellenger,
G. Baranton, and I. Saint Girons.
1993.
Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.
J. Clin. Microbiol.
31:2873-2877[Abstract/Free Full Text].
|
| 5.
|
Berger, B. W.,
R. C. Johnson,
C. Kodner, and L. Coleman.
1992.
Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin.
J. Clin. Microbiol.
30:359-361[Abstract/Free Full Text].
|
| 6.
|
Burgdorfer, W.,
A. Barbour,
S. Hayes,
J. Benach,
E. Grunwalt, and J. Davies.
1982.
Lyme disease a tick-borne spirochetosis?
Science
216:1317-1319[Abstract/Free Full Text].
|
| 7.
|
Cartter, M. L.,
J. L. Hadler,
V. J. Rhodes, and V. J. Peterson.
1989.
Reinfection with Borrelia burgdorferi.
Conn. Med.
53:376-377[Medline].
|
| 8.
|
Golde, W. T.,
T. R. Burkot,
S. Sviat,
M. Keen,
L. W. Mayer,
B. Johnson, and J. Piesman.
1993.
The major histocompatibility complex-restricted response of recombinant inbred strains of mice to natural tick transmission of Borrelia burgdorferi.
J. Exp. Med.
177:9-17[Abstract/Free Full Text].
|
| 9.
|
Golde, W. T., and M. C. Dolan.
1995.
Variation in antigenicity and infectivity of derivatives of Borrelia burgdorferi, strain B31, maintained in the natural, zoonotic cycle compared with maintenance in culture.
Infect. Immun.
63:4795-4801[Abstract].
|
| 10.
|
Gustafson, R.,
B. Svenungsson,
M. Forsgren,
A. Gardulf, and M. Granström.
1992.
Two year survey of incidence of Lyme disease and tick-borne encephalitis in a high risk population in Sweden.
Eur. J. Clin. Microbiol. Infect. Dis.
11:894-900[Medline].
|
| 11.
|
Guttman, D. S.,
P. Wang,
I.-N. Wang,
E. Bosler,
B. Luft, and D. Dykhuizen.
1996.
Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand confirmation polymorphism.
J. Clin. Microbiol.
34:652-656[Abstract].
|
| 12.
|
Hongyo, T.,
G. S. Buzard,
R. J. Calvert, and C. M. Weghorst.
1993.
'Cold SSCP': a simple, rapid, and nonradioactive method for optimized single strand conformation polymorphism analysis.
Nucleic Acids Res.
21:3637-3642[Abstract/Free Full Text].
|
| 13.
| Kuiper, H., A. P. van Dam, L. Spanjaard, B. M. de Jongh, A. Widjojokusumo, T. C. P. Ramselaar, I. Cairo, K. Vos, and J. Dankert. Isolation of Borrelia burgdorferi
from biopsy specimens taken from healthy-looking skin of patients with
Lyme borreliosis. J. Clin. Microbiol. 32:715-720.
|
| 13a.
|
Nowakowski, J.,
I. Schwartz,
R. Nadelman,
D. Liveris,
M. Aguero-Rosenfeld, and G. Wormser.
1997.
Culture-confirmed infection and reinfection with Borrelia burgdorferi.
Ann. Intern. Med.
127:130-132[Free Full Text].
|
| 14.
|
Padula, S. J.,
A. Sampieri,
F. Dias,
A. Szczepanski, and R. W. Ryan.
1993.
Molecular characterization and expression of p23 (OspC) from a North American strain of Borrelia burgdorferi.
Infect. Immun.
61:5097-5105[Abstract/Free Full Text].
|
| 15.
|
Peisman, J.,
M. C. Dolan,
C. M. Happ,
B. J. Luft,
S. E. Rooney,
T. M. Mather, and W. T. Golde.
1997.
Duration of immunity to tick-transmitted Borrelia burgdorferi in naturally infected mice.
Infect. Immun.
65:4043-4047[Abstract].
|
| 16.
|
Persing, D. H.,
D. Mathiesen,
D. Podzorski, and S. W. Barthold.
1994.
Genetic stability of Borrelia burgdorferi recovered from chronically infected immunocompetent mice.
Infect. Immun.
62:3521-3527[Abstract/Free Full Text].
|
| 17.
|
Pfister, H. W.,
U. Neubert,
B. Wilske,
V. Preac-Mursic,
K. M. Einhaupl, and G. D. Bosario.
1986.
Reinfection with Borrelia burgdorferi.
Lancet
ii:984-985.
|
| 18.
|
Preac-Mursic, V.,
K. Weber,
W. Pfister,
B. Wilske,
B. Gross,
A. Bauman, and J. Prokop.
1989.
Survival of Borrelia burgdorferi in antibiotically treated patients with Lyme borreliosis.
Infection
17:355-359[Medline].
|
| 19.
|
Rose, C. D.,
P. T. Fawcett,
J. D. Klein,
S. C. Epps,
G. M. Caputo, and R. A. Doughty.
1993.
Reinfection in pediatric Lyme borreliosis.
Ann. Rheum. Dis.
52:695-696.
|
| 20.
|
Sinsky, R. J., and J. Piesman.
1989.
Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents.
J. Clin. Microbiol.
27:1723-1727[Abstract/Free Full Text].
|
| 21.
|
Steere, A. C.
1989.
Lyme disease.
N. Engl. J. Med.
3219:586-596.
|
| 22.
|
Stobierski, M. G.,
W. N. Hall,
B. Robinson-Dunn,
H. Stiefel,
S. Shiflett, and V. Carlson.
1994.
Isolation of Borrelia burgdorferi from two patients in Michigan.
Clin. Infect. Dis.
19:944-946[Medline].
|
| 23.
|
Strand, J.,
E. D. Walker, and R. Merritt.
1992.
Vector control bulletin.
North Central States
1:111-118.
|
| 24.
|
Strle, F.,
Y. Cheng,
J. Cimpermsn,
V. Maraspin,
S. Lotric-Furlan,
J. A. Nelson,
M. M. Picken,
E. Ruzic-Sabljic, and R. N. Picken.
1995.
Persistence of Borrelia burgdorferi sensu lato in resolved erythema migrans lesions.
Clin. Infect. Dis.
21:380-389[Medline].
|
| 25.
|
Strle, F.,
V. Preac-Mursic,
J. Cimpermsn,
E. Ruzic,
V. Maraspin, and M. Jereb.
1993.
Azithromycin versus doxycycline for the treatment of erythema migrans: clinical and microbiological findings.
Infection
21:83-88[Medline].
|
| 26.
|
Walker, E. D.,
T. W. Smith,
J. DeWitt,
D. C. Beaudo, and R. G. McClean.
1994.
Prevalence of Borrelia burgdorferi in host seeking ticks (Acari:Ixodidae) from a Lyme disease endemic area in northern Michigan.
J. Med. Entomol.
31:524-528[Medline].
|
| 27.
| Wang, I.-N., J. Dunn, B. J. Luft, and D. Dykhuizen. Genetic diversity of OspC in a local population of
Borrelia burgdorferi sensu stricto. Genetics, in press.
|
Journal of Clinical Microbiology, April 1998, p. 1015-1019, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
