With the
microhematocrit
technique, in which the
cellular elements at the interface between plasma and packed
erythrocytes are examined, the limit of detection might be as low as 10
3
spirochetes (71).
...
By either phase-contrast or
dark-field
microscopy of live organisms or standard light microscopy
of stained,
fixed organisms, B. burgdorferi can usually be
distinguished from other
borreliae by its looser and more irregular coiling.
... Tinctorially,
borreliae are
gram
negative. However, the Gram stain is not nearly as
sensitive as
Giemsa
(39, 40) and silver (31, 58, 61) stains for
demonstrating the organisms.
Acridine
orange was used to detect
spirochetes in phagocytic cells (29) and in the CSF of a patient with
Bannwarth's syndrome (36). This dye was also used to stain what
appeared to be spirochetes in the urine of field mice (35).
Warthin-Starry
and
modified Dieterle
silver stains have been used to
reveal the spirochetes in a variety of biopsy and autopsy materials
that have been Formalin fixed and embedded in paraffin (31, 61, 62, 67,
99, 123, 130, 146). According to Duray and Johnson,
a modified Dieterle
stain is easier to perform than the Warthin-Starry stain
(61). In
various reports,
silver
stains have been used successfully to detect
spirochetes in <1 to 100% of ECM lesion biopsies (31, 32, 63,
67,
130); for most investigators the success has been about 40 to 50%. The
spirochetes are most easily found if the biopsy is taken from the
advancing edge of the ECM lesion and the papillary dermis is examined.
Usually fewer spirochetes are located in the center of the lesion and
in the epidermis. A "positive control" slide prepared from B.
burgdorferi cells suspended in an agar block should be included when
biopsy material is examined by silver stains (50).
In exceptional
cases, spirochetes have also been detected in synovial tissue biopsies
with either the standard or modified Dieterle silver stain (63, 98).
When seen, the numbers of spirochetes present were very low.
The
borreliae were seen within and close to small vessels displaying
microangiopathic changes (98). A
modification of the Steiner
silver
stain is reported to further improve the sensitivity of
histologic
detection (58). In this method the tissues are treated with amylase
after they have been fixed in Formalin and before the immersion in
silver nitrate. When compared with the Warthin-Starry stain, this
method has been reported to provide greater contrast between the
spirochetes and the background tissues. Using this modified Steiner
stain (Bosma-Steiner), de Koning et al. reported 100% sensitivity in
demonstrating spirochetes in skin biopsies of ECM patients and in
synovial biopsies of Lyme arthritis patients (58).
Polyclonal
antibodies have been used successfully in immunohistologic studies to
demonstrate spirochetes in tissues (35, 102). However, with monoclonal
antibodies not only are spirochete structures demonstrated, but also
the particular type of spirochete can be determined (18, 20, 21).
Using
a monoclonal antibody to a borrelial flagellar antigen, Park et al.
demonstrated spirochetes in a frozen section of skin biopsy from a
patient with ECM (133).
Monoclonal
antibodies were also used by
MacDonald and Miranda to reveal spirochetes in touch preparations of
unfixed human brain tissues from an autopsy specimen (115),
by
Magnarelli et al. to identify B. burgdorferi in the kidney tissues of a
dog with renal disease (118), and by Burgess et al. to show borreliae
in organs of an infected cow (47). Direct and indirect
immunofluorescence assays with antiborrelial antibodies have been used
to determine the prevalence of infected ticks in different geographic
areas (7, 43). Although this approach has proved useful in field
studies, laboratory experiments with ticks have shown that some
borreliae in the ticks may either not react at all with certain
monoclonal antibodies or react more weakly than they usually do with
polyclonal antisera (43, 110). This phenomenon suggests that
antigenic variation
occurs.
In
Vitro Cultivation.
Koch's postulates are partially fulfilled by isolating the offending
organism from the affected patient; this has been done in several cases
of Lyme borreliosis as described below. The culture medium is complex
and expensive and has a short shelf life. Only a minority of cultures
from definite cases of Lyme borreliosis yield spirochetes. Under these
circumstances, B. burgdorferi cultivation can hardly be considered the
diagnostic method of choice, but
this approach
remains the only way to confirm a diagnosis.
Recovery of
B. burgdorferi from a patient indicates an active or latent
disease state and not simply an inconsequential
colonization.
We first recovered a spirochete from I. dammini ticks by using
Stoenner's version of Kelly's medium (39). This, or a closely related
formulation, was then used to recover identical spirochetes from the
blood, skin, and CSF of patients with
Lyme disease (26, 161). By
additional modifications of Stoenner-Kelly medium to improve the
buffering capacity and make preparation easier (BSK medium), we were
able to isolate a borrelia from I. ricinus ticks of Europe and to grow
B. burgdorferi from a single organism (15). This culture capability
allowed us to clone the newly isolated spirochetes by limiting
dilution. For details of the current medium formulation used in our
laboratory, BSK II, see reference 11. Kanamycin and 5-fluorouracil have
been added to BSK
medium for the selective isolation of the spirochetes from ticks (96).
Others have used neomycin, gentamicin, rifampin, or kanamycin alone to
reduce contamination (9, 42, 43, 130, 161). We currently use rifampin
(50 pug/ml) and phosphomycin (100 pg/ml) to prevent the growth of other
bacteria (A. Barbour and A. MacDonald, unpublished results). ...
B.
burgdorferi is grown at temperatures between 30 and 37 °C in the
laboratory. [see also
http://lymerick.net/Borrelia-growth-optimum.html].
At temperatures above 38
°C, borrelial growth slows
substantially (11). Most investigators use temperatures of 32 to 34
°C.
The cap or lid of the
culture vessel is usually tight or sealed to
prevent loss of carbon dioxide from the medium. The
generation time is
8 to 24 h, and culture-adapted strains achieve cell densities of about
108 spirochetes per ml (11).
The
microaerophilic
character of the
borrelia is indicated by its preference for the bottom portion of the
culture medium during initial growth (11, 92). Addition of
low
concentrations (0.1 to 0.2%) of agarose to further thicken the medium
improves the recovery of B. burgdorferi from animal fluids and tissues
(4, 6, 94, 95).
... Our early studies showed that B. burgdorferi
would grow as a lawn on BSK medium containing 0.8% agarose (11). Kurtti
and colleagues subsequently grew these organisms as isolated colonies
by increasing the agarose concentration to 1.3% and doubling the amount
of gelatin (109). The plates were incubated for 2 to 3 weeks in a
candle jar. Using this solid medium, these investigators demonstrated
growth of at least two different colony types of B. burgdorferi.
...
B.
burgdorferi can survive in citrated blood stored at 4 °C for 25 days
(G. Baronton and I. Saint-Girons, Abstr. Int. Conf. Lyme Dis., abstr.
no. 55, 16 Sept. 1987), but most cultures have been inoculated soon
after blood collection.
Citrated
or heparinized blood is lightly
centrifuged to separate the plasma from the cellular blood elements.
The plasma is then centrifuged at a higher force, and the plasma pellet
is suspended in growth medium. The plasma
pellet should
contain
platelets as
borrelia
are usually found in the platelet-rich fraction
of blood. Cells in the CSF have also been concentrated by
centrifugation to improve the odds of recovery (136, 138, 139).
The
frequency of recovery of borrelia from
skin biopsies of ECM
lesions has
ranged from about 6 to 45% (8, 32, 139, 140, 160). Most biopsies
yielding positive cultures have been taken from the expanding edge of
the ECM lesion where histologic stains have shown that the
spirochetes are in highest numbers. B. burgdorferi has also been
isolated from skin biopsies of patients with acrodermatitis chronica
atrophicans of several years duration (9, 130) and patients with
lymphocytoma
(86).
A skin biopsy was
stored frozen at -80'C for 2 years before
successful recovery of borreliae in culture medium (130).
The etiologic
agent has been isolated only infrequently from affected joints (140,
153); synovial fluid cultures of humans and dogs with Lyme arthritis
have usually been negative (83, 104, 160). Nevertheless, the
ameliorating effect of antibiotics on Lyme arthritis strongly suggests
that viable organisms are required for disease progression (64, 83,
159).
B. burgdorferi has been isolated from
Ixodes spp. ticks
(2,
7, 15, 39, 43, 96, 161). The culture success rate is close to
prevalence of infection in the tick population as established by
immunofluorescent
detection. Although organisms have been isolated
from whole ticks ground up and inoculated into culture medium, the
midgut is the site most likely to contain cultivable spirochetes (39,
40, 43). Dissection of the midgut out of the tick reduces the chance of
contamination of the cultures (15, 39). Preparing serial dilutions
of the inoculated medium can also reduce the chance of contamination;
enough spirochetes are usually present in the tick to permit up to
1,000-fold dilutions of the original inoculum (15). In some studies,
antibiotics have been added to the medium to prevent overgrowth of
bacteria colonizing the exterior and interior of the tick (43, 96).
Once
in culture the borreliae may undergo change in one or more traits.
Alteration in both the size of outer membrane proteins and the
reactivity of these proteins with monoclonal antibodies has been seen
after an isolate has been passaged as few as 10 to 20 times (70 to 140
generations) in the laboratory (20, 151, 176).
Cell-Mediated
Immunity
Several
studies have been carried out on the role of cell-mediated immunity in
Lyme disease and, specifically, the interaction between B. burgdorferi
cells and the cellular elements of the immune system. Although much of
this work
does not have immediate relevance for clinical
microbiology, a brief consideration of pertinent immunologic findings
might provide useful background for understanding the disease and
future developments.
The B.
burgdorferi cells associate with macrophages and polymorphonuclear
leukocytes even in the absence of immune serum (29, 135).
Phagocytosis has been demonstrated under these conditions, but it is
still unclear whether the association between the borreliae and the
macrophages in this situation is entirely due to uptake of the
spirochetes into the phagocyte. The spirochetes that are taken up may
be those already damaged during the centrifugation and washing steps of
cell preparation.
Moreover,
the
finding that killed B. burgdorferi cells adhere to T- and B-cell
lymphocytes suggests that the spirochete has binding sites on its
surface for eucaryotic
cell ligands (68).
The addition of specific antiserum significantly increases the
association of the spirochetes with macrophages and other phagocytic
cells; this appears to be due to phagocytosis of the borreliae
(29, 135). The
borreliae appear to be killed once inside a phagocytic cell (29).
[killing rate not 100% PMID:
8423346] Uptake appears not to be
mediated
by
heat-labile plasma components such as complement (29, 135).
Investigations
of cell-mediated immunity showed a specific response by T cells of Lyme
borreliosis patients to B. burgdorferi antigens (57, 124, 132, 152).
This response has been demonstrated for T cells obtained from the
peripheral blood, the diseased synovium, and the CSF. The specific
proliferative response of T cells from either CSF or synovial fluid is
greater than that of peripheral blood T cells obtained at the same time
from a Lyme disease patient (132, 152). The greatest reactivity is at
the site of the localized infection, be it the nervous system or a
joint. Antigen-specific T-cell responses have also been demonstrated in
the synovial fluids of patients with postinfectious Reiter's syndrome
(66).
T-cell clones that
respond
specifically to B. burgdorferi antigens have been recovered from the
CSF of a patient with Lyme borreliosis (124).
Patient's T cells were
significantly stimulated by whole cells as well as soluble components
of the spirochete (57, 132, 152).
T-cell blastogenesis was more vigorous in the presence of whole
borreliae than when a sonicated supernatant was used (R. J. Dattwyler,
D. J. Volkman, J. Thomas, P. A. Falldorf, and M. G. Golightly, Ann.
N.Y. Acad. Sci., in press).
The
active response to whole cells of B. burgdorferi indicates that
patients are responding to cell surface components.
The stimulation index of T-cell responsiveness to whole borreliae has
been used at one institution to confirm the diagnosis of Lyme
borreliosis (57).
Some
patients demonstrate a significant cell-mediated immune response to the
borreliae when they have only borderline or slightly elevated antibody
titers to the organisms (57). [probably due to formation
of
immune complexes that traps many/most/all formed antibodies which are
becoming bound to antigen only whenever there are many Borreliae
currently active]. Family members of Lyme disease patients
have
higher stimulation indices than unrelated controls, indicating either a
hereditary predisposition or shared exposure to the infectious agent
(152).
The
T-cell
responses of patients show less apparent cross-reactivities than the
corresponding antibody responses to relapsing fever borreliae antigens
(152).
In affected tissues and
organs, there is usually a prominent lymphocytic infiltration. Among T cells, there are more
helper/inducer cells than cytotoxic/suppressor cells (57, 127,
152);
this is also the case in joint fluids in rheumatoid arthritis patients
(72). In the skin, large numbers of dendritic Langerhans cell have also
been seen (37). In biopsies of ECM lesions, HLA-DR markers have been
found on keratinocytes; patients acrodermatitis chronica atrophicans
have both HLA-DR and HLA-DQ markers displayed on keratinocytes in their
chronic skin lesions (172).
Infected humans produce
IgM, IgG (14, 54, 82, 116, 161, 168, 178), and
IgE antibodies (30)
that recognize B. burgdorferi
antigens. There does not seem to be a significant specific
IgA response (82). The bulk of the IgG-reactive antibodies are of the
IgG1 and IgG3 subclasses
(B. Vandvik, Abstr. Int. Conf. Lyme Dis., abstr. no. 43, 16 Sept. 1987;
K. E. Hechemy, H. L. Harris, and M. J. Duerr, Abstr. Int. Conf. Lyme
Dis., abstr. no. 18, 14 Sept. 1987).
In the IFA [indirect
immunofluorescent antibody], whole borrelial cells are invariably used.
The cells are dried on the slide with or without yolk sac material. Our
laboratory uses washed sheep erythrocytes mixed with the borreliae to
evenly distribute the spirochetes on the smear and to provide a
convenient reference point for microscope focusing (14, 21). Once dried
on the slide, the spirochetes are fixed with methanol or acetone; some
investigators freeze the slides without the organic solvent fixation
step.
While many laboratories use the original Shelter Island,
N.Y., isolate of B. burgdorferi, strain B31 (ATCC 35210), several other
isolates are also used. This practice of using different strains may
have little consequence for testing
within North America, where
strains are very similar to one another in their antigenic makeup (12,
20, 43). On the other hand, European strains are more heterogeneous in
the types of major outer membrane proteins they possess (Fig. 3) (19,
155, 176; B. Wilske, V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G.
Barbour, and M. Kramer, Ann. N.Y. Acad. Sci., in press). The
differences between strains are not great enough to completely
invalidate use of one strain, even a North American one, like B31, in
all geographic areas. This strain has been used successfully in Europe
for serologic testing (2).
Nevertheless,
a one- or two-tube difference between the reactivity of a particular
immune serum against one strain versus another could, in some
instances, mean that a sample could be called falsely negative.
There is also the question whether continuous passage of the test
strain could result in loss of certain critical antigens for IFA and
ELISA. Changes in the major outer membrane proteins OspA and OspB (Fig.
3) have been noted during serial in vitro cultivation (20, 33, 151,
l51a, 176; Wilske et al., in press).
False-positive IFA and
ELISA reactions can occur in patients with syphilis or
relapsing fever, two other spirochetal diseases (89, 116, 117).
Lyme
borreliosis patients occasionally have reactive fluorescent treponemal
antibody absorption and treponemal agglutination tests for syphilis
(89, 117, 128); in these as well as treponemal antibody
negative
cases, reagin antibody-assays, such as the Venereal Disease Research
Laboratory and rapid plasma reagin tests, are negative.
As
demonstrated by IFA, syphilis, yaws, and pinta patients have had high
titers of antibodies that react with B. burgdorferi (14, 117, 126).
The immunofluorescence
reactions
of sera from syphilis patients are qualitatively different from those
seen with sera from Lyme disease patients. The cross-reactive
antibodies of syphilis patients do not bind to the outer membrane blebs
of the spirochete,
and, consequently, the stained spirochetes appear
thinner and less irregular than fixed organisms bound by antibodies
recognizing outer membrane antigens (14). In serologic tests, some Lyme
disease patients have equivalent titers to B.
burgdorferi and B.
hermsii, a relapsing fever agent in North America (117). Patients with
tick-borne and louse-borne relapsing fever have cross-reactive
antibodies to B. burgdorferi (117). Although this is a potential
problem, the clinical
presentation and the epidemiologic features of
the case would usually allow discrimination between Lyme borreliosis
and relapsing fever. There is much less cross-reactivity between B.
burgdorferi and the leptospires (14, 117). Only a
few serovars of Leptospira interrogans seem to be weakly cross-reactive
with B. burgdorferi (118).
Some
patients with rheumatic diseases, such as rheumatoid arthritis and
systemic lupus erythematosus, which the physician may be trying to
distinguish from Lyme borreliosis, have false-positive reactions in B.
burgdorferi serologic
tests (14, 53, 116, 144). These reactions
appear due to the nonspecific sticking of rheumatoid factor aggregates
or immune complexes to the borrelial antigens. One indicator of a
false-positive IFA reaction among rheumatic disease patients is the
beaded appearance of the spirochetes (128). This type of staining
reaction is also seen in false-positive fluorescent treponemal
antibody-absorption test results of patients with systemic lupus
erythematosus and other autoimmune diseases (125).
IgM antibody
determinations tend to be less specific than those for IgG antibodies,
but may be useful in early disease or when reactivation or reinfection
is suspected (54, 126). Patients with infectious mononucleosis often
have falsepositive IgM tests (126, 161, 168). Magnarelli and Johnson
found that 5 of 16 patients with Rocky Mountain spotted fever and three
of 7 patients with rheumatoid arthritis had a positive IgM-specific
ELISA for B. burgdorferi (116).
While patients with second- or
third-stage Lyme borreliosis almost always have elevated IgG titers,
those with early disease often have serum antibody titers below the
diagnostic threshold for 6 weeks or more after onset (2, 54). Only
about 50 to 60% of patients with early disease, i.e., ECM, have
diagnostic titers as measured by either IFA or ELISA (2, 14, 54, 126).
Antibiotic therapy of first-stage disease may blunt the immunoglobulin
response to the point that diagnostic thresholds are never
reached
(161). In cases of reinfection, the antibody titers to B. burgdorferi
may show a fourfold rise from the previous convalescent value (137).
In
patients with a neurologic disorder attributable to Lyme borreliosis,
the antiborrelia antibody concentrations in the CSF are usually higher
than could be accounted for by leakage of circulating antibodies into
the CSF. Any CSF titer above 5 is probably significant (168). However,
antibodies may be present in the CSF as a consequence of disturbance of
the blood-brain barrier. One indication of nervous system involvement
is the presence of oligoclonal immunoglobulin peaks in the
CSF
but not in the serum (82, 84, 129, 143). To further establish that the
antiborrelia antibodies were produced intrathecally, a comparison of
serum antibodies and CSF antibodies can be carried out.
CSF/serum-specific antibody ratios can be adjusted by using factors
that take
into account the total IgG, IgM, or albumin concentrations
in the CSF and serum. The resultant indices serve to identify those
patients with antibody produced locally in the central nervous system
(82, 84, 85, 105, 168, 170, 178). Calculations of such an index may be
needed, for example, to accurately diagnose the disease in a patient
who has a neurologic disorder resembling multiple sclerosis and an
elevated titer to B. burgdorferi in the serum.
Western blot (immunoblot)
assays have been performed on a research basis to determine to which
protein antigens patients are responding with antibody (12, 14, 51, 53,
74, 176, 178; Wilske et al., in press). These studies have confirmed
the finding of IFA and ELISA studies that there is a delay in
production of detectable amounts of antibody to the borreliae. Once
antibody production begins, it is usually in the form of IgM antibody
to flagellin (18), a 41,000-dalton (41-kilodalton [kDa]) protein that
is the predominant component of the flagella (53, 74). With
time,
both IgM and IgG antibodies to a variety of other antigens appear;
these include proteins with apparent molecular weights of 15,000,
27,000, 55,000, 60,000, 66,000, and 83,000 (12, 14, 53, 177,
178).
The 66-kDa protein appears to be another protein associated with the
outer membrane (20, 51). The more chronic and complicated the disease,
the greater the number of antigens to which the patients respond.
Almost all patients with Lyme disease of more than a few weeks duration
have IgG antibody to the 41-kDa flagellin protein (14, 51, 53, 74).
Some patients have IgE antibodies to the 41-kDa protein (30).
Other
abundant proteins of the B. burgdorferi cell are the major
surface-exposed proteins OspA and OspB (87, 88). In most North American
strains, the apparent molecular weights of these proteins are 31,000
and 34,000, respectively (Fig. 3) (20, 21). These proteins
appear
to be highly immunogenic in experimental animals that have been
injected with whole organisms (20, 21; unpublished observations).
Paradoxically, humans with Lyme borreliosis develop antibody against
OspA and OspB, if they develop them at all, only late in the course of
the disease (14, 51, 53). Sera from patients with other spirochetal
disease have shown cross-reactions in Western blots to the 41- and
60-kDa proteins of B. burgdorferi (14, 74, 176). Considering the known
antigenic relatedness between the flagella of the different Borrelia
spp. (18), one might expect some degree of cross-reactivity to the
flagellin protein. Epitopes of the 60-kDa protein antigen appear to be
conserved among various spirochetes (75a, 176).
Qualitative
as well as quantitative differences may be seen in Western blots and
other immunoblotlike assays that use serum and CSF obtained from the
same patient with neurologic involvement (129, 178). To
date,
these differences have not been noted when paired serum and synovial
fluid specimens from patients with arthritis have been examined (53;
unpublished observations). The B-cell response to B. burgdorferi has
been demonstrated by studies of immunoglobulin synthesis and
specificity on an individual B-cell level (111).
The finding of
almost universal responsiveness to the 41-kDa flagellar protein has
been used by investigators as a point of departure for studies of
subunit components of B. burgdorferi. Coleman and Benach used purified
flagellin protein eluted from sodium dodecyl sulfate-polyacrylamide
electrophoresis gels (51); the protein presumably was denatured during
purification. These investigators found that an ELISA based on a cruder
but undenatured "flagellin-enriched" fraction was more sensitive than
an ELISA that used purified flagellin eluted from a gel.
Hansen
et al.
isolated whole flagella through mild detergent disruption of the cells
and subsequent density gradient ultracentrifugation (76); flagella
remain intact by this method (18).
This
group used the isolated flagella as the antigen in ELISA testing and
found improved sensitivity in serologic tests of patients with early
disease when compared with a standard ELISA (76). The
heightened sensitivity was due in part to lowering of the cutoff point
between positive and negative reactions.
The assay of Hansen and co-workers appeared to provide greater
discrimination between patients with Lyme borreliosis and those either
without disease or with nonspirochetal disorders.
The outer membrane
OspA and OspB proteins are other isolated components of the borrelial
cell that have been examined by Coleman and Benach for use in
immunoassays (51). This study, which used eluted proteins in an ELISA,
confirmed the Western blot analyses that showed antibodies against
these antigens appearing later in the course of the disease. Although
these outer membrane proteins may not be useful for immunodiagnosis of
early disease, they could have a role as components of a very specific
assay in secondary or tertiary disease in humans. A patient with Lyme
arthritis had antibodies that bound to recombinant OspA and OspB
proteins (88), and, thus, it is likely that patients are responding to
the proteins themselves and not carbohydrate or glycolipid moieties
that might be associated with them.
The
Western blot analysis has been proposed as a practical clinical
laboratory test for Lyme borreliosis (74). The advantage to this
procedure is that the response to individual components can be examined.
Grodzicki and Steere found
the
Western blot to be the most sensitive test in early Lyme borreliosis
(74). Kirsch et al. used the Western blot to diagnose Lyme disease in a
patient with a fatal illness (102). Almost all immunodiagnostic assays
reported on have used culture-grown borreliae that were washed and
centrifuged at least twice before use in the assay. Whether or not
loosely associated spirochetal antigens, such as a slime layer, could
be dislodged from the cell surface during antigen preparation is not
known. Neubert and colleagues used borreliae obtained directly
from the blood of an infected mouse for their IFA; however, the
Borrelia species used in the test was not B. burgdorferi (130). Another
area that has been little investigated is whether there are
important nonproteinaceous antigens of B. burgdorferi.
TYPING STRAINS
As
the number of isolates of B. burgdorferi from different human and
animal sources and from different parts of the world increases, greater
attention is being paid to strain distinctions. Several options are
available, including poly-acrylamide gel electrophoresis profiles of
cellular proteins, reactivities of monoclonal antibodies, and plasmid
analysis. The initial isolates of B. burgdorferi from the United States
were almost identical in their polyacrylamide gel electrophoresis
profiles (12, 20, 21, 43). They all had major proteins of 31 (OpsA) and
41 (flagellin) kDa. A large majority had an abundant 34-kDa surface
protein, OspB, but some isolates either lacked this protein or had an
OspB with a slightly different electrophoretic migration (12, 20). As
more isolates from Europe were examined, differences in the
OspA
and OspB proteins were noted (19, 155, 176; Wilske et al., in press).
The OspA-like proteins varied from approximately 30 to 33 kDa in
apparent size. OspB-like proteins also varied; some European
strains had no major protein that could be considered the equivalent of
OspB. Some strains, especially those from regions of Germany, Austria,
and Scandinavia, lacked even an OspA-like protein. Instead, they had a
major protein of about 22 kDa. This protein has been designated "pC" by
Wilske et al. until its surface localization can be confirmed (176; in
press). A single United States strain with a major surface protein of
about the same size as pC has been isolated from a tick in California
(33).
When antisera prepared
against whole cells of different strains have been compared by IFA, too
few differences in the reactivities of the various isolates have been
seen to justify a serologic typing scheme based on use of antisera
against whole cells (7). Polyclonal antibodies to isolated
cell
components, such as OspA and pC protein, offer better discrimination
between strains (Wilske et al., in press), as do monoclonal antibodies.
The monoclonal antibodies are directed against single epitopes in one
protein, usually either OspA- or OspB-like proteins (20, 21; Wilske et
al., in press). Using criteria of polyacrylamide gel electrophoresis
profiles, polyclonal antisera reactivities, and monoclonal antibody
binding, Wilske et al. (in press) identified seven distinct types of B.
burgdorferi among a panel of European strains.
Another way to
characterize B. burgdorferi isolates is to analyze their plasmid
content; both circular and linear plasmids have been identified (13,
16, 90, 151a). A relatively simple extraction procedure can be used to
enrich for plasmids in the DNA preparation (13, 16). The plasmid
species are then separated on low-percent agarose gels. Analyses have
shown considerable heterogeneity in plasmid profiles among strains,
even those from North America (13).
Plasmids
either undergo rearrangement or are lost from the cell during serial in vitro
cultivation (13, 90, 151a).
DNA hybridization of whole chromosomal DNA has shown that B.
burgdorferi is a distinct species in the genus Borrelia (90, 92, 93,
148) and that strains within the species differ in the amount of DNA
relatedness. These differences may not be great enough, however, to use
genomic DNA hybridization as a routine typing procedure for B.
burgdorferi. Its most appropriate use is still as a tool for
determining whether an unknown arthropod-associated spirochete is a
member of the genus Borrelia and to what species it is most closely
related. Use of DNA probes for specific genes, such as the ospA gene,
may offer more advantages for distinguishing between strains within the
species (19).
LABORATORY SAFETY
B. burgdorferi is a
blood-borne pathogen of humans and domestic animals that can cause
significant and prolonged disease. It may be confused with a variety of
other chronic, noninfectious disorders. B. burgdorferi, like the
relapsing fever borreliae, has been considered a biocontainment level 2
[
Wiki]
organism, and it is appropriate to continue to treat it as such.
Although there have been no documented examples of laboratory-acquired
Lyme borreliosis in humans*, there clearly has not been enough
experience with the organism to be complacent about its risk to
laboratory workers.
The
most likely routes of infection would be through a break in the skin,
the conjunctiva*, and the oral mucosa; experience with the
closely related relapsing fever borreliae indicates
that infection can occur through these routes (17).
Infected
blood and cultures pose the greatest potential risk, but animal and
laboratory workers may also be infected through contact with urine of
infected animals (35, 46) and through handling live ticks.
There is probably little chance of infection through aerosolization or
contact with spirochetes that have dried on animate or inanimate
materials.
*) 1996 PMID: 8817171 describe
2 cases of direct transmission of Borrelia into the eye
via blood spatter accidents i.e. in the laboraty setting - so
wear protection like glasses, mask and gloves, when handling
possibly infected blood samples and other material, both for
own safety reason and to avoid contamination of the sample! In
six patients, a skin biopsy specimen was taken at the site of a
previous erythematous skin lesion 1 to 6 months after disappearance of
the lesion. Four of them presented with early disseminated Lyme
borreliosis. In one additional patient with early disseminated Lyme
borreliosis, the site of a previous tick bite was biopsied.
None of these patients
had been treated with antibiotics before presentation.
The
cultures of the skin biopsy specimens of the seven patients showed
growth of Borrelia species. By rRNA gene restriction analysis and
genospecies-specific PCR, six isolates were classified as
Borrelia garinii
and one as Borrelia group VS461 [
Borrelia
afzelii].
Excerpt on procedure:
...
A 4-mm-diameter biopsy was taken with a biopsy punch (Disposable Biopsy
Punch; Stiefel, Wachtersbach, Germany) after the skin was treated with
either 1% iodine (wt/vol) or 0.5% chlorhexidine in 70% (vol/vol)
alcohol.
...
Culture method for B. burgdorferi. Skin biopsy
specimens were transferred to
5.5
ml of modified Kelly's medium
(28) in 7-ml Duran borosilicate glass disposable culture tubes (Schott,
Mainz, Germany). Prior to use, the tubes were rinsed 10 times with
distilled water and subsequently sterilized with heated air at 180 °C
for 2 h. Rifampin (final concentration of 50 mg/liter) and fosfomycin
(final concentration of 100 mg/liter) were added to the medium to
inhibit the growth of contaminating bacteria. Preliminary experiments
had shown that these concentrations of antibiotics did not inhibit the
growth of
B. burgdorferi.
For
culturing of CSF specimens, approximately 2 to 4 ml of CSF was
centrifuged at 5,000 x g for 20 min. The pellet was transferred to 5.5
ml of modified Kelly's medium without antimicrobial agents. Incubation
at
33 °C
was done
for 8 weeks. The presence of spirochetes was determined by dark-field
microscopy weekly. A culture was considered positive when motile
spirochetes were seen. All isolates were preserved at - 70 °C with 50%
glycerolpeptone for cryoprotection.
... 57 consecutive patients
referred to our hospital. All these patients were examined by a
dermatologist (Toni Ramselaar or Irina Cairo) and met the Centers for
Disease Control case definition criteria for EM.
The recovery rate of B. burgdorferi from skin biopsy specimens from
patients with EM was 86% (36).
...
Four of them (patients 1, 3, 4, and 5) had neuroborreliosis, as shown
by an increase of the leucocyte count and protein level in the CSF.
Patient 2 had clinical symptoms of arthritis and cardiac involvement.
... Three of the four patients with neuroborreliosis had also
antibodies
against B. burgdorferi in the CSF; one patient (patient 4), who only
had immunoglobulin M (IgM) antibodies against B. burgdorferi in her
serum, had no CSF antibodies against B. burgdorferi.
...
Two other patients were included in the study, since they reported
previous erythematous lesions and had antibodies against B. burgdorferi
in their sera.
One
of
them (patient 6) [sampled 60 days after disapperance of erythema
IgM+IgG serum antibody positive] had mild myalgia at the time of skin
biopsy, whereas the other patient (patient 7) [sampled 180 days after
disappearance of erythema, IgM negastive, IgG positive] was
asymptomatic.
...None of the cultures became contaminated
with other microorganisms. From patient 7, a culture from a second skin
biopsy specimen taken at a site without a previous skin lesion remained
negative.
... It was concluded that the strains from patients 4 and 6 were
B. garinii
and the strain from patient 7 was group VS461and that an additional
typing method was necessary for the other four strains. Therefore,
isolates were also genotyped by genospecies-specific PCR (18).
Using the primer pair specific for
B. garinii,
a 527-bp fragment was amplified from the strains recovered from
patients 1 through 6 (Fig. 4). A 591-bp fragment was amplified from the
isolate from patient 7 by the primer pair specific for group
VS461.
Therefore,
we conclude that the strains from patients 1 through 6 were B. garinii and the
strain from patient 7 belonged to group VS461 [~Borrelia afzelii].
Detection
of antibodies to B. burgdorferi. Antibodies to B. burgdorferi in serum
and CSF samples were detected by flagellum-enzyme-linked immunosorbent
assay (ELISA) (
Dakopatts
A/S, Glostrup, Denmark) (13).
Table
1: One patient (#4) with radiculopathy and CSF pleocytosis of
207
and Sp-protein 0.69 g7l was IgM positive, but IgG negative for
Borrelia antibodies in serum at time of biopsy 32 days after
disappearance of the skin lesion; all the others
were IgG
serum antibody positive.
... Interestingly,
at
42 days after the onset of disease one of the seven patients (patient
4) had developed only IgM but no IgG antibodies against B. burgdorferi. This
is unusual, although such patients were also described by Hansen and
Lebech (14), who reported that 44 of 187 patients with neuroborreliosis
had only IgM antibodies against B. burgdorferi; the range of their
disease duration was 6 to 54 days, with a median of 19 days.
14:
Hansen K, Lebech AM. The clinical and epidemiological profile
of
Lyme neuroborreliosis in Denmark 1985-1990. A prospective study of 187
patients with Borrelia burgdorferi specific intrathecal antibody
production. Brain 1992;115:399-423.
We cultured B. burgdorferi from CSF samples from two of
four patients included in this study
(50%), but CSF cultures from 13 other patients with neuroborreliosis
seen in our hospital remained negative. Our total recovery rate from
CSF specimens from patients with neuroborreliosis was therefore 3 of 17
(18%), which is similar to the recovery rate reported in the
above-cited studies. The rather high recovery rate from CSF specimens
from the four patients included in this study could possibly be
explained by the recent onset of neuroborreliosis in them; also,
Karlsson et al. reported that CSF cultures were most successful for
patients with a recent onset of neurological symptoms (15).
Usually,
patients with EM are treated with an antibiotic, since such treatment
is generally effective in preventing the onset of early disseminated or
chronic LB (4).
Our
patients had no antibiotic treatment because their skin lesions had not
been recognized as EM by themselves or by their general physicians.
Whether antibiotic treatment always leads to elimination of
B. burgdorferi
from the skin has been studied by culturing posttreatment skin
biopsy specimens taken from healthy-looking skin from the site
from which
B.
burgdorferi had
been cultured before treatment (8, 23). In total, 26 patients were
involved in these studies, and cultures were negative in all cases.
However, B.
burgdorferi
has been cultured from skin biopsy specimens obtained at the sites of
previous erythematous lesions from three patients after antibiotic
therapy (27).
27:
Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross
B, Baumann A, Prokop J. 1989. Survival of Borrelia burgdorferi
in antibiotically treated patients with Lyme borreliosis. Infection
17:355-359. PMID: 2613324
In
vitro studies showed that human skin fibroblasts can protect B.
burgdorferi from antibiotic treatment (11). Invasion of spirochetes
into fibroblasts has been documented and could serve as a possible
mechanism by which spirochetes
can escape from antimicrobial actions of antibiotics (16).
Intracellular
persistence of spirochetes
could also lead to a decrease of the inflammatory skin reaction and to
fading of the erythema. Alternatively, a down-regulation of the cellular
immune
response, which has been documented for the related microorganism
Treponema pallidum (10), could explain the disappearance of the
erythema during persistence of microorganisms in the skin.
Several
pathogens of humans and domestic animals
depend on hematophagous arthropods to transmit them from one vertebrate
reservoir host to another
and maintain them in an environment. These pathogens use antigenic
variation to prolong their circulation in the blood and thus increase
the likelihood
of transmission. By convergent evolution, bacterial and protozoal
vector-borne pathogens have acquired similar genetic mechanisms for
successful
antigenic variation. Borrelia spp. and Anaplasma
marginale (among bacteria) and African trypanosomes, Plasmodium
falciparum, and Babesia bovis
(among parasites) are examples of pathogens using these mechanisms.
Antigenic variation poses a challenge in the development of vaccines
against
vector-borne pathogens
Results:
Compared with Slovenian patients, U.S. patients had erythema migrans
for a briefer duration (median duration, 4 days compared with 14 days;
P , 0.001) but were more likely to have systemic symptoms (P 5 0.01),
abnormal findings on physical examination (P , 0.001), and
seroreactivity (P , 0.001). Central clearing of erythema migrans
lesions was more likely in Slovenian patients (P ,0.001).
Conclusions:
Erythema migrans caused by B. afzelii in Slovenia and erythema migrans
caused by B. burgdorferi in New York have distinct clinical
presentations. Caution should be used when clinical and laboratory
experience from one side of the Atlantic is applied to patients on the
other.