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,
*
Section of Rheumatology, Department of Internal Medicine,
Section of Immunobiology, and
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520;
§
Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, Davis, CA 95616; and
¶
Department of Tropical Public Health, Harvard University School of Public Health, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. burgdorferi N40 preferentially synthesize BBK32 and BBK50 during murine and human Lyme disease. These proteins were first simultaneously identified by differentially probing a B. burgdorferi N40 expression library with sera from B. burgdorferi-infected mice and sera from mice hyperimmunized with killed spirochetes (11, 16). RT-PCR and immunofluorescence studies then directly showed that bbk32 and bbk50 were expressed in vivo but not in vitro (11, 20). BBK32 and BBK50 Abs were also found in the sera of B. burgdorferi-infected mice and individuals with Lyme disease, further indicating that these Ags were synthesized during infection of the vertebrate host (11, 21). Studies by Probert and Johnson (13) then showed that BBK32 is a fibronectin-binding protein and plays an important role in the attachment of spirochetes to the extracellular matrix. These studies also confirmed that BBK32 was not synthesized by B. burgdorferi N40 during in vitro cultivation (13). It was also demonstrated, however, that five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates synthesized BBK32 or other fibronectin-binding proteins when cultured in BSK II medium (13, 22).
Humoral responses to BBK32 and BBK50 can influence the course of Lyme borreliosis. Murine arthritis and carditis develops at 2–4 wk and regresses during the subsequent 1–2 mo. The resolution of disease correlated with the genesis of high-titer BBK32 and BBK50 Abs (11). The ability of BBK32 and BBK50 Abs to afford immunity was therefore directly tested. Active immunization with BBK32 and BBK50, or passive immunization with BBK32 and BBK50 antisera, partially protected mice from challenge with a syringe inocula of spirochetes (11). Partial or complete immunity following vaccination has been observed with several other B. burgdorferi proteins, most notably OspA, OspB, OspC, OspF, and decorin-binding protein (Dbp) A (23, 24, 25, 26, 27, 28, 29). OspA has been the most extensively studied immunogen, has successfully completed phase III clinical trials, and has been approved by the Federal Drug Administration for use as a vaccine against Lyme disease (30, 31).
The administration of BBK32 and BBK50 Abs to mice that had been infected with spirochetes for 24 h also eradicated B. burgdorferi infection (11). Postinfection clearance of B. burgdorferi has not been observed with OspA Abs and has only been described in experimental syringe-challenge studies with Abs to OspC and DbpA (29, 32, 33, 34). To extend these observations to human infection, the Ag-specific humoral responses in a cohort of patients with untreated Lyme disease were correlated with progression from early- to late-stage disease (21). High levels of BBK32 Abs were associated with early-stage disease that resolved, whereas the lack of significant BBK32 Abs resulted in an increased likelihood of the development of late-stage Lyme arthritis. These studies suggest that BBK32 and BBK50 Abs, and in particular BBK32 Abs, can affect the course of Lyme disease in mice or humans.
An investigation of bbk32 and bbk50 expression throughout the spirochete life cycle should provide a more realistic understanding of the importance of these proteins. Research on the selective expression of bbk32 and bbk50 in vivo has relied upon comparing spirochetes cultured in BSK II medium with B. burgdorferi in mice following experimental challenge with inocula of in vitro-cultivated organisms. Obviously, spirochetes are not normally exposed to BSK II medium, but rather persist in a tick-mouse-tick cycle (1). More specifically, spirochetes reside in particular tissues in the vector or host at precise intervals during infection. B. burgdorferi are located in the gut of flat I. scapularis and migrate to the salivary glands of feeding ticks before moving to a reservoir host. Spirochetes are then deposited into the host skin. After several days, B. burgdorferi disseminate via the bloodstream to distant tissues. Spirochetes must then return from the host to the ticks that feed on B. burgdorferi-infected mice because the efficiency of transovarial transmission is low (35). Temporal and tissue-specific B. burgdorferi gene expression should consider these diverse niches.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A clonal isolate of B. burgdorferi N40 that is infectious and pathogenic in mice was used throughout (36). Spirochetes were cultivated in BSK II medium at 33°C (37).
Animals
Three-week-old female C3H/HeNCr (C3H) and C.B-17-scid mice were obtained from the Frederick Cancer Research Center (Frederick, MD) and housed in filter-frame cages. Mice were given food and water ad libitum and euthanized with CO2.
Two hundred- to 500-g female Hartley guinea pigs were housed in individual hanging cages at Yale University animal facilities.
Ticks
Mated adult female I. scapularis were collected in the field. The egg mass was then laid in the laboratory. Hatched larvae were fed on uninfected C3H mice to produce pathogen-free nymphs. Alternatively, to generate B. burgdorferi-infected nymphal ticks, larvae were allowed to engorge to repletion upon B. burgdorferi N40-infected mice and then molt to the nymphal stage. B. burgdorferi infection rates within the nymphs were >90%. All tick rearing was performed in an incubator at 26°C with 85% relative humidity and a 12-h:12-h light:dark photo period regimen.
Infection and immunization
For infection, 4- to 6-wk-old C3H mice were intradermally challenged with 104 B. burgdorferi N40 and sacrificed at 14, 30, and 60 days. In an experiment, groups of 10–20 mice were inoculated with B. burgdorferi and 4–5 mice were sacrificed at each time point. All studies were repeated, and in each experiment, tissue samples from each mouse were collected and stored individually.
For the postinfection clearance studies, mice were first infected with 104 spirochetes by intradermal syringe inoculation and administered 200 µl of rabbit BBK32 and BBK50 antisera on days 1, 2, 4, or 8 (11). Control mice were administered glutathione transferase (GT) antisera in an identical manner (11). The BBK32 and BBK50 antisera had been previously produced (11). BBK32 antisera were prepared by immunizing animals with the p35 portion of the BBK32 protein and have been shown to be immunogenic in the murine model (11). The full-length BBK32 protein from B. burgdorferi N40 has also been produced in our laboratory, but is not highly antigenic in its current form (data not shown) and was therefore not used for these studies. Mice were killed at 14 days. At necropsy, blood, spleen, urinary bladder, and skin specimens were collected aseptically, cultured in BSK II medium, and examined by dark-field microscopy for spirochetes. The tibiotarsal joints and the hearts were Formalin-fixed, paraffin-embedded, sectioned, and examined microscopically for inflammation (36). Arthritis was graded by blinded analysis on a scale from 0, no inflammation, to 3, severe disease (36).
For the larval acquisition studies, groups of four to five C.B.17-scid mice were infected with 104 B. burgdorferi by intradermal challenge. At 20 days, mice were passively immunized with 200 µl of BBK32 and BBK50 antisera or a control GT antiserum. Twenty-four hours after passive immunization, mice were anesthetized with ketamine/xylazine and 50 larvae were placed on each mouse. The larvae fed to repletion and were examined for spirochetes. Individual larvae were tested by immunofluorescence (see below), using B. burgdorferi-antisera, for the presence of spirochetes.
For the tick-borne transmission studies, groups of four to five mice were first administered 200 µl of BBK32 and BBK50 antisera. Twenty-four hours later, 4 or 20 I. scapularis nymphs, infected with B. burgdorferi N40, were allowed to feed to repletion on each of the mice. Engorged ticks usually fell from the animals within 4 days and were collected in water baths below the cages. Two weeks later, all mice were sacrificed and examined for infection and disease, as previously stated.
In the guinea pig infection studies, a 5- to 10-cm section of the animals’ posterior mid thorax was shaved and then three to four adult ticks were applied per guinea pig and allowed to attach. The adult ticks were obtained from groups of nymphal ticks that had fed upon mice immunized with BBK32, BBK50, or GT antisera and then allowed to undergo the molting process. Guinea pigs were sacrificed 2 wk after tick challenge. Sera were collected and tested for Abs to B. burgdorferi by immunoblot (38).
PCR
PCR amplification of B. burgdorferi DNA was performed for 30 cycles with denaturing, annealing, and extension temperatures of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. 5' and 3' primers were: 5'-CAATCAGGTAACGGC-3' and 5'-TCTATTTTGGAAAGC-3' for flaB, 5'-GC GGTGAAAGTGGTGAATTGG-3' and 5'-CTTTAGCAAAGTTGTCAAGGCGTG-3' for bbk32, and 5'-GACAACAACAGAAGTGGTCGCAC-3' and 5'-TAGCATCGGAA TGAGCTGTACC-3' for bbk50. flaB, which has one copy on the B. burgdorferi chromosome, was used to quantify the organism load (18).
For RT-PCR, total RNA was isolated from the spleens, skin, tibiotarsal joints, bladders, and hearts of B. burgdorferi-infected mice by acid guanidinium thiocyanate phenol-chloroform extraction (16). RNA was treated with RNase-free DNase (Promega, Madison WI) for 3 h at 37°C. A total of 2 µg of RNA was then reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA). PCR was then conducted for 30 cycles.
For quantitative PCR, a competition assay was performed based on established techniques (12, 39). Competitor constructs were created in which a 540-bp fragment of unrelated DNA was flanked by the flaB, bbk32, or bbk50 primers. The unrelated DNA was a fragment of the gene encoding the 44-kDa Ag of the agent of human granulocytic ehrlichiosis, amplified using the Ehrlichia primers 5'-TTTGCAGGACGAAGAAGAATGG-3' and 5'-CCAACGCAAGCATAAGGAACAAC-3', which have previously been used in constructs for competitive PCR (40). Varying concentrations of competitor were added to the RT-PCR reactions, and the concentration at which the amplified competitor product and the gene of interest were similar in intensity was used to calculate the quantity of mRNA or DNA.
Immunofluorescence of B. burgdorferi within ticks
Groups of B. burgdorferi N40-infected ticks, including unfed ticks and ticks that fed to repletion on mice, were checked for spirochetes, and the presence of specific B. burgdorferi Ags, within the vector by indirect immunofluorescence (IFA). Each tick was dissected to separate the internal organs from the cuticle and lightly homogenized in 100 µl of PBS. Then triplicate 10-µl aliquots were spotted on silylated glass slides. The slides were air dried and fixed with 4% paraformaldehyde. The specimens were incubated in rabbit BBK32, BBK50, OspC, or OspA antisera (1:100 dilution) for 1 h, washed, followed by incubation in anti-rabbit IgG coupled with FITC at a 1:500 dilution for another hour, and viewed under a Zeiss Axioskop fluorescence microscope (Oberkochen, Germany) (11, 27, 41). B. burgdorferi antisera or FlaB antisera, which recognizes B. burgdorferi within both unfed and engorged ticks, was used as positive controls (4, 40). GT antisera was used as the negative control. Ten high-power fields (x400) were examined, and the total number of spirochetes was calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bbk32 and bbk50 have been shown to be
induced during murine infection and in several patients with erythema
migrans or Lyme arthritis (11, 20). We have now
quantitatively examined the temporal and tissue-specific expression of
B. burgdorferi bbk32 and bbk50 during
experimental murine Lyme borreliosis. Following intradermal challenge,
spirochetes are deposited in the skin and then begin disseminating
(4–10 days) to the joints, heart, spleen, and bladder (36, 42). Arthritis and carditis then become prominent (2–4 wk) and
regress over 1–2 mo (36, 42). At later intervals, mice
remain infected but disease is no longer apparent (36, 42). bbk32 and bbk50 mRNA levels were
therefore first measured at the cutaneous site of inoculation from days
1 to 14 (Fig. 1
) and then at different
disseminated sites on days 14, 30, and 60, intervals that reflect the
various stages of disease (Table I).
flaB mRNA, which is constitutively expressed, was used for
comparison (12). Representative examples of the
flaB, bbk32, and bbk50 quantitative
RT-PCR are shown in Fig. 2.
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). At this time point, quantitative DNA-PCR revealed that 2 x
104 ± 0.5 x 104
spirochetes were present at the site of inoculation, representing the
original inocula and perhaps some initial spirochete growth. By day
6, flaB mRNA levels increased 10-fold (37 ± 10
femtograms/tissue specimen) from the initial values before declining on
day 14. Quantitative DNA-PCR demonstrated that the spirochete number
increased up to 3 x 105 ± 0.6 x
105 on day 6 before declining, reflecting the
changes in flaB mRNA detection, and further demonstrating
that flaB mRNA levels can be used to follow spirochete
burden in infected mice (12). bbk32 ands
bbk50 mRNA levels were not significant on day 1, consistent
with previous studies that B. burgdorferi N40 does not
immediately express these genes following intradermal syringe
challenge. Low levels of bbk50 were detected at the local
site of inoculation on days 4–14 and reached levels of up to 6.0
± 2 femtograms/tissue specimen on day 5. As the number of total
spirochetes declines at the local site of inoculation on day 14, more
bbk50 mRNA is expressed per spirochete at this later
interval than at earlier time points. bbk32 mRNA levels
greatly increased from days 3 to 7 and then declined. The change in
bbk32 expression is consistent with the study of Probert and
Johnson (13) demonstrating the BBK32 is a
fibronectin-binding protein and may therefore required during the
initial stage of infection.
Gene expression in the skin, joints, heart, spleen, and bladder was
then assessed during disseminated murine infection (Table I). Skin
specimens were obtained from the murine mid-posterior thorax, away from
the site of inoculation. flaB mRNA was detected in all of
the tissues and at all intervals, confirming that spirochetes were
present at these locations and that B. burgdorferi mRNA had
been successfully isolated. flaB mRNA levels increased from
days 14 to 30 and then decreased at 60 days, consistent with studies
suggesting that the number of spirochetes decrease in mice as the host
immune response to B. burgdorferi expands. At 14, 30, and 60
days, flaB mRNA levels were generally greater in the joints,
heart, and spleen than in the skin and bladder, suggesting that the
spirochete burden is higher in these tissues. bbk32 and
bbk50 mRNA levels were lower than flaB mRNA
levels at each location. At 14 days, bbk32 and
bbk50 mRNA levels were generally similar in the tissues
examined. At 30 days, bbk32 mRNA levels in the skin, heart,
and spleen were greater than the bbk50 mRNA levels,
particularly in the spleen. bbk32 and bbk50 mRNA
levels in the joints and bladder at 30 days were not different. At 60
days, bbk32 and bbk50 mRNA levels declined as the
number of spirochetes decreased in all tissues; however, spirochetes in
the spleen continued to produce more bbk32 than
bbk50 mRNA.
B. burgdorferi bbk32 and bbk50 expression in ticks
Previous research demonstrated that B. burgdorferi N40
expressed bbk32 and bbk50 during mammalian
infection but not when cultured in vitro (11). B.
burgdorferi are, however, maintained in a natural
vector-host-vector cycle and not usually exposed to laboratory medium.
bbk32 and bbk50 expression in ticks was therefore
investigated to further understand when these genes are normally
expressed. Individual spirochetes within ticks are readily discernible
by IFA, unlike B. burgdorferi within murine tissues.
Therefore IFA rather than RT-PCR was primarily used to examine
spirochetes within the vector. BBK32 and BBK50 Ag on spirochetes within
flat and engorged I. scapularis were examined by
immunofluorescence using BBK32 or BBK50 antisera. BBK32 and BBK50 were
not evident on spirochetes within unfed ticks but were apparent on
B. burgdorferi in engorged ticks (Fig. 3). The number of BBK32- and
BBK50-producing spirochetes was determined by probing B.
burgdorferi within flat and fed ticks with selected antisera.
Fields containing 
1000 spirochetes were first examined by dark-field
microscopy. As the number of B. burgdorferi within ticks
increases exponentially during engorgement, a larger number of fields
from flat tick extracts were required to identify 1000 organisms.
Following engorgement, 57 ± 3% of B. burgdorferi
synthesized BBK32 and 78 ± 4% produced BBK50 (Fig. 4). As expected, B.
burgdorferi- or FlaB antisera (controls) readily identified
spirochetes in flat and engorged ticks. OspA-producing spirochetes were
only abundant in the flat ticks, and OspC-synthesizing organisms
increased in number during feeding, consistent with previous reports
(4, 5, 7). RT-PCR substantiated the IFA results (Fig. 5). bbk32 and bbk50
mRNA were detected in engorged, but not flat ticks, whereas
flaB mRNA (control) was evident in both groups of ticks.
These studies demonstrated that bbk32 and bbk50
are expressed on B. burgdorferi within feeding
I. scapularis ticks.
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BBK32 and BBK50 antisera have previously been shown to protect
mice from experimental challenge with an intradermal inocula of
spirochetes and also to clear B. burgdorferi from mice
infected with spirochetes for 24 h (11). We have now
performed studies to examine whether the postinfection immunity
phenomenon extends to later intervals after infection (Table II). Results were similar when
102 rather than 104B. burgdorferi were used as the challenge inocula (data not
shown). Partial eradication was observed when BBK32 and BBK50 antisera
were administered up to 2 days after infection. At 4 and 8 days after
infection, when spirochetes had begun to disseminate from the skin to
distant organs, B. burgdorferi clearance was no longer
evident. In addition, passive immunization with BBK32 and BBK50
antisera at these intervals did not influence the severity of disease
in the B. burgdorferi-infected mice. Arthritis was graded on
a scale from 0 to 3, with 1, 2 and 3 representing mild, moderate, and
severe inflammation, respectively. Tibiotarsal joints from BBK32- and
BBK50-immunized mice had mild to moderate arthritis (1.7 mean ±
0.6 SD, similar to control mice (1.5 mean ± 0.5 SD).
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). Passive transfer of murine BBK32 antisera to
naive mice partially prevented B. burgdorferi infection
transmitted by tick bite. Murine BBK50 antisera did not provide
immunity and the combination of murine BBK32 and BBK50 antisera did not
afford greater protection than murine BBK32 antisera alone. Although
the passive transfer of murine BBK32 antisera was partially protective
against B. burgdorferi infection when 4 ticks engorged on
mice, protection was not obtained when 20 ticks were placed on each
animal. Influence of BBK32 and BBK50 antisera on B. burgdorferi within ticks
Spirochetes synthesize BBK32 and BBK50 within ticks during
engorgement and BBK32 antisera provides partial protection against
tick-borne infection. Therefore, the effects of BBK32 and BBK50
antisera on spirochetes within engorging ticks were examined. The
number of spirochetes within ticks that fed on mice administered BBK32
or BBK50 antisera were assessed by IFA (Table III). Spirochetes were abundant in
nymphal ticks that fed on mice given GT antisera (control). B.
burgdorferi within ticks that engorged on mice administered BBK50
antisera also had substantial numbers of spirochetes. Ticks that fed on
mice given BBK32 antisera had markedly fewer spirochetes (90% less)
and the combination of BBK32 and BBK50 antisera did not result in a
greater reduction in spirochete number.
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). The number of spirochetes in
adult control ticks was less than that in engorged control nymphs
because B. burgdorferi increase in number during tick
feeding. We then performed studies to determine whether this reduction in B. burgdorferi due to BBK32 antisera could prevent spirochete transmission from ticks to a mammalian host. Guinea pigs were used for these experiments because adult ticks do not engorge on mice and guinea pigs can be infected with B. burgdorferi (38, 43). Four or five adult ticks that had been exposed to GT, BBK32, or BBK50 antisera during nymphal engorgement were allowed to feed to repletion on guinea pigs. The guinea pigs were then assessed for exposure to B. burgdorferi infection by immunoblot (38). Guinea pigs fed upon by GT (three of four animals) or BBK50 (three of three animals) antisera-exposed ticks, but not ticks exposed to BBK32 (zero of four animals) or BBK32 and BBK50 (zero of three animals) antisera had evidence of B. burgdorferi infection. These data demonstrate that BBK32 antisera inhibited spirochete transmission by adult ticks.
BBK32 and BBK50 antisera inhibits larval acquisition
BBK32 Abs play an important role in B. burgdorferi
survival within ticks. Therefore, the effects of BBK32 and BBK50
antisera on the larval acquisition of spirochetes were examined because
these Ags are synthesized within engorging ticks and during mammalian
infection. SCID mice were infected with B. burgdorferi and
at 14 days the animals were administered BBK32 and BBK50 antisera.
Larvae were allowed to engorge on the mice and spirochete transmission
from the host to larvae was examined by IFA (Fig. 6). Larvae that engorged on control
animals had large numbers of spirochetes. In contrast, ticks that fed
on BBK32- and BBK50-immunized mice had reduced numbers of B.
burgdorferi.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We then performed a detailed analysis of the expression of bbk32 and bbk50 during murine Lyme borreliosis. In contrast with erpT, an in vivo-expressed gene which appears to be most prominently expressed in extracutaneous tissues, bbk32 and bbk50 expression were apparent in diverse tissues, including the skin, joints, heart, spleen, and bladder of infected mice (12). Expression was most abundant on day 30 and then declined. At this later interval (60 days), mice have developed high titer BBK32 and BBK50 Abs, and the lower levels of expression may represent either the destruction of spirochetes expressing bbk32 or bbk50 or the down-regulation of gene expression. Our early time points studies also demonstrate that bbk32 and bbk50 are expressed at low levels early after intradermal inoculation and that bbk32 expression then rapidly increases at a greater rate than bbk50 at the local site of inoculation.
BBK32 antisera influenced spirochete survival at various intervals in the B. burgdorferi life cycle, most prominently in the tick. We demonstrated that the "postinfection clearance" of BBK32 and BBK50 is not apparent after 2 days. One hypothesis to account for this short-lived effect is that the accessibility of these proteins to Ab during the course of infection may influence protective immunity (44). Various Abs have borreliacidal effects at different intervals of infection. OspA Abs are only protective when administered before spirochete challenge, presumably because of the substantial down-regulation of ospA expression in vivo. In contrast, BBK32 Abs have an early clearance effect similar to what has been seen with DbpA Abs (29). Since both BBK32 and DbpA mediate spirochete attachment to the extracellular matrix, these Abs may influence B. burgdorferi at this location, perhaps before spirochetes have been able to coat themselves with fibronectin or decorin. In contrast, Abs to OspC, when repeatedly administered in high doses, have the capacity to clear spirochetes after several weeks of infection, a phenomenon that has not been demonstrated with other antisera (33, 34). Therefore, Abs to different in vivo-expressed proteins, when used together, and at different intervals may have synergistic effects on preventing spirochete survival and persistence.
BBK32 antisera has a significant impact on the survival of spirochetes within ticks and in the ability of ticks to maintain spirochetes throughout the molt. Spirochete numbers are reduced, but not eradicated, within ticks that engorged on BBK32-immunized mice and adult ticks could then not readily transmit B. burgdorferi to guinea pigs. These data show that the Abs interfere with the capacity of ticks to retain organisms and transmit infection. This effect is also evident during larval acquisition, for BBK32 and BBK50 antisera interfered with the ability of larval ticks to acquire spirochetes when feeding on immunized SCID mice. The ability of these antisera to affect spirochetes in the vector may account for enhanced protection against tick-borne challenge rather than needle inoculation.
BBK32 and BBK50 are two B. burgdorferi genes that are expressed during tick feeding and then also during systemic murine infection. Abs to these proteins, in particular BBK32, can partially protect mice from tick-borne infection with B. burgdorferi and may therefore be used as alternative vaccines that destroy B. burgdorferi at different time points. The diversity, and expression, of BBK32 by different B. burgdorferi isolates will help determine whether BBK32 will have broader utility as an immunogen. BBK32 antisera also had the capacity to affect spirochete survival within ticks, especially during the molting process. Therefore, another strategy to control B. burgdorferi would be to selectively immunize the natural reservoir, i.e., Peromyscus leucopus, with BBK32, thereby reducing the larval acquisition of spirochetes, and the ability of engorged nymphs and adults to harbor the pathogen. A detailed understanding of the temporal expression of B. burgdorferi genes and the specific location in which Abs can exert pressure on the spirochete may lead to new rational approaches to multivalent vaccines that can effect the spirochete during different stages of the B. burgdorferi life cycle.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard A. Flavell, Section of Immunobiology, Yale University School of Medicine, 310 Cedar Street, FMB 412, P.O. Box 208011, New Haven, CT 06520-8011.
3 Abbreviations used in this paper: Osp, outer surface protein; Dbp, decorin-binding protein; GT, glutathione transferase; IFA, indirect immunofluorescence.
Received for publication October 8, 1999. Accepted for publication March 2, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. Lahdenne, J. Panelius, H. Saxen, T. Heikkila, H. Sillanpaa, M. Peltomaa, M. Arnez, H.-I. Huppertz, and I. J.T. Seppala Improved serodiagnosis of erythema migrans using novel recombinant borrelial BBK32 antigens J. Med. Microbiol., July 1, 2003; 52(7): 563 - 567. [Abstract] [Full Text] [PDF]
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T. R. Crother, C. I. Champion, X.-Y. Wu, D. R. Blanco, J. N. Miller, and M. A. Lovett Antigenic Composition of Borrelia burgdorferi during Infection of SCID Mice Infect. Immun., June 1, 2003; 71(6): 3419 - 3428. [Abstract] [Full Text] [PDF]
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